HK1015693A - Inhibin compositions and methods of enhancing production performance - Google Patents

Description

Statin compositions and methods of improving productivity

the technical field to which the invention belongs

In general, the invention relates to methods of increasing the production capacity of a bird by administering to the bird a heterologous protein comprising a statin protein or fragment thereof and a carrier protein. The invention also relates to methods of increasing the productivity of a bird by administering to the bird a fusion gene product comprising a gene encoding the expression of an alpha-subunit avian inhibin protein, or a fragment thereof, and a gene encoding the expression of a carrier protein. The invention also relates to the heterologous protein and fusion gene products described above and to methods of producing these products.

Background

The ratite species is non-flying, and is generally a large walking (running) bird that is involved in several orders, including the following species: ostrich, pyro, Rhea, turkey, and wingless birds. Bird eating (Dromicius novaehollandiae) is an Australian ratite bird characterized by a primary wing and a pinnate head and neck. Adult avians average about 6 feet in height and weigh about 150 pounds. Ostrich (Struthio camelius) is a large walking bird with very small wings but well-developed legs. A standard adult camel bird is about 8 feet tall and weighs about 325 to 375 pounds. The term "rhea" is a generic term for members of Rheiformes of the order Oreoidea. Rheiformes is a category of southern American walkers called American ostriches which are otherwise characteristically distinguished from true ostriches by their small size, with a pinnate head and neck and three toe feet (feet).

Ostriches and pyrotechnics have long been of significant commercial value in south africa and australia, respectively, where they naturally grow. The demand for camel bird products has been around for over 100 years, and in essence their skins, meats and feathers have a broad market. For example, camel skin is used for boots, handbags, coats, handbags, wallets, and many other items. Ostrich feathers are used in fashion clothing, make-up wear, and feather dusters.

In contrast, the pyrotechnics is a new commodity in the market. It is valuable for the same product to incorporate essential oils used in the cosmetic industry. The thick subcutaneous fat layer provides pyroxylin oil with a strong permeability that makes it useful in cosmetic creams (e.g., wrinkle-removing emollients). Also, experimental studies are being conducted on the possible medical uses of the oil of avians, such as the treatment of arthritis. Typical adult avians may reach heights of 1.6 to 1.9 meters or more and weights of 30 to 45kg or more. The pyro-avians mature for about one year, and the pyro-avians before and after the puberty never show sex-specific phenotypic differences. Similar to camels, the avians population in the united states has also experienced a period of dramatic growth over the past few years. In the united states, the total number of pyrotechnics in 1994 was approximately 150,000 (including 15,000 pairs of breeding-stage pyrotechnics). It is expected that the total number of pyrotechnics will increase further to 500,000 to 750,000 in 1995, with an estimated 45,000 pairs of breeding-stage pyrotechnics.

There is an increasing demand for ratite type products in countries including australia, belgium, israel, canada, the netherlands, nanobira, south africa and zimbabwe. Thus, over the past few years, the domestic market for camel birds, pyro birds, and to a lesser extent rhea has grown dramatically. In the last 5 years, the number of pairs of ostriches and the total number of ostriches which can breed in the United states have increased by 7.5 and 20 times respectively. It is estimated that in 1995, 200,000 ostriches will be present in the united states, including 20,000 pairs of fertile ostriches. The great benefit of raising these animals is the apparent value of adult birds as well as young birds, especially fertile ostriches up to $75,000.OO or more per pair, and fertile pyrotechnics up to $30,000.00 or more per pair. Young camels, three to four months old, are about $7,500.00 expensive, and young turkeys are about $5,000.00 expensive. Most of these birds are purchased three to six months of age.

In addition, it is of great interest to use the ratite as a replacement for traditional forms of animal husbandry. Several factors associated with ratites have given priority to the breeding of ratites as a replacement for some traditional forms of animal husbandry (i.e., breeding beef cattle, swine and sheep). These factors include: higher food conversion rate, greater ability to concentrate, large body size, increased reproductive capacity and specific meat nutritional value of these animals.

For example, red camel bird meat like beef contains very little fat, calories, and cholesterol compared to chicken or turkey meat. More specifically, 85 grams of camel bird meat contains 2 grams of fat, 58 milligrams of cholesterol, and 97 calories. In contrast, 85 grams of turkey has 3 grams of fat, 59 milligrams of cholesterol, and 135 calories. 86 grams of chicken contained 3 grams of fat, 73 milligrams of cholesterol and 140 calories. 85 grams of beef (steak) contains 15 grams of fat, 77 milligrams of cholesterol, and 240 calories. Finally, 85 grams of pork contained 19 grams of fat, 84 milligrams of cholesterol, and 275 calories. (values for camel bird meat are from AMSI quality laboratory report # 0800100. values for other meat are from U.S. D.A. manual No.8, "nutritional value of food"). Like camels, the pyrotechnics meat is a low-fat red meat. More specifically, 100 grams of turkey meat contained 1.7 grams of fat, 57.5 milligrams of cholesterol, and 109 calories (the values for the turkey meat are from the Silliker laboratory of Texas corporation).

Furthermore, ratites such as ostriches can provide approximately 100 pounds of meat at 12 months of age, and thus provide considerable meat over a relatively short period of time.

An illustration of the ratite type, which is an alternative to the traditional form of animal husbandry, is the following comparison of ostriches and cattle. First, camel birds have a 42 day gestation or hatching period, while cattle require 280 days. Second, on average, each camel produces 20 more offspring per year, while cattle produce one offspring per year. Third, the food conversion rate of camel birds is less than 2: 1, while that of cows is 5: 1. Fourth, the ostrich takes about 407 days from conception to slaughter, while the cow takes 645 days. Finally, ostriches have feathers in addition to meat and leather, while cattle cannot produce products other than meat and leather.

Considering these attributes and the increasing demand for meat by the world population (meat has nutritional value and low fat and cholesterol content, and can be produced efficiently with minimal negative impact on the environment), the future growth of the ratite industry has a high potential.

The current demand for the flat chest category is much greater than the supply. However, in most female breasts of reproductive age, flat-breast producers are limited by undesirable egg production. Depending on the species, the production capacity of most ratites today is 10-20 eggs per year, and their genetic potential for egg production is considered to exceed 60 per year. For example, a wild high-producing ostrich lays one egg every 48 hours during the breeding period, and a wild high-producing turkey lays one egg every 72 hours during the breeding period. In contrast, ostriches are generally known to produce one egg in 5-10 days, and pyrotechnics typically produce one egg in 4-8 days.

Only if a sufficient number of offspring of the ratite class are produced each year, the slaughter market for the ratite class can be developed. According to certain predictions, at least 250,000 birds per year are necessary to maintain supply in the slaughter market. Thus, a method of increasing productivity would greatly facilitate the development of the slaughter market. Thus, there is a need for compositions and methods for increasing the productivity of birds, particularly ratites such as ostriches and pyrotechnics.

In addition to the recently emerging flat-breast industries, there are also many industries involved in poultry species that have developed well, mainly laying hens (Single Comb White leghorns), broilers (broilers suitable for roasting), turkeys, ducks, geese, quails. Worldwide demand for poultry meat and egg products is enormous and has been steadily increasing over the past 10 years.

The following attempts are made to describe the scale and complexity of the poultry industry (except for ratite production) in the united states. For ease of discussion, it should be understood that current population numbers (U.S. census, 1990 data) indicate approximately 250 million residents in the united states. Furthermore, the population plan in the united states was about 275 million by 2000, a figure representing a 25 million population growth from 1990 to 1995 (using the U.S. d.a. 1995 plan figure). Broiler chicken and turkey suitable for broiling were consumed by each individual at average 76.7 and 18.1 pounds respectively (see, not signed, poultry processing reference manual, meat processing, vol.33 (9): 22-25.(1994) and Bowman, m., beef and pork: competition for food production value, meat processing, vol.33 (12): 16-25, (1994)). The average poultry meat consumed over the last 5 years was 94.8 pounds per person.

In addition, poultry meat consumption has been reported to be on a steadily increasing trend. For example, chickens consumed per person have increased by approximately 28 pounds (from 55.6 pounds in 1985 to 83.5 pounds in 1995). Likewise, turkeys consumed per man has increased by approximately 2.5 pounds (from 15.9 pounds in 1989 to 18.4 pounds in 1995). It is expected that this trend towards increased consumption of chicken and turkey meat products will increase with the expected growth of the human population (see numbers cited above). If these two predictions are correct, then by 2000, approximately 16.6 million tons of chicken and turkey will be consumed annually in this country alone. This increasing trend in poultry consumption is directly related to the fact that poultry meat is considered a "heart-beneficial" food (low animal fat content) and, due to its inexpensive price (pricewise), it has advantages in competition with more expensive red meat such as beef, pork and lamb.

Due to the ever increasing demand for products, increasing the production capacity of chickens and turkeys to breed hens has important economic value for the rapidly growing industries today. Meeting consumer demand and maintaining the competitive advantage of meat price, the task of chicken and turkey breeders is to continue producing as much hatching eggs as possible. Therefore, any method that can increase egg production (even by a small amount) would produce significant economic benefits.

For example, a single-breed hen (based on rearing of one hen) that produces another 15 eggs in one production cycle (about 1 year) is scheduled as follows. Selling these extra hatched chickens at the current market chicken price ($ 0.16/chicken) will receive a revenue of $2.00, continuing to raise these chickens, selling the chicken will receive a revenue of $10-12.00 (a suitable inference considering feed costs and fixed costs). Since it is estimated that hens giving birth in the united states exceed 60 million hens, the economic gain is about $750+ million. All revenues from the commercial meat side (meat-side) of poultry (including all poultry with increased meat consumption, not including ratites) are conservatively estimated to be between $1 and 2 billion (including revenues that can be predicted by increased productivity of turkey breeders (as well as hens, both the specialized poultry business of crow, geese and quail and the females of chicken discussed above).

It should be noted that the poultry industry was first developed in this country primarily as the egg-eating industry. This genetic selection is then detrimental to egg production rates when selecting for increasing the weight of the poultry (broiler chicken). In other words, egg production is inversely related to the genetic changes found after selecting for increased body weight. Thus, the potential to increase the production capacity of the hen and turkey females (broiler birds) through endocrine manipulation (which the present invention considers to be) is greater in broiler birds than in single crown white leghorn (birds bred for enhanced egg production since the end of the 20 th 19 th century). On the other hand, as described below, laying birds are 3 to 4 times more abundant in the united states than do chickens. Thus, even small improvements in egg production capacity are enormous when considering the size of the flock that may be affected.

The scale and importance of the egg industry in this country is discussed below. Homo-egg consumption remained at a fairly constant level since 1989 to 1992, varying from 30.0 to 30.4 pounds (see table 653: homo-egg consumption of main foods, us 1984-92; agro-statistical table, 1993, u.s.d.a. national agro-statistical table, u.s.govt. print office, washington, d.c., p.457; 1993 and 1994). The rate of growth in consumption of table eggs has been stalled in the united states may be primarily related to public concern about consuming too much egg yolk, which has a high cholesterol content.

Despite the steady trend in average human egg consumption rates, the number of laying birds has steadily increased, from 228.8 million females in 1990 to 240.7 million in 1994 (see Bell, D.D., California university statistics reports monthly, Table 28: laying birds: number in farm in this month, 1980-93, UC Rivers). The trend towards increasing numbers of laying hens simply reflects an increase in egg demand to meet the increase in population, the increase in export of eggs and egg products, or other uncertain factors. For whatever reason, since almost 80% of egg production in the world is not in the united states, this suggests that any perceived positive impact on the us egg-on-demand market may be expanded 5-fold to achieve its impact on the world-wide economy. However, if this calculation is limited to the United states only, it is based on the average human consumption being stabilized at about 30.2 pounds and the population of the United states will increase to 275 million in the next 5 years, so that by 2000, 4.2 million tons of eggs will be produced annually in this country only.

Due to the existence of many complex characteristics of the production cycle of the leghorn which affect the decision-making, such as the cost of renewing the pullets by pulltrusion, the adverse effect of pulltrusion on the production performance after pulltrusion, the loss of egg sales during pulltrusion, etc., it is difficult to obtain economic benefits in improving the egg production performance of laying birds. However, on a per hen basis that is only half the size discussed above, it is unreasonable to conclude that an increase in laying eggs by laying hens means several million economic benefits.

The phrase "increasing production capacity" is understood by those of ordinary skill in the art to mean that the female birds are increased in one or more of the following ways: promoting the onset of egg production; facilitating the start of a maximum egg production phase; the egg laying period is prolonged; increasing egg production intensity; or increase the total egg production throughout life. This phrase also includes improving feed conversion; the quality of the eggshell is improved; or to improve its resistance to adverse egg laying conditions such as heat stress, overcrowding, malnutrition and noise. This phrase is meant to increase the males in one or more of the following ways: accelerating the onset of puberty or the production of sperm; promoting the onset of the maximum sperm production phase; prolonging the production of sperm; increase the production intensity of sperm (sperm count); increasing ejaculation volume; the survival ability of the sperms is improved; increase testosterone production; or to increase libido.

Recently, the hormone statins have been investigated as potential means of increasing ovulation in mammals. Inhibin is a peptide hormone produced primarily by the gonads, specifically by the growing follicles and testes. In mammals, it functions as an inhibitory feedback regulator of the follicle stimulating hormone ("FSH") secretion from the pituitary gland. Although the presence of statins was first proposed more than 60 years ago, its chemical isolation was only recently completed.

Inhibin in mammals is a dimeric protein hormone composed of an alpha-subunit (MW 18,000) and a beta-subunit (MW 14,000). The α -subunit is unique to inhibin, while the β -subunit dimer forms activin (an FSH released from the pituitary gland). The beta-subunit is in two forms (beta)_AAnd beta_B) There are two forms that are different but very similar. Thus, according toThe related beta-subunit, inhibin is inhibin-A or inhibin-B. When the α -subunit and β -subunit are linked by a disulfide bond, their biological activity is to inhibit secretion of follicle stimulating hormone ("FSH") from the pituitary. The amino acid sequence of the inhibin alpha-subunit exhibits approximately 80-90% similarity in porcine, bovine, human, murine and domestic chicken species. Isolation, production, measurement and biological effects of statins can be found in "modern statin biology" by Risbrid et al, the report on Endocrinology (Copenh), 122: 673-682, (1990); and Rivier, c, et al, "study of inhibin hormone family": for review, hormone studies 28: 104-118(1987), the entire contents of which are incorporated herein by reference.

In mammals and birds, FSH has an effect on the growth and development of follicles, while luteinizing hormone ("LH") is thought to induce ovulation. Several brain and gonadal factors (peptides and steroid hormones) interact to control gonadotropin release. Among these factors, gonadotropin releasing hormone ("GnRH") and inhibin have opposite control effects on the secretion of pituitary FSH in mammals. Gonadotropin releasing hormone is brain decapeptide, which acts to stimulate secretion of FSH and LH; inhibin is a gonadal protein, and its apparent action is to selectively inhibit secretion of mammalian FSH.

Understanding the role of statins in avian ovulation endocrine control requires a basic understanding of the avian ovulation process. In the functionally mature ovaries of domestic females, the growing follicles are present in tissues of different sizes. A typical ovary comprises 4 to 6 large, 2-4 cm diameter, yolk filled follicles (F)₁To F₄，F₆) (ii) a There are also a large number of smaller, 2-10 mm, yellow follicles and a number of very small white follicles. Largest pre-ovulating follicle (F)₁) Ovulation the next day, the next largest follicle (F)₂) Ovulation the next day (after approximately 26 hours), and so on. Little is understood about the recruitment and development of follicles in this layer of tissue. The intervention of pituitary gonadotropins has been demonstrated, however the role of statins in the control of the secretion of avian gonadotropins and in the control of ovulationIt is unclear.

Recent strategies for inducing excessive ovulation in mammalian species have been developed as a method in connection with the neutralization of the endostatin activity. For example, mammals have been investigated for active immunity to various statin-containing compounds. Immune neutralization of inhibin in heifers, sheep, gilts and rats is associated with increased ovulation rate.

The increase in ovulation rate found in mammals following administration of an somatostatin preparation is believed to be the result of increased plasma FSH levels (promoting the development of ovarian follicles). The increase in ovulation rate in mammals has been demonstrated using various antigens as vaccines in the study. Some antigens tested in mammals include: recombinant DNA derived from a fragment of the inhibin alpha-subunit (Wrathall et al, J. Productivity Progredien 95: 175-182, 1992; and Meyer et al, antisera to inhibin alpha-chain peptide neutralize biological activity and increase ovulation rate of inhibin in sheep; Minnesota agricultural laboratory science series, article No. 17, 103, 1991); synthetic peptide replicas of the N-terminal sequence of the α -subunit of bovine inhibin in combination with ovalbumin (Glencross et al, Effect of active immunity of heifers to inhibin on plasma FSH concentration, ovarian follicular development and ovulation rate, J.Endocrinology, 134, 11-18, 1992); synthetic peptide sequences of the bovine inhibin alpha-subunit which bind to human serum albumin (Morris et al, influence of immunization with synthetic peptide sequences of the bovine inhibin alpha-subunit on ovulation rate and fertility twins rate in heifers, J. Prolifera 97: 255-261, 1993); inhibin partially purified from bovine follicular fluid (Morris et al, use of inhibin partially purified from bovine follicular fluid for immunization of pre-pubertal ewes with low and high ovulation genotypes, Theriogenology, Vol.35 No.2, 1991).

In all periodic mammals studied, although conflicting with data on how FSH levels fluctuate during the ovulation cycle, immune neutralization of the endoproteolytic inhibin, regardless of the antigen used or the species of mammal challenged, continues to promote ovarian follicular development and ovulation rate.

As mentioned above, the relationship of inhibin to the regulation of reproductive function in avian species is not known. To date, published papers have focused on the reproductive function of statins in domestic poultry. The extensive literature supports the theory that inhibin exhibits physiological effects in poultry comparable to those reported in the literature in mammals: in the female bird, statins may be regulators of follicle recruitment and/or development. However, inhibin may or may not be involved in the control of ovulation rate in avians by inhibiting pituitary FSH secretion. For example, although lower laying hens were found to have higher laying hens and higher levels of inhibin in plasma and the granulosa cell layer of the prearranged ovarian follicles, no difference was found in the correlation of plasma FSH levels with laying rates. Wang et al, increased expression of the α -inhibin gene and plasma immunoreactive inhibin levels in the ovaries of domestic females correlated with decreased ovulation rates, general and comparative endocrinology, 91, 52-58, (1993). Thus, this reference suggests that in females, the expression of the inhibin alpha-subunit gene and the variation in plasma immunoreactive inhibin levels associated with ovulation rate cannot directly affect ovulation rate by modulating plasma FSH levels. Furthermore, Johnson, P.A. (inhibin in female birds, poultry science 72: 955 + 958, (1993)) has shown that successful evaluation of immunoreactive inhibin in the plasma of female birds using the bovine inhibin RIA system, however, despite the push of LH on preovulation, no significant peak of immunoreactive inhibin is detected throughout the ovulation cycle. Thus, the role of inhibin in avian follicular production remains unclear.

Recently, the α -subunit of chicken inhibin was successfully cloned and sequenced. Wang and Johnson, complementary deoxyribonucleic acid cloning and sequence analysis of the α -subunit of chicken ovarian granulocytic inhibin, reproductive biology, 49, 1-6, (1993), the entire contents of which are incorporated herein by reference. Avian inhibin sequences exhibit 86-89% homology to known mammalian inhibin alpha-subunit sequences. Northern subunit analysis using two separate probes (cINA6 and cINA12) indicated that the inhibin alpha-subunit was expressed in chicken ovarian granulosa membrane cells, but not in chicken brain, kidney, liver or spleen tissue.

Thus, inhibin biology in avians is poorly understood and no attempt or test has been made to respond to antigen inhibin challenge in avians. Thus, since the ratite market is substantially limited by the undesirable productivity of many camels, pyrotechnics and rhea, there is a need for a composition and method for increasing the productivity of these birds.

Furthermore, there is a need to improve the production capacity of all poultry in order to increase the number of poultry produced for consumption, to increase the efficiency of such production or the feed conversion ratio. Thus, there remains a need for a composition and method that increases or improves productivity of all poultry, including chickens, turkeys, ducks, quail and geese.

In addition, there is a need for a composition and method for improving the productivity of exotic birds, such as psittaciformes. The order psittaciformes includes parrots, which are the orders of monomias in birds, with a toe-in and a powerful hooked beak. Parrots are defined as any member of the family avian psittacifidae (the only family of the order psittacifidae) and are characterized by short, strong, hooked mouths.

The need for compositions and methods to increase productivity is not limited to birds. There is also a need for effective compositions and methods to improve productivity in many animals. For example, there is a continuing need to improve productivity in most agriculturally fed animals (e.g., pigs, cattle and sheep). There is also a continuing need to improve the productivity of furred animals (mink, fox, otter, ferret and raccoon) and rodents (such as rats, mice, gerbils and hamsters) used as pets and experimental research subjects, and there is an increased need for improved productivity of hides of other animals for decorative purposes.

In addition, there is a need for compositions and methods that improve productivity in order to increase the population of many animals (e.g., exotic and endangered species) and avoid their extinct. There is a continuing need to improve the productivity of animals such as horses, dogs, cats, zoo animals and circus animals for competition, entertainment or performance (competition). There is also a need to improve productivity in humans, as indicated by an increased need to treat infertility in humans. Thus, there is also a need for compositions and methods that improve productivity in many animals.

Summary of The Invention

In general, the invention relates to methods for improving the productivity of such animals by administering to said animals a heterologous protein comprising a statin protein or fragment thereof and a carrier protein. The invention also relates to methods for improving the productivity of such animals by administering to said animals a fusion gene product comprising a gene encoding the expression of an alpha-subunit inhibin protein, or a fragment thereof, and a gene encoding the expression of a carrier protein. Administering to the animal an effective amount of the heterologous protein or fusion gene product to generate an immune response in the animal against the heterologous protein. It will also be appreciated that the methods of the invention promote the production capacity of inhibin-producing animals. Preferably, the animal is a bird. More preferably, the avian is a chicken. Another preferred bird is a ratite (e.g., a turkey, ostrich, rhea, or turkey).

The invention also relates to the heterologous proteins and fusion gene products described above, and methods for producing them. More specifically, the present invention is directed to compositions and methods for producing a heterologous protein comprising a statin or fragment thereof and a carrier protein. The inhibin protein or fragment thereof can be avian inhibin, mammalian inhibin, pisatin, or reptilian inhibin. Carrier proteins include, but are not limited to: maltose binding protein, thyroglobulin, keyhole limpet hemocyanin or bovine serum albumin. The preferred carrier protein is maltose binding protein.

The heterologous protein may be a statin bound to said carrier protein or a statin fused to said carrier protein. The method of producing such a fusion heterologous protein comprises inserting a cDNA encoding for the expression of inhibin, or a fragment thereof, into a vector containing the information encoding for the production of the carrier protein. After the vector is inserted into an expression system, the fusion heterologous protein is expressed by the system. Preferably, the heterologous protein comprises a ratistatin (e.g., ostrich statin, pyrostatin, and rhestatin). Another preferred heterologous protein includes chicken inhibin.

The present invention is also directed to a method of improving productivity of an animal by administering a heterologous protein of the invention comprising a statin protein or fragment thereof and a carrier protein. In one embodiment, the method comprises administering to the female an effective amount of such a protein. In another embodiment, the method comprises administering to a male animal an effective amount of such a protein. Ideally, an immune response is generated in these animals directly against these heterologous proteins. More desirably, the immune response generated in the animal is also directed against a statin protein (endostatin) produced by the animal.

The invention is also directed to a fusion gene product comprising a gene encoding an α -subunit inhibin protein, or a fragment thereof, and a gene encoding the expression of said carrier protein. The gene encoding inhibin protein or its fragment can encode and express avian inhibin, mammalian inhibin, pisatin or reptilian inhibin. The gene encoding the expression of said carrier protein may encode maltose binding protein or bovine serum albumin expressed therein. Preferred genes encoding expression of the carrier protein encode expression of maltose binding protein.

The invention also relates to methods for improving the productivity of such animals by administering to said animals a fusion gene product comprising a gene encoding the expression of an alpha-subunit inhibin protein, or a fragment thereof, and a gene encoding the expression of a carrier protein. More particularly, the invention also relates to methods of gene therapy, i.e., introducing DNA sequences encoding inhibin, or a fragment thereof, and a carrier protein into an animal. The fusion gene product of the present invention can be administered directly to the animal, or can be administered to the animal via a vector or a vector-containing cell (such that the cell contains the fusion gene product).

The methods of the invention increase the productivity of inhibin-producing females (e.g., mammals, reptiles, fish and birds). More specifically, the method improves the productivity of female galliformes and ratites. More specifically, the method increases the productivity of chickens, turkeys, ostrich, turkey, and rhea. This method also improves the productivity of turtles, including endangered species of turtles. Unexpectedly, the method of the present invention promotes the onset of puberty or egg production in animals. In addition, the method of the present invention unexpectedly facilitates the start of the maximum laying period for the animal. In addition, the methods of the invention increase the strength of the egg produced by the animal. It is also surprising that the method of the invention extends the maximum egg production period of the animal. In addition, the methods of the invention unexpectedly increase the total egg production of an animal throughout its life. In birds, the methods of the invention also improve feed conversion ratio of birds. In addition, the methods of the present invention unexpectedly reduce or eliminate the effect of adverse egg laying conditions on the egg production rate of animals exposed to such adverse conditions. Such adverse conditions include elevated temperature, crowding, nutrient deprivation and noise.

Surprisingly, the method of the invention also improves the productivity of inhibin-producing males, such as mammals, reptiles and birds. More specifically, the methods of the invention increase testosterone levels in males. Similarly, the method of the invention increases the onset of puberty or sperm production in the male. In addition, the method of the invention promotes the onset of the maximum sperm production phase in males. Furthermore, the method of the present invention unexpectedly increases the intensity of sperm production (sperm count) in males. Also, the methods of the invention extend the maximum spermatozoa production phase of the animal. In addition, the methods of the invention improve the viability of animal sperm. In addition, the methods of the present invention unexpectedly reduce or eliminate the effects of adverse conditions on animal sperm cells exposed to such adverse conditions. Such adverse conditions include elevated temperature, overcrowding, nutrient deprivation and noise. It is also surprising that the method of the invention increases libido and thus the reproductive potential of male birds.

As described above, any inhibin-producing animal (including, but not limited to, most agriculturally-reared animals such as pigs, cows, sheep, turkeys, quail, ducks, geese, chickens, and fish) can be enhanced using the methods of the present invention; periderm animals (mink, fox, otter, ferret, rabbit, and raccoon); experimental animals (e.g., rats, mice, gerbils, and guinea pigs); animal skins can be used for decorative animals (e.g., alligator and snake produced in the americas); foreign or anomeric species; animals used for competition, entertainment or shows (competitions) (e.g., horses, dogs, cats, zoo animals, and circus animals); and human productivity. Further, birds whose productivity can be increased by the method of the present invention include ratites, psittaciformes, picoformes, strigiformes, passeriferomes, corraciformes, raliformes, cuculeiformes, columbiformes, galliformes, anseriformes and herodiones. More specifically, the method of the present invention can be used to increase the productivity of camels, turkeys, rhea, chickens, turkeys, ducks, geese, quail, wingless birds (partridge kiwi), turkeys, parrots (parrot), parrots (parakeet), makaw, falcon, eagles (eagle), hawks (hawk), pigeons, canrowigars, birds, jay birds, mountain birds, pencils, bird birds, canaries, giant cuckoos, octopus or sparrows.

Accordingly, it is an object of the present invention to provide statin compositions that induce an immune response in an animal after administration to the animal.

It is another object of the invention to provide a heterologous protein comprising a statin protein or fragment thereof and a carrier protein.

It is another object of the present invention to provide a composition comprising a fusion heterologous protein comprising inhibin, or a fragment thereof.

It is another object of the invention to provide a method for producing a fusion heterologous protein comprising a statin protein or fragment thereof and a carrier protein.

It is another object of the invention to provide a fusion gene product comprising a gene encoding the expression of an α -subunit inhibin protein, or a fragment thereof, and a gene encoding the expression of a carrier protein.

It is another object of the invention to generate an immune response against the heterologous protein of the invention by injecting said fusion gene product into an animal.

It is another object of the present invention to provide compositions and methods useful in gene therapy for modulating inhibin levels.

It is another object of the present invention to provide a method for increasing the productivity of an animal.

It is another object of the present invention to provide a method of increasing the production capacity of birds.

It is another object of the present invention to provide a method for improving throughput in the thorax category.

It is another object of the present invention to provide a method for increasing the productivity of chickens.

It is another object of the present invention to provide a method for increasing the productivity of a crawler.

It is another object of the present invention to provide a method of increasing productivity in a mammal.

It is another object of the invention to provide a method for increasing the production capacity of fish.

It is another object of the present invention to provide a method for increasing human productivity.

These and other objects, features and advantages of the present invention will become apparent upon reading the following detailed description of the disclosed embodiments and appended claims.

Brief description of the drawings

FIG. 1 is an SDS-PAGE gel in which A is a camel anti- (chicken inhibin-maltose binding protein) antibody and B is the plasmid pMAL^TMC a vector standard, C a protein molecular weight standard, D pMAL used in said fusion heterologous protein preparation^TM-c vector, E is the purified fusion chicken inhibin-maltose binding protein (heterologous protein) of the invention, and F is the eluate of the purification process without said heterologous protein.

FIG. 2 specifically illustrates the use of cINA₅₂₁The effect of neutralizing effect of inhibin alpha-subunit immunity on daily egg production ("HDEP") of the female birds following fusion of the encoded protein to maltose-binding protein. More specifically, FIG. 2 is a graphical illustration of the data for example 8.

Detailed Description

In general, the invention relates to methods for improving the productivity of such animals by administering to said animals a heterologous protein comprising a inhibin protein or a fragment thereof carrier protein. The invention also relates to methods for improving the productivity of such animals by administering to said animals a fusion gene product comprising a gene encoding the expression of an alpha-subunit inhibin protein, or a fragment thereof, and a gene encoding the expression of a carrier protein.

Administering to the animal an effective amount of the heterologous protein or fusion gene product to generate an immune response in the animal against the heterologous protein. It will also be appreciated that the methods of the invention promote the production capacity of inhibin-producing animals. Preferably, the animal is a bird. More preferably, the avian is a chicken. Another preferred bird is a turkey. Further preferred birds are ratites (e.g. avians, ostriches, rheas or turkeys). The invention also relates to the heterologous proteins and fusion gene products described above, and methods for producing them.

Following the following definitions, the compositions of the present invention will be described in detail, followed by a detailed description of the methods of the present invention. Definition of

The term "avian" or "poultry" as used herein is defined as a member of the class avida (Aves) animals, characterized by warm-blooded, egg-laying vertebrates primarily suitable for flight. The term "ratite" as used herein is defined as a family of non-flying, mostly large walking birds, which includes the following items: avians, ostriches, wingless birds and turkeys. The term "parrot" as used herein includes parrots and is a monokout in birds, the toe and powerful hooked beaks. "parrot" is defined as any member of the family avifamily psittataceae (the only family in order psittaciformes) and is characterized by a short, strong, hooked mouth. As used herein, "chicken" refers to chickens that are used for egg production (e.g., monoguan white-legehne chickens) and raised for consumption or chickens that are suitable for baking.

The term "egg" is defined herein as a large female cell produced by birds and reptiles that is encapsulated in a porous calcium-containing or cortical shell. As used herein, "bird or reptile egg production" is the behavior of an egg-laying bird, otherwise known as "egg laying". The term "egg" is defined as a female gamete, also known as an egg. Thus, egg production in all animals used herein, with the exception of birds and reptiles, is defined as the production and ejection of eggs by the ovaries, otherwise known as "ovulation". As such, it should be understood that the term "egg" as used herein, when produced by a bird or a crawler, should be defined as a large female cell encased in a porous calcium-or cortical shell; and an egg when it is produced by all other animals.

For birds, the text may use the terms "onset of egg production", "first egg production" and "puberty" interchangeably, which refer to the first egg produced by a bird. Thus, as used herein, "accelerated onset" of egg production or puberty in a bird refers to the induction of an earlier date of first egg production as compared to the date of first egg production in a typical bird. Similarly, "puberty" and "onset of sperm production" are used interchangeably for males.

The phrases "increase productivity", "improve productivity" and "increase productivity" are used interchangeably and refer to improvements in one or more of the following: accelerating the onset of puberty (egg production or ovulation in females, sperm production in males); accelerating the onset of the maximum egg production or ovulation phase in females, or accelerating the onset of the maximum sperm production phase in males; increasing the egg production intensity of females or the sperm production intensity of males; extending the egg production period of a female or the sperm production period of a male; increasing total egg production or ovulation in the female lifetime; the feed conversion rate is improved; the quality of the eggshell is improved; increased resistance to adverse conditions (such as elevated temperature, overcrowding, malnutrition, and noise); improving the viability of male sperm; increase in production of male testosterone; increasing semen volume; increase male sexual desire.

The phrase "egg production intensity" is intended to be known to those of ordinary skill in the art and refers to the frequency of eggs produced.

Birds "total eggs for life" is defined as the total number of eggs a bird produces over the entire life of the bird. The phrase "daily egg production by a hen" or "HDEP" as used herein is defined as the number of eggs produced per day by a particular group of hens.

As used herein, the phrase "promoting the onset of maximum egg production" or "promoting the onset of maximum egg production" refers to a period from birth to the time when the animal lays or ovulates at 50% of the peak laying rate or ovulation rate that is shorter than the normal period from birth to maximum laying.

As used herein, a method of "cholesterol level reduction" of an egg refers to a method of inducing a bird of the same species to hatch one or more eggs having a lower cholesterol content as compared to the average cholesterol content of eggs hatched from such bird.

As used herein, the term "mammal" is defined as a member of the class mammalia, a large class warm-blooded vertebrate animal characterized by the mammary gland, body coat, three periosteum (oscicles) of the middle ear, the muscular diaphragm separating the thoracic and abdominal cavities, red blood cells without nuclei, embryonic development in the allantois and amnion, in contrast to the terms poultry or fowl.

The term "reptile" as used herein is defined as any member of the class Reptilia, a class of terrestrial vertebrates characterized by the absence of hairs, feathers and mammary glands, their skin covered with scales, and the presence of a three-compartment heart, whose pleural and peritoneal cavities are in communication.

A heterologous protein, as used herein, is defined as a protein comprising a statin protein or fragment thereof and a carrier protein. It is understood that the terms "inhibin" and "inhibin fragment" are used interchangeably in heterologous protein compositions, methods of producing heterologous proteins, and methods of using the heterologous proteins of the present invention.

It should also be understood that "cINA" is used herein₅₂₁"refers to a521 base pair sequence (SEQ ID NO: 1). cINA₅₂₁The part of the alpha-inhibin subunit of the ostrich is shown as SEQ ID NO: 2, respectively. As used herein, "MBP-cINA₅₂₁"is in the process of converting cINA₅₂₁Cloned into recombinant host cells and expressing a recombinant host cell comprising maltose binding protein ("MBP") and cINA₅₂₁The encoded inhibin protein alpha-subunit fragment is fused to a heterologous protein and the heterologous protein is expressed by a recombinant host cell. Preferably, the commercially available vector pMAL is used^TMC cloned cINA₅₂₁After expression, MBP-cINA is produced in host E.coli cells₅₂₁. Thus, "cINA₅₂₁"refers to a nucleotide sequence, and" MBP-cINA₅₂₁"refers to a fused heterologous protein.

As used herein, a fusion heterologous protein refers to two different proteins fused together. For example, a protein comprising a statin protein or fragment thereof fused to a carrier protein. The fused heterologous protein is expressed by an expression system comprising a fused gene product comprising a gene encoding a inhibin protein, or a fragment thereof, fused to a gene encoding a carrier protein. As used herein, a "fused gene product" is defined as the product of the fusion of a gene encoding the expression of a inhibin protein, or a fragment thereof, with a gene encoding the expression of a carrier protein.

A conjugated heterologous protein, as used herein, is defined as a protein comprising a statin protein or fragment thereof conjugated to a carrier protein. The conjugated heterologous protein is produced by a chemical reaction that links the statin protein to the carrier protein by a covalent bond.

As used herein, an immune response of an animal to a substance administered thereto is defined as a cell-mediated and/or humoral response of the animal which is specific to the substance.

The term "selective interaction" as used herein is defined as the binding of two substances to each other by covalent bonds, non-covalent bonds, hydrogen bonds, electrostatics, receptor-ligand interactions, enzyme-substrate interactions, or by other binding or adsorption means. The selectivity of such binding is that the two substances interact with each other in a specific manner, in a specific location or specifically. Inhibin composition

The present invention relates generally to compositions for use in methods of increasing productivity in animals, including birds. The composition consists of a heterologous protein comprising a statin protein or fragment thereof and a carrier protein. The inhibin can be inhibin of any animal species that produces inhibin. These statins include, but are not limited to: avian inhibin, mammalian inhibin, reptilin, amphibian inhibin, or fish inhibin. More specifically, mammalian statins include, but are not limited to: bovine inhibin, human inhibin, equine inhibin, feline inhibin, canine inhibin, rabbit inhibin, ovine inhibin, mink inhibin, fox inhibin, otter inhibin, ferret inhibin, raccoon inhibin, donkey inhibin, rat inhibin, mouse inhibin, hamster inhibin, and porcine inhibin. Avian statins include, but are not limited to: ostrich inhibin, avidine, rhea inhibin, turkey inhibin, wingless avian inhibin, turkey inhibin, quail inhibin, chicken inhibin, duck inhibin, goose inhibin, and inhibin of members of the order Psittaciformes.

Preferred statins are avian (avian) or avian (bird) statins. More preferred statins are ratiostatins, such as camel, pyro or rhea statins. A particularly preferred statin is camel inhibin. Another preferred statin is chicken statin. Most preferably, the heterologous proteins of the invention include an alpha-subunit inhibin protein, or a fragment thereof, and a carrier protein.

The inhibin or a fragment thereof can be isolated from animal body fluid, expressed by genetically engineered cells in an expression system, or synthesized through a series of chemical reactions. More specifically, fragments of inhibin include, but are not limited to, the following components: an alpha-subunit inhibin; a β -subunit inhibin; recombinant DNA derived from an alpha-subunit inhibin or a fragment of beta-subunit inhibin; a synthetic peptide copy of an alpha-subunit inhibin or a beta-subunit inhibin fragment; a synthetic peptide copy of the N-terminal sequence of α -subunit inhibin or β -subunit inhibin; a fragment of inhibin partially purified from follicular fluid; an endogenous α -subunit inhibin or a fragment of β -subunit inhibin; exogenous alpha-subunit inhibin or a fragment of beta-subunit inhibin. As noted above, the most preferred inhibin fragment is alpha-subunit inhibin, or a fragment thereof. In the case of statins, it will be understood by those of ordinary skill in the art that they include statins with amino acid substitutions that may provide more immunogenicity or more activity at the recipient.

Inhibin in the heterologous protein is either fused or conjugated to a carrier protein as described below. When the inhibin is fused to a carrier protein, the heterologous protein is a "fusion heterologous protein". When the inhibin is bound to a carrier protein, the heterologous protein is a "binding heterologous protein". Preferred heterologous proteins are fusion heterologous proteins.

The identity of the carrier protein in the heterologous protein is not a critical aspect of the invention. Any carrier protein known in the art may be used in the heterologous protein. Carrier proteins for use in the present invention include, but are not limited to: maltose binding protein "MBP"; bovine serum albumin "BSA"; keyhole limpet hemocyanin "KLH"; egg white protein; flagellin; thyroglobulin; serum albumin of any species; gamma globulin from any species; a sexual germ cell; sexual germ cells containing Ia antigen; and polymers of D-and/or L-amino acids. The preferred carrier protein is MBP. Another preferred carrier protein is BSA if the heterologous protein is not administered to cattle or horses. Another preferred carrier protein is ovalbumin if the heterologous protein is not administered to the bird. The most preferred carrier protein is MBP. Preferably, the carrier protein is immunogenic to the animal to which it is to be administered.

The invention also relates to methods of producing the binding heterologous proteins of the invention. Methods for producing binding proteins are well known in the art. The binding of proteins to proteins is well described in antibody-laboratory manuals (edited by Harlow and David Lane, Cold spring harbor laboratory (1988)), the contents of which are incorporated herein by reference. Additional methods for producing binding heterologous proteins (including binding reagents such as dialdehyde, carbodiimide, bis-azobenzidine and others; carrier proteins and immunological processes) are described in detail in sections 38 (page 605-618) and 42 (665-678) of neuroendocrine peptide methodology (P.Michael Conn, ed. academic Press, New York, 1989, the contents of which are incorporated herein by reference), section 4, "preparation of antibodies".

Although binding proteins may be used in the methods of the invention, fusion proteins are preferred. More specifically, fused heterologous proteins produce homologous products in which different parts of the protein are always fused at the same position and the number of fused protein fragments is the same. In addition, the fusion heterologous protein can be produced consistently, inexpensively, and in large quantities. In contrast, binding to heterologous proteins is not as consistent as for fusion proteins. For example, depending on the proteins bound, the binding reaction may result in a mixture of proteins with one or several binding, proteins that bind at different positions, or proteins that are not yet bound. In addition, some binding may sterically hinder the intended use of the heterologous protein (e.g., sterically hindering the immunogenic portion of the protein). In addition, binding reaction conditions and reagents can degrade the produced protein. For example, glutaraldehyde is commonly used in binding reactions, which can alter the conformation of proteins. Also, it is more expensive to produce binding proteins in large quantities than fusion proteins.

The invention is also directed to fusion gene products comprising a gene encoding the expression of an alpha-subunit inhibin protein, or a fragment thereof, and a gene encoding the expression of a carrier protein. The gene encoding inhibin protein or its fragment can encode and express avian inhibin, mammalian inhibin, pisatin or reptilian inhibin. The gene encoding the expression of said carrier protein may encode maltose binding protein or bovine serum albumin expressed therein. Preferred genes encoding expression of the carrier protein encode expression of maltose binding protein. The fusion gene products and methods for producing such fusion gene products are described more fully below.

Briefly, the method of producing the fusion heterologous protein of the invention comprises the following steps: the fusion gene product is inserted into the coding region of a plasmid, the plasmid is used to transform a host cell from which the fusion heterologous protein is expressed by methods well known in the art. More specifically, the method for producing said fusion heterologous protein comprises inserting a cDNA encoding an expression of inhibin or a fragment thereof into a vector containing information encoding the production of the carrier protein. After the vector is inserted into an expression system, the fusion heterologous protein is expressed by the system.

Many methods of producing fusion heterologous proteins are known in the art. Thus, any method known in the art can be used to produce the fusion heterologous protein of the invention. The fusion heterologous proteins of the invention can be prepared using a number of commercially available vector kits and expression systems. An example of such a commercially available vector kit and expression system is pMAL from New England Biolabs (Beverly Massachusetts)^TM-c. In pMAL^TMCytoplasmic expression of said fusion heterologous protein can occur in the system-c. Examples 1 and 2 below describe the synthesis of pMAL^TMC a method for producing the fusion heterologous protein of the present invention. Other sources of vector kits and expression systems that can be used to produce the fusion heterologous proteins of the invention include, but are not limited to: pharmacia Biotech company of Piscataway (New Jersey); clonetech corporation of Palo Alto (Calif.).

The invention also relates to fusion gene products comprising a gene encoding the expression of a inhibin protein, or a fragment thereof, and a gene encoding the expression of a carrier protein. The inhibin gene can be from any inhibin-producing animal species. Such a repressor gene may be a bird repressor gene, a mammalian repressor gene, a reptile repressor gene, an amphibian repressor gene, or a fish gene. More specifically, mammalian inhibin genes include, but are not limited to: bovine inhibin gene, human inhibin gene, equine inhibin gene, feline inhibin gene, canine inhibin gene, ovine inhibin gene, mink inhibin gene, fox inhibin gene, otter inhibin gene, ferret inhibin gene, raccoon inhibin gene, rat inhibin gene, mouse inhibin gene, hamster inhibin gene, donkey inhibin gene, and porcine inhibin gene. Avian inhibin genes include, but are not limited to: ostrich inhibin gene, avidine inhibin gene, rhea gene, turkey inhibin gene, wingless avian inhibin gene, turkey inhibin gene, quail inhibin gene, chicken inhibin gene, inhibin gene from any member of the order psittaciformes, inhibin gene from any member of the order falconatales, inhibin gene from any member of the order piciformes, a statin gene from any member of strigiformes, a statin gene from any member of coraciform, a statin gene from any member of raliformes, a statin gene from any member of passeriformes, a statin gene from any member of cucules, a statin gene from any member of columbiform, a statin gene from any member of galliformes, a statin gene from any member of anseriformes (geese, ducks and other aquatic birds), a statin gene from any member of herodiones.A avian inhibin gene selected from the group consisting of: falcon, eagle (eagle), eagle (hawk), pigeon, parrot (parakeet), crown parrot, makaw, parrot (parrot), canary, yagi, giant-mouth, and perching bird (e.g., whooping bird, mezzo, hawk, parrot, etc.),Birds, birds of mountain, birds of bird, twitter birds and sparrows).

Preferred inhibin genes are avian inhibin genes. More preferably, the inhibin gene is a ratiocidin gene. Particularly preferred inhibin genes are the camel inhibin genes. Another preferred inhibin gene is the pyroaurin gene. Another preferred inhibin gene is the rhea inhibin gene. Yet another preferred inhibin gene is the chicken inhibin gene.

The chicken inhibin α -subunit eDNA clone inserted into the EcoR 1 site of Bluescript (Stratagene, La Jolla, cA) (cINA 6; Wang and Johnson, complementary deoxyribonucleic acid cloning and sequence analysis of the α -subunit of chicken ovarian granulosa cytostatin, reproductive biology 49: 453,458, 1993, the entire contents of which are incorporated herein by reference) was given as P.A. Johnson (university of Cornell). The cINA6 clone specifically hybridized with ostrich genomic DNA in Southern assay, indicating significant DNA homology between the two species (choujenko et al, expression and purification of chicken α -inhibin as a fusion protein with maltose binding protein of E.coli, poultry science, 73 (supl.1): 84, 1994)). Excision of a DNA fragment from the cINA6 clone ("cINA") using PstI digestion₅₂₁”)。cINA₅₂₁The DNA fragment comprises the majority of the mature chicken inhibin a-subunit. Despite excision of cINA from the clone cINA6 reported by Wang and Johnson₅₂₁However, the resulting sequence (SEQ ID NO: 1) is still different from the DNA sequence published by Wang and Johnson.

The camelina inhibin alpha-subunit sequence was obtained by Polymerase Chain Reaction (PCR) methods well known in the art. More specifically, the following primers were constructed based on the sequence reported by Wang and used in PCR reactions with camel bird genomic DNA: 5'-CTCAGCCTGCTGCAGCGCCC-3', respectively; and 5'-GTGTCGACCGCGCGACGCCGAC-3'. More specifically, the primers described above are directed to the chicken inhibin α -subunit cDNA clones, 778-798 and 1348-1326 Bingji pairs of cINA6 reported by Wang and Johnson, respectively. The resulting PCR product was digested with the restriction endonuclease Pst1 and subcloned into the commercially available vector PUC19 (New England Biolabs). The sequence of the ostrich Pst1 fragment inhibin gene is identical to the corresponding portion of chicken α -inhibin.

As noted above, it should be understood that the carrier protein is not a critical aspect of the invention. Thus, genes encoding the expression of any carrier protein may be used in the present invention. The carrier protein gene includes but is not limited to the genes encoding the following proteins: maltose binding protein "MBP"; bovine serum albumin "BSA"; keyhole limpet hemocyanin "KLH"; egg white protein; flagellin; thyroglobulin; serum albumin of any species; gamma globulin from any species; a sexual germ cell; sexual germ cells containing Ia antigen; and polymers of D-and/or L-amino acids. Preferred carrier protein genes are genes encoding for expression of MBP. If the resulting heterologous protein is not to be administered to cattle or horses, another preferred carrier protein gene is the BSA gene. Another preferred carrier protein gene is the ovalbumin gene if the resulting heterologous protein is not administered to an avian. The most preferred carrier protein gene is the gene encoding MBP or a derivative thereof. Preferred carrier protein genes encode proteins that will increase the intensity and duration of the host immune response to the inhibin protein.

The invention also relates to methods of producing fusion gene products comprising fusing a gene encoding the expression of a inhibin protein, or a fragment thereof, to a gene encoding the expression of a carrier protein. Briefly, the method of producing the fusion gene of the present invention comprises the steps of: isolating the desired inhibin-complementary DNA (cDNA) to produce double-stranded inhibin DNA, obtaining double-stranded carrier protein DNA, and fusing the double-stranded inhibin DNA to the double-stranded carrier protein DNA in such a manner that the fused DNA is capable of expressing a fused heterologous protein comprising the inhibin protein or a fragment thereof and the carrier protein.

Many methods of isolating genes and producing fusion gene products are known in the art. See, for example, Sambrook, Fritsch and Maniatis for molecular cloning, A laboratory Manual, second edition, Cold spring harbor laboratory Press, 1989, volumes I, II, III. Thus, any method known in the art can be used to produce the fusion gene products of the invention. The fusion gene products of the present invention can be prepared using a number of commercially available vector kits. An example of such a commercially available vector kit is the pMAL of New England Biolabs (Beverly Massachusetts)^TM-c. Example 1 below is a full description of the synthesis of pMAL^TMC a method for producing the fusion gene product of the present invention. Other sources of vector kits that can be used to produce the fusion gene products of the invention include, but are not limited to: pharmacia Biotech company of Piscataway (New Jersey); clonetech corporation of Palo Alto (Calif.).

As described above, the chicken inhibin a-subunit cDNA clone (crina 6) inserted into the EcoR 1 site of Bluescript was donated by p.a. johnson (university of Cornell). Excision of a DNA fragment ("cINA") from the cINA6 clone using PstI digestion₅₂₁"to be used herein). The fragment (cINA)₅₂₁) Cloning in frame to plasmid p-MAL with maltose binding protein ("MBP")^TMIn-c, a fusion protein of appropriate size was detected after IPTG (isopropyl. beta. -D-thiogalactopyranoside) induction and SDS-PAGE (lane E of FIG. 1). The resulting protein conjugate ("MBP-cINA)₅₂₁") used as an antigen to immunize against circulating inhibin levels against prepubertal female Japanese quail (Coturnix cathurnix japonica), as fully described in example 8. Method for increasing production capacity

It has surprisingly been found that the compositions of the invention improve the productivity of animals, in particular poultry. Thus, the present invention is also directed to a method of increasing productivity in an animal by administering a heterologous protein of the invention. In one embodiment, the method comprises administering to a female animal an effective amount of the protein to increase the productivity of the animal. In another embodiment, the method comprises administering to a male animal an effective amount of the protein to increase the productivity of the animal. Preferably, an immune response directed against the protein occurs in said animal. More preferably, the immune response that occurs in the animal is also directed against a statin protein (endogenous statin) produced by the animal.

More specifically, the methods of the invention comprise administering to an animal an effective amount of a heterologous protein of the invention (comprising a statin or fragment thereof and a carrier protein) such that the productivity of the animal is increased. Preferably, the animal is a bird. It is understood that a "treated" bird refers to a bird to which a heterologous protein of the invention has been administered.

The methods of the invention can be used to increase the productivity of any inhibin-producing female avian species. Female birds include, but are not limited to: ratites, psittaciformes, falciparules, piciformes, strigiformes, passeriferomes, coraciformes, raliformes, cuculeiformes, columbiformes, galliformes (domestic poultry), anseriformes (geese, ducks and other water poultry) and herodiones. More specifically, female birds include, but are not limited to: ostrich, aviary, rhea, wingless, aviary, turkey, quail, chicken, falcon, eagle, hawk, pigeon, parrot, canker, makaw, parrot, rooster (e.g. bird, etc.),Birds, mountain birds, twitter birds and sparrows) and any member of the order psittaciformes. Preferred birds are ratites. More preferably the bird is a camel bird. Another preferred ratite is a bird. Yet another preferred class of thorax is rhea. Another preferred bird is any member of the order psittaciformes. Another preferred bird is a chicken. In addition, another preferred bird is a quail. The method of the invention can be used to accelerate the onset of egg production in endangered avian species. These endangered species include, but are not limited to: eagles (eagle), hawks (hawk), bald hawks, and owls.

The inhibin and carrier proteins of the heterologous protein compositions of the present invention will vary with the avian species to which the composition is administered. When the composition is administered to birds, the use of avian inhibin and maltose binding protein is preferred. When the composition is administered to the ratite, the preferred statin is a domestic chicken or ratite. More preferably, when the composition is administered to an ostrich or ratite, the preferred statin is a domestic chicken or ostrich statin. Another preferred statin is a domestic chicken or ostrich statin when the composition is administered to a chicken. It will be appreciated that the inhibin of the heterologous protein need not be from the same species to which it is to be administered. For example, the heterologous protein administered to a camel bird may consist of chicken inhibin and a carrier protein.

It is also understood that the compositions also include adjuvants, preservatives, diluents, emulsifiers, stabilizers and other components known and used in prior art vaccines. Any adjuvant system known in the art may be used in the compositions of the present invention. These adjuvants include, but are not limited to: freund's incomplete adjuvant, Freund's complete adjuvant, heterogeneous dispersion of beta-1, 4 linked acetylated mannan ("Acemannan"), Titermax  (a polyoxyethylene-polyoxypropylene copolymer adjuvant from CytRx), a modified lipid adjuvant from Chiron, a saponin derivative adjuvant from Cambridge Biotechnology for the eradication of Bordetella pertussis, a Lipopolysaccharide (LPS) from gram-negative bacteria, a macroanion (e.g., dextran sulfate), an inorganic gel (e.g., alum, aluminum hydroxide, aluminum phosphate). The preferred adjuvant system is Freund's incomplete adjuvant. Another preferred adjuvant system is Freund's complete adjuvant.

The heterologous protein composition of the invention can be administered to birds by any method known in the art. For example, the composition may be administered subcutaneously, intraperitoneally, intradermally, or intramuscularly. Preferably, the composition is injected subcutaneously. The composition may be administered to the bird in one or more doses. Preferably, the composition is administered to the bird in multiple doses, such that a booster immunization can be performed after the initial immunization.

The composition may be administered to the animal at any time before it stops ovulation or produces sperm due to disease or age. The desired age for administration of the compositions of the present invention to an animal depends on the species of the animal of interest, the mating season (if any) of the animal, and the purpose for which the composition is to be administered.

For example, if the composition is administered to accelerate egg production or sperm production, the composition of the present invention should be administered to the bird prior to egg production or puberty. As noted above, the desired age for the first administration of the compositions of the invention to an animal depends on the species of the animal in question, the mating season (if any) of the animal, the size of the bird, and the identity of the components (statin and carrier protein) of the composition.

As another example, when the composition is applied to increase the productivity of an agricultural animal during a reproductive period, the composition is ideally applied prior to the start of the reproductive period. Conversely, when the composition is administered to a mature animal whose laying rate and sperm production rate are inhibited, the composition should be administered prior to the determination of such inhibition.

With respect to animals that are in breeding age, although the heterologous protein of the invention can be administered to an avian (e.g., a ratite of any age), it is desirable to immunize the avian within 6 months prior to the first breeding stage of the avian. It will be understood by those of ordinary skill in the art that female birds typically begin laying eggs during the first reproductive period. It is more preferred that the bird be immunized about 6 months prior to its first reproductive stage and then boosted at monthly intervals prior to its first reproductive stage. It is preferred that the birds be immunized about 6 months prior to the first breeding period and then boosted at monthly intervals during the 6 month period.

For example, the best result of increasing the production of female camel eggs is to first immunize them approximately 6 months prior to their first reproductive period, and then to boost them every other month during these 6 months. The amount of first immunization is about 0.5-4.5mg of the heterologous protein of the invention. The amount of booster is about 0.30-3.0mg of the heterologous protein of the invention. Preferably, the amount for priming is about 1.5-3.0mg of the heterologous protein of the invention and the amount for boosting is about 0.75-1.5mg of the heterologous protein of the invention. It is also preferable to emulsify the heterologous protein in Freund's complete adjuvant (Sigma chemical Co., St. Louis MO) in the first immunization and emulsify the heterologous protein in Freund's incomplete adjuvant (Sigma) in the booster immunization. More preferably, the heterologous protein composition is injected subcutaneously. Most preferably, the heterologous protein composition is injected subcutaneously in three locations in the upper thigh region of a camel bird.

The amount of the heterologous protein of the invention administered to the bird will vary depending on the species of the bird, the age and weight of the bird, and the time of administration of the protein will be related to the reproductive stage (if the bird has a reproductive stage), and the number of times the protein is administered. In addition, the start of the application plan (or treatment plan) varies depending on the species of the bird, the average adolescent age of the species of the bird, the family history of the bird (related to the family history of adolescent age), when the bird has hatched during the year, the nutritional status of the bird (highly nutritious birds have earlier adolescence than less nutritious birds), the overall health status of the bird at the start of dosing, the immunocompetence of the bird, the long term health history of the bird, the presence of extreme weather conditions (prolonged excessively severe weather, such as rain, high temperature or wind to which the bird is not adapted), the dwelling conditions (overcrowding), and the lack of activity.

As a result of the description of the present invention, one of ordinary skill in the art will be able to determine by routine experimentation how much of a heterologous protein is necessary to elicit an immune response in the bird to that protein.

Another example of a method of increasing productivity is as follows. Administering to the mammal an immunologically effective amount of the heterologous protein-binding composition such that the animal generates an immune response directed against the heterologous protein. The heterologous protein preferably comprises a mammalian inhibin conjugated to a maltose binding protein. Another preferred binding heterologous protein comprises avian or reptilian inhibin and maltose binding protein.

For example, the following is a brief overview of the method of the present invention for improving the productivity of Japanese quail, which is discussed in detail in example 8. The average adolescent age of untreated quails is about six to eight weeks. The following is a treatment plan for japanese quails having a body weight of about 0.1 to 0.25 pounds: the first (first) injection of 0.75mg of the heterologous protein of the invention at its 25 th day of age; boosters of 0.375mg were performed at day 32, 39, 46, 53, 60 and 90 days of age, followed by a booster every 35 days for a total of 3 times (i.e., at day 95, 130 and 165).

More specifically, at 25 days of age, 50 female quails were randomly divided equally into two injection groups (25 per group) as follows: (1) MBP-cINA₅₂₁Freund's adjuvant solution ("MBP-cINA")₅₂₁FRN "), or (2) freund's adjuvant (adjuvant control; "FRN"). Birds immunized with anti-inhibin (group 1) were injected with approximately 0.75mg of MBP-cINA521 per bird (dissolved in the appropriate control vehicle). Group 2 was injected with the same vehicle volume (0.2 ml). All injections were performed subcutaneously using a tuberculin syringe fitted with a 25 gauge needle. Each bird was immunized subcutaneously with about 0.375mg MBP-cINA521 of a booster inhibin or injected with the appropriate control, as described above, and observed for a total of 20 weeks.

Daily egg production ("HDEP") and mortality ("MORT") were recorded for females for 20 consecutive weeks starting at 41 days of age (considered as the first day of the egg-laying cycle). In addition, the mean age of the FIRST eggs (FIRST) and the age at which the birds lay 50% (f ify) were calculated for each treatment group. Example 8 discusses these in detail, and analysis of variance was performed on the HDEP, MORT, FIRST and FIFTY data.

The immune neutralization of inhibin obviously accelerates the puberty of female quails. As shown in Table 2, the mean age reduction (P <. 0088) of the first eggs from inhibin-treated females was approximately 6 days. Also, as shown in Table 3, the age of laying was significantly reduced in 50% (FIFTY) inhibin-treated females (12 days; P <.01).

As shown in FIG. 2, the positive effect of statin treatment on egg production intensityAlso present, is most apparent at the beginning and end of the active period. For example, MBP-cINA was observed compared to FRN control₅₂₁the/FRN-treated females had significantly increased average HDEP rates at week 1 (16.5vs 2.6%), week 2 (50.0vs 28.6%), week 4 (96.6vs 79.7%), week 15 (98.8vs 86.9%), week 16 (96.9vs 86.3%), week 18 (85.7vs 66.1%), week 20 (96.8% vs 73.8%), again significant increases were observed (P <. 05). At 20 weeks of total egg production, the inhibin-treated females had a total HDEP rate of 83.5% and a control of 75.4% (P <.14).

In addition to accelerating puberty, prolonging egg production, and increasing total egg production strength, statin treatment reduced the time required to reach peak egg production by about 3 weeks. Referring to FIG. 2, MBP-cINA was compared₅₂₁FRN (which has a HDEP at week 4 of 96.6%) and FRN (which has a HDEP at week 7 of 96.6%). Although the difference in HDEP peaks was not statistically evaluated, the difference in mean age at which the female reached 50% HDEP level (FIFTY) reflected a different peak.

Since only 8 birds died (3 controls, 5 treatments), mortality was not a factor in this study. A mortality rate of 16% was expected for quails that had reached 180 days of age.

The effect of this treatment on the onset, extent and duration of most timed biological responses was investigated. The data presented herein are mostly data of the entire egg laying period (i.e., 20 weeks after puberty or egg laying period) of japanese quails. Thus, the following effects on the onset, extent and duration of egg production in this species with respect to the neutralizing effect of inhibin immunity were demonstrated.

These data support the conclusion that the onset of puberty in inhibin-immune-neutralized groups is accelerated. This is evidenced by the apparent processing differences seen in both the FIRST and fty variables and the differences seen in the HDEP data of the FIRST few weeks.

The acceleration of puberty associated with the prolongation of egg production in inhibin-treated birds resulted in an increase in labeled total HDEP (8.1%). For example, on an individual female basis, inhibin treatment results in approximately 0.081 eggs per day for each day during the laying period when the female is fertile (i.e., capable of laying). That is, during 20 weeks of the examination, 11 more eggs were obtained from each female bird that was raised (0.081 eggs/hen × 140 days of laying period — 11.34 eggs per laying period per female bird).

Results found in chickens and turkeys similar to those found in Coturnix have an important strategic relationship with the poultry industry. It should be noted that Japanese quails were selected for egg production intensity and that the egg production potential of Coturnix was considered to be much greater than that of commercially raised female chickens (monobromoney white-skinned chickens) that had been fed eggs only. Thus, the increase in egg production intensity resulting from statin vaccination may be greater in chickens selected for meat production (e.g., broiler breeders raised for consumption of their meat) rather than for egg production.

Thus, the above data indicate that the production capacity of the inhibin composition of the present invention is improved because it accelerates the onset of puberty, increases the intensity of egg production, and accelerates the onset of maximum egg production in Japanese quails. Since Japanese quail is an accepted animal model for the breeding system of chickens, the above data indicate that the method of the invention will also accelerate the onset of egg laying in chickens. Thus, the method of the present invention will result in an egg-laying person being able to produce more eggs at lower feeding costs.

The above data also show that the statin compositions of the invention improve the productivity of Japanese quail eggs because they minimize the adverse effect of increased temperature on their laying rate. More specifically, at week 18 of the study described in example 8, such quails were exposed to elevated temperatures without adverse effects. As shown in FIG. 2, group 1 birds (with MBP-cINA)₅₂₁FRN treatment) was reduced by about 5%. In contrast, the laying rate of birds in group 2 (control: FRN) decreased by approximately 26%. Thus, the method of increasing production capacity of the present invention can ameliorate the adverse effects of adverse egg laying conditions on egg production. This aspect of the invention is of great significance because poultry is often kept in an open, uncontrolled environment. As such, breeder birds are often exposed to adverse conditions (e.g., high temperatures and other extreme weather conditions to which they cannot adapt), which reduces the egg production rate of the poultry industry.

The following is a brief summary of the method for improving the productivity of an ostrich according to the present invention which is discussed in example 9. The average adolescent age of the untreated ostriches is about 28 to 32 months. The following is a treatment plan for ostriches having a body weight of about 150 to 300 lbs: the first (first) injection of 5.0mg of the heterologous protein of the invention at its 26 months of age; 2.5mg boosters were performed at 27, 28, 30, 32, 34, and 36 months of age.

The following is a brief summary of the method of improving productive capacity of a pyrotechnical bird of the present invention discussed in example 10. The average adolescent age of untreated avians is about 20 months. The following is a treatment program for a pyrotechnical bird having a body weight of about 50 to 90 pounds: the first (first) injection of 3.0mg of the heterologous protein of the invention at its 18 months of age; 1.5mg boosters were performed at 19, 20, 22, 24, 26 and 30 months of age.

The following is a brief overview of the method of improving chicken productivity of the present invention discussed in example 11. The average adolescent age of untreated chickens was approximately 20 weeks. The following is a treatment plan for chickens having a body weight of about 2.0 to 3.5 pounds: the first (first) injection of 1.5mg of the heterologous protein of the invention at its 15 weeks of age; 0.75mg of boost was performed at 17, 20, 24, 30, 40 and 50 weeks of age.

The following is a brief overview of the method of improving the productivity of turkeys of the present invention, discussed in detail in example 12. The average adolescent age of untreated turkeys was approximately 30 weeks. The following is a turkey treatment program with a body weight of about 9.0 to 12 pounds: the first (first) injection of 2.0mg of the heterologous protein of the invention at 28 weeks of its age; 1.0mg of boost was performed at 29, 30, 34, 38, 46 and 54 months of age.

The following is a brief overview of the method of improving the production capacity of parrots of the present invention discussed in example 13. The average adolescent age of untreated parrots is about 30 months. The following is a treatment plan for parrots having a body weight of about 0.5 to 1.25 pounds: a first (first) injection of 0.75mg of the heterologous protein of the invention at 28 months of its age; 0.375mg of boost was performed at 29, 30, 32, 34, 36 and 38 months of age.

As noted above, the method of the present invention increases productivity by accelerating the onset of puberty in the animal to which it is administered. The term "accelerated" with respect to egg production onset means that egg production by a treated bird begins at least about 3% earlier than egg production by an untreated bird typically begins. Preferably, the egg begins at least about 5% early, more preferably, the egg begins at least about 7% early. Even more preferably, the eggs begin at least about 10% earlier, and most preferably the eggs begin at least about 13% earlier than untreated birds.

In addition, as described above, the methods of the present invention increase the productivity of an animal by increasing the intensity of egg production or sperm production. The term "increase" with respect to egg production means an increase in egg production by at least about 3% in treated birds as compared to untreated birds. Preferably, egg production is increased by at least about 7%, and more preferably, egg production is increased by at least about 12%.

Furthermore, as mentioned above, the method of the invention improves its productivity by accelerating the start of the maximum laying period. The term "accelerated" with respect to the onset of maximum egg production refers to treated birds beginning at least about 3% earlier in maximum egg production than untreated birds typically begin laying eggs. Preferably, the maximum egg production period begins at least about 5% earlier, more preferably at least about 7% earlier. Even more preferably at least about 10% earlier, and most preferably at least about 13% earlier than the time at which the maximum egg production period typically begins in untreated birds.

Surprisingly, the compositions of the present invention can also be used to increase the total egg production of an avian throughout its life. The term "increase" with respect to total egg production means an increase in total egg production by the treated birds of at least about 3% over total egg production by the untreated birds. Preferably, total egg production is increased by at least about 7%, more preferably, total egg production is increased by at least about 12%. Most preferably, the total egg production is increased by at least about 13%.

Unexpectedly, the compositions of the present invention can also be used to reduce or eliminate the need for moulting in females, such as by providing a second laying cycle to extend the laying period. More specifically, if the above-described compositions are administered continuously to female birds (as disclosed in the above-described methods), the egg production of these birds will remain sufficiently high that they do not require moulting to increase their egg production as compared to birds not treated with the compositions of the present invention. When the egg production of a female bird, such as a hen (monoguan white legehan, a producer of edible eggs), is reduced such that the economic cost of maintaining the female egg production exceeds the economic return of the egg it produces, the art typically re-breeds the female. Hens are "feathered," i.e., the females are allowed to undergo a fasting period of about 4-14 days until they begin to feather (e.g., lose feathers). During moulting, the birds stop laying eggs. Some period after these birds return to normal feed levels, eggs begin to lay. The whole moulting period is about two months from fasting to the next laying period. In fact, the laying rate of the birds is restored. However, after a chicken has moulted, its laying rate in the next cycle is different from the laying rate in the first (pre-moulting) laying cycle. Handbook of production of commercial chickens, 4 th edition, chapter 19, new york, published by Van normal hold.

For example, chickens begin laying eggs at about 20 weeks, with an economically significant egg laying period of about 40 to 50 weeks. In the peak of laying eggs, the chicken can lay 8-9 eggs every 10 days. However, after about 50 weeks of laying, the laying rate drops to about 60% of the peak of laying. At this point, the cost of raising chickens is higher than the value of their eggs. It is common practice to re-molt the chickens at this point so that when they again lay eggs, the laying rate increases. In these birds, the "egg production period" of chickens and quails is extended, i.e., the egg production period is extended for about 1-4 weeks.

Thus, the composition of the present invention can reduce or eliminate the need to moult birds because it can maintain a higher level of egg production than birds that have not been treated with the composition. Reducing or not requiring moulting is a great saving. More specifically, during or before a bird's moulting period, the birds cannot produce their feeding costs until their moulting is complete, and then they cannot produce their feeding costs for a period of time after feeding begins. Maintaining egg production at an increased level can eliminate or reduce these periods of non-production by the birds, thereby reducing the cost of the producer and increasing the profit of the producer. Because the laying rate after moulting is not equal to the laying rate of the first laying period as described above, maintaining the laying rate at an increased level also increases the profits of the egg producer.

Briefly, the egg production rate of birds is increased by administering an effective amount of a heterologous protein of the invention to induce an immune response in the bird, thus eliminating the need to moult the bird, and then administering the heterologous protein (boost) in order to maintain a higher level of egg production.

Thus, the methods of the invention increase the productivity of inhibin-producing females, such as mammals, reptiles, and birds (e.g., ratites). More specifically, the method improves the productivity of female ratites (such as camels, pyrotechnics and rhea) and chickens. Unexpectedly, the method of the present invention promotes the onset of puberty or first egg production in animals. In addition, the method of the invention accelerates the onset of the maximum laying period of the animal. In addition, the methods of the invention increase the egg production of the animal. Also, the methods of the invention extend the maximum egg production period of the animal. Also, the method increases the total egg production of the animal throughout its life. For birds, the method of the invention also improves feed conversion ratio of the bird. In addition, the methods of the present invention unexpectedly reduce or eliminate the effect of adverse egg-laying conditions on egg-laying rates in animals exposed to such conditions. These adverse conditions include elevated temperature, overcrowding, nutrient deprivation and noise.

While not wishing to be bound by the following, it is theorized that the present methods of increasing productivity provide higher egg production in species that have not been genetically selected for egg prolificacy. This is particularly true for certain birds. For example, since the late 20 s of the 19 th century, laying hens were genetically selected for their maximum productivity (see, e.g., Jull, M.A, 1932, poultry breeding, John Wiley and Sons). Given the short life cycle of chickens, a large selection for this trait has been made from about 1928 to the present. In contrast, ratites and parrots, most other exotic birds and to a lesser extent broiler chickens (broilers suitable for baking) have not been genetically selected for prolific egg production. In addition, endangered birds have not been genetically selected for prolific egg production. Thus, laying hens have been genetically superior egg producers, and the amount of increase seen with treatment with the method of the invention is limited compared to birds that are genetically poor to medium egg production. Thus, birds that are genetically unselected for egg prolificacy (e.g., ratites, psittaciformes, other exotic birds, endangered birds, turkeys, and broiler chickens) are treated with the methods of the invention in greater amounts to increase productivity.

Immunization of an animal with a heterologous protein of the invention induces the animal to produce antibodies that are selective against the heterologous protein. Preferably, immunization also induces the production of antibodies in the animal that are selective against endogenous inhibin. This antibody produced by birds can reduce the onset of puberty or egg production. The production of such antibodies by the animal also increases the egg-laying capacity or sperm-producing capacity of the animal because the antibodies neutralize the biological activity of inhibin in the blood of the animal.

While not wishing to be bound by the following theory, it is believed that the α -subunit of inhibin binds to the FSH receptor and thus competitively inhibits binding of FSH to this receptor site. Decreasing the level of inhibin that can bind to the receptor site increases the biological effects of FSH in the animal due to decreased competition for the FSH receptor site. It is believed that these antibodies neutralize these inhibins by interacting with circulating inhibins, thereby sterically (stearically) interfering with the binding of the interacting inhibins to the FSH receptor site.

Unexpectedly, the methods of the invention also improve the productivity of inhibin-producing males (e.g., mammals, reptiles, and birds). More specifically, the methods of the invention increase testosterone levels in a male animal. Similarly, the methods of the invention increase the onset of puberty or sperm production in a male animal. In addition, the method of the invention promotes the onset of the maximum sperm production phase in males. In addition, the method of the present invention increases the intensity of sperm production (sperm count) in males. Also, the methods of the invention extend the maximum spermatozoa production phase of the animal. In addition, the method of the invention increases the semen volume of the male. Also, the methods of the invention improve the viability of animal sperm cells. In addition, the methods of the present invention unexpectedly reduce or eliminate the effects of adverse conditions on animal sperm cells exposed to such adverse conditions. Such adverse conditions include elevated temperature, overcrowding, nutrient deprivation and noise. Surprisingly, the method of the invention increases libido and thus the reproductive potential of male birds.

Another unexpected and surprising aspect of the present invention is that the compositions of the present invention also produce more eggs with lower cholesterol levels than untreated bird eggs. More specifically, if the above-described composition is administered to a female bird in the manner described above, it produces eggs with lower cholesterol levels over a longer period of time than birds not treated with the composition of the present invention. Thus, the compositions of the present invention increase or produce greater amounts of low cholesterol eggs.

The term "increase" or "greater amount" refers to an increase in the number of low cholesterol eggs produced by a treated bird of at least about 2% as compared to the number of low cholesterol eggs produced by an untreated bird. Preferably, the number of low cholesterol eggs produced is increased by at least about 5%, more preferably by at least about 10%. "treated" birds are understood to be birds to which the heterologous protein of the invention has been administered. The term "lower cholesterol" or "low cholesterol" means that the cholesterol content of an egg is at least about 10% lower than the average cholesterol content of eggs laid during the life of such bird. Preferably, the cholesterol content of the low cholesterol eggs is at least about 20% lower than this average. More preferably, the cholesterol content of the low cholesterol eggs is at least about 30% lower than this average.

It is known that five to six eggs laid first after the female (hen) reaches puberty are lower in cholesterol content than eggs laid later in chickens. The compositions of the invention induce females to produce eggs with lower cholesterol levels over an extended period of time. Egg products with low cholesterol content are still needed for health reasons associated with high cholesterol levels in the blood. The composition of the present invention thus provides good layers to egg producers. Gene therapy using said fusion gene product

The invention also relates to methods for improving the productivity of such animals by administering to said animals a fusion gene product comprising a gene encoding the expression of an alpha-subunit inhibin protein, or a fragment thereof, and a gene encoding the expression of a carrier protein. The fusion gene product of the present invention can be administered directly to the animal, or can be administered as a vector or a cell containing a vector having the fusion gene product therein.

In vivo gene transfer into mammalian somatic cells (N.Yang, review of Biotechnology standards 12 (4): 335-356(1992), the contents of which are incorporated herein by reference) describes various methods for transferring or delivering DNA into cells for expression of gene product proteins (also known as gene therapy). For use of DNA sequences in ex vivo or in vivo therapies, gene therapy involves the addition of DNA sequences to somatic or germ line cells. The function of gene therapy is to replace genes and to increase normal or abnormal gene function.

Strategies for gene therapy include therapeutic strategies such as identifying defective genes and then adding functional genes to replace the function of the defective gene or to slightly increase the function of the gene; or preventive strategies such as the addition of genes for said product proteins. An example of a prophylactic strategy is to place a fusion gene product encoding a statin or fragment thereof and a carrier protein in an animal and reduce the level of the statin in the animal secondarily as a result of the immune response.

Any protocol for transferring the fused gene product of the invention is contemplated as part of the invention. Transfection of promoter sequences (rather than any other promoter sequences normally found to be specifically associated with inhibin) or other sequences that would reduce production of inhibin protein is also envisioned as a means of gene therapy. An example of this technique is found in Cambridge Transkarya, Massachusetts, which is a "gene switch" that has been demonstrated to be an erythropoietin gene in cells using homologous recombination insertions. See the report of genetic engineering, 4, 15, 1994.

Transgenic approaches for gene therapy include three major classes: physical methods (such as electroporation, direct gene transfer, and particle bombardment), chemical methods (lipid-based vectors or other non-viral vectors), and biological methods (viral-derived vector and receptor uptake). For example, non-viral vectors comprising liposomes coated with DNA may be used. Such liposome/DNA complexes can be injected directly intravenously into animals. It is believed that the liposome/DNA complex is accomplished in the liver to deliver the DNA to macrophages and astrocytes. These cells have a long life span, which allows long term expression of the delivered DNA. Alternatively, the vector or "naked" DNA gene can be directly injected into the desired organ, tissue or tumor to achieve targeted delivery of the therapeutic DNA.

Methods of gene therapy can also be described in terms of delivery sites. Basic methods of gene delivery include ex vivo gene transfer, in vivo gene transfer, and in vitro gene transfer. In ex vivo gene transfer, cells are obtained from the animal and cultured in cell culture. The cells are transfected with said DNA, the transfected cells are expanded to a certain number and then reimplanted in the animal. In vitro gene transfer, transformed cells are cells grown in culture, such as tissue culture cells, and are not specific cells of a particular animal. These "experimental cells" are transfected, and the transfected cells are selected and expanded for implantation into an animal or other use.

In vivo gene transfer involves introducing the DNA into an animal cell (when the cell is in an animal). The methods include the use of virus-mediated gene transfer, i.e., the use of a non-infecting virus to deliver the gene to the animal, or the injection of naked DNA into a site in the animal and the uptake of the DNA by cells that partially express the protein of the gene product. In addition, other methods described herein (e.g., using a "gene gun") can be used for in vitro insertion of inhibin DNA or inhibin regulatory sequences.

The chemistry of gene therapy involves lipid-based compounds (not necessarily liposomes) that allow the DNA to pass through the cell membrane. Lipofectins or cytofectins, lipid-based positive ions that bind to negatively charged DNA form complexes that can cross the cell membrane and provide DNA on the inside of the cell. Another chemistry uses receptor-based endocytosis, which involves binding of specific ligands to receptors on the cell surface, enveloping and transporting them across the cell membrane. The ligand binds to the DNA and transports the entire complex into the cell. The ligand gene complex is injected into blood, and then target cells having the receptor specifically bind to the ligand, and the ligand-DNA complex is transported into the cells.

Many gene therapy methods use viral vectors to insert genes into cells. For example, altered retroviral vectors have been used in ex vivo methods to introduce genes into peripheral and tumor infiltrating lymphocytes, hepatocytes, epidermal cells, muscle cells, or other somatic cells. These altered cells are then introduced into the animal to provide the gene product from the inserted DNA.

Viral vectors are also used to insert genes into cells via in vivo protocols. To direct tissue-specific expression of foreign genes, cis-acting regulatory elements or promoters known to be tissue-specific may be used. Alternatively, this can be achieved by delivering the DNA or viral vector in situ to sites of specific constructs in vivo. For example, gene transfer to blood vessels is accomplished in vivo by implanting transduced endothelial cells at selected arterial wall sites in vitro. The virus is able to infect surrounding cells that also express the gene product. For example, viral vectors can be delivered directly to a site in the body via a catheter, such that only a specific site is infected by the virus, and long-term site-specific gene expression is provided. In vivo gene transfer using retroviral vectors has also been achieved in breast and liver tissues by injecting the altered virus into blood vessels and to organs.

Viral vectors that have been used in gene therapy protocols include, but are not limited to: retroviruses, other RNA viruses (such as polio or sindbis viruses), adenoviruses, adeno-associated viruses, herpes viruses, SV 40, vaccinia and other DNA viruses. Replication-defective murine retroviral vectors are the most widely used gene transfer vectors. Murine leukemia retroviruses are composed of single-stranded RNA complexed with a nucleoprotein and polymerase (pol), which is enveloped by a protein core (gag) and surrounded by a glycoprotein envelope (env) that defines the host range. The genomic structure of retroviruses includes the gag, pol and env genes, accompanied by 5 'and 3' Long Terminal Repeats (LTRs). The use of retroviral vector systems is based on the fact that if the viral structural proteins are provided transmembrane (in trans) in the packaging cell line, the basic vector containing the 5 'and 3' LTRs and a packaging signal is sufficient for the vector to be packaged, infected and integrated into the target cell. The fundamental benefits of retroviral vectors for gene transfer include efficient infection and gene expression in most cell types, integration of precise single copy vectors into the chromosomal DNA of the target cell, and ease of manipulation of the retroviral genome.

Adenoviruses are composed of linear double-stranded DNA complexed with a core protein and surrounded by viral capsid proteins. Advances in molecular virology have enabled the use of the biology of these organisms to create vectors capable of transducing novel gene sequences into target cells in vivo. Adenovirus-based vectors will express gene product peptides at high levels. Adenovirus vectors have high infectivity even at low titers of virus. Furthermore, these viruses are completely infected as acellular viral particles, so that injection of the producer cell line is not necessary. Another potential advantage of adenoviral vectors is the ability to achieve long-term expression of heterologous genes in vivo.

Mechanical methods of DNA delivery include fusogenic lipid vesicles (such as liposomes or other vesicles used for membrane fusion), DNA lipid particles combined with cationic lipids (such as lipofectins), polylysine-mediated DNA transfer, direct injection of DNA (such as microinjection of DNA into embryonic or somatic cells), airborne delivery of DNA coated particles (such as gold particles used in "gene guns"), and inorganic chemical methods (such as calcium phosphate transfection). Another ligand-mediated gene therapy method involves complexing the DNA with a specific ligand to form a ligand-DNA conjugate, and introducing the DNA into a specific cell or tissue.

It has been found that injection of plasmid DNA into muscle cells produces a very high proportion of transfected cells which are able to achieve expression of the marker gene. The plasmid DNA may or may not be integrated into the genome of these cells. Non-integrated transfected DNA will allow the final differentiated non-proliferative tissue to transfect and express the gene product protein for longer periods of time without fear of mutational insertions, deletions or alterations in the cellular or mitochondrial genome. Long term (but not necessarily permanent) transfer of therapeutic genes into specific cells can allow genetic diseases to be treated or for prophylactic use. The DNA may be re-injected periodically to ensure that no mutations have occurred in the gene product in the genome of the recipient cell. Non-integrated exogenous DNAs allow several different exogenous DNA constructs to be present in a cell, so that all of the constructs express different gene products.

Particle-mediated gene transfer methods were first used to transform plant tissues. Using a particle bombardment device or "particle gun," the force to accelerate a high density of particles (e.g., gold or tungsten) coated with a layer of DNA to a high velocity that allows penetration of the target organ, tissue or cell can be generated. Particle bombardment can be used in vitro systems or with ex vivo or in vivo techniques to introduce DNA into cells, tissues or organs.

Electroporation methods of gene transfer use an electrical current in order to sensitize cells or tissues to electroporation-mediated gene transfer. The use of short electrical pulses with a given field strength increases the permeability of the membrane so that DNA molecules can penetrate into the cells. This technique can be used in an in vitro system or with ex vivo or in vivo techniques to introduce DNA into cells, tissues or organs.

Exogenous DNA can be transfected into cells using vector-mediated gene transfer in vivo. The vector-DNA complex is usually introduced into body fluid or blood and then specifically introduced into a target organ or tissue in vivo at a site. Liposomes and polycations (e.g., polylysine, lipofectins, and cytofectins) can be used. Cell-or organ-specific liposomes can be produced such that the exogenous DNA carried by the liposome will be taken up by the target cells. Injection of immunoliposomes directed against specific receptors of certain cells can be used as a routine method for inserting the DNA into cells containing the receptors. Another carrier system used is the asialoglycoprotein/polylysine conjugate system for carrying DNA into hepatocytes for in vivo gene transfer.

The transfected DNA may also be complexed with various other vectors, which transport the DNA into the recipient cell and then colonize the cytoplasm or nucleoplasm. The DNA can be bound to the carrier nuclear protein in specific engineered bleb complexes and transported directly into the nucleus.

Gene regulation of inhibin can be achieved by administering a compound that binds to the inhibin gene, or to a control region associated with the inhibin gene, or its corresponding RNA transcription region, to alter the rate of transcription or translation. In addition, cells transfected with DNA sequences encoding inhibin, or a fragment thereof, and a carrier protein can be administered to an animal to provide a source of the heterologous protein of the invention in vivo. For example, a cell can be transfected with a vector comprising a fusion gene product of the invention encoding a inhibin, or a fragment thereof, and a carrier protein.

The term "vector" as used herein is a vector that contains or is associated with a specific nucleic acid sequence and functions to transport the specific nucleic acid sequence into a cell. Examples of vectors include plasmids and infectious microorganisms such as viral or non-viral vectors (e.g., ligand-DNA conjugates, liposomes, lipid-DNA complexes). The recombinant DNA molecule comprising the fusion gene product of the invention is operably linked to an expression control sequence to form an expression vector capable of expressing the heterologous protein of the invention. The transfected cells may be cells derived from normal animal tissue and diseased animal tissue, or may be non-animal cells.

For example, cells obtained from an animal are transfected with a vector capable of expressing the heterologous protein of the invention and reintroduced into said animal. The transfected cells then produce the heterologous protein of the invention, thus inducing an immune response against said inhibin. Cells can also be transfected by non-vector or physical or chemical methods known in the art, such as electroporation, ion-perforation (ionophoresis), or by "gene gun". Alternatively, the fusion gene products of the invention can be injected directly into animals without vector assistance. In particular, the fusion gene product of the present invention can be injected into skin, muscle or blood.

The gene therapy regimen for transfecting the inhibin or a fragment thereof into an animal can be by integration of the fused gene product into the genome (i.e., minichromosome) of said cells, or as an independently replicating or non-replicating DNA construct in the cytoplasm or nucleoplasm of such cells. Expression of the heterologous protein can be sustained for an extended period of time, or the fusion gene product of the invention can be periodically reinjected to maintain the desired level of the heterologous protein in these cells, tissues or organs, or in established blood.

The fusion gene products of the invention can be administered to birds by any method known in the art. For example, the composition may be administered subcutaneously, intraperitoneally, intradermally, intravascularly, or intramuscularly. Preferably, the composition is injected subcutaneously. Another desirable method of administration is intravascular infusion near the desired treatment site. The composition may be administered to the bird in one or several doses. Preferably, the composition is administered to the bird in several doses, such that a booster immunization can be performed after the first immunization. The preferred amount of fusion gene product administered is 50-300 micrograms per kilogram of body weight. Preferably, the fusion gene product is administered in a carrier (e.g., buffer or Freund's adjuvant).

The method of improving avian productivity of the present invention will greatly promote the growth of populations of ratites (such as camel birds and pyrotechnics), and thus satisfy market needs, since the method of the present invention will improve the undesirable egg production rates of these birds. The process of the present invention will also meet the growing need for poultry (e.g., domestic chickens) and their eggs.

The increase in productivity using the method of the present invention is not limited to increasing the productivity of birds. The method of the invention for increasing productivity can be used in many animals. As noted above, the methods of the invention can be used to increase the productivity of any inhibin-producing animal, including, but not limited to: most animals raised in agriculture (e.g., pigs, cows, sheep, turkeys, quail, ducks, geese, turtles, fish, and chickens); furred animals (such as mink, fox, otter, ferret, rabbit, and raccoon); experimental rodents (e.g., mice, rats, hamsters, guinea pigs, and gerbils); animals whose skins are used for decorative purposes (e.g., alligator and snake produced in america); foreign or endangered species; animals used for competitions, entertainment and shows (competitions) (e.g., horses, dogs, cats and zoo animals); circus animals and humans. Other birds whose productivity can be increased by the method of the invention include: ratite, nautita, falconate,piciformes, strigiformes, passeriformes, coraciformes, ralliformes, cuculelformes, columbiformes, galliformes, anseriformes and herodiones. More specifically, the method of the invention can be used to increase the productivity of: ostrich, aviator, rhea, wingless bird, turkey, parrot, parakeet, makaw, falcon, eagle, hawk, pigeon, canker, bird, etc,Bird, bird of mountain, bird of bird twitter, canary, giant beak bird, blackbird or sparrow. Qualitative or quantitative methods of the invention

Another aspect of the invention is the production of antibodies against the heterologous proteins of the invention. In general, the method of producing an antibody against the heterologous protein of the invention comprises the following steps: administering to an animal an effective amount of a heterologous protein of the invention comprising a statin protein or fragment thereof and a carrier protein, such that an immune response can occur in the animal against the heterologous protein; obtaining a blood sample from the animal; any antibodies against inhibin are then isolated from the serum of the blood sample. Preferably, said antibodies are isolated from the serum of said blood sample by passing the serum through a column containing an effective amount of said carrier protein to isolate the antibodies from the serum. In addition, the column contains the heterologous protein of the invention. In another technique, the antibody is isolated by first passing the serum through a column containing the carrier protein and then through a column containing the heterologous protein of the invention. Techniques for generating and purifying antibodies against the heterologous proteins of the invention are well known to those of ordinary skill in the art.

It is understood that the heterologous protein of the invention can be administered to any animal, depending on the type of antibody desired. Also, it is understood that the statins may be exogenous or endogenous. Thus, the type of inhibin in the heterologous protein of the invention and the animal species to which the composition is administered is determined by the type of antibody desired. For example, a heterologous protein comprising chicken inhibin and maltose binding protein can be administered to the ostrich in order to produce camel anti-chicken inhibin antibodies. In addition, a heterologous protein comprising ostrich inhibin and maltose binding protein can be administered to the ostrich in order to produce camel anti-camel inhibin antibodies.

The present invention is also directed to a method for quickly, easily, reliably, and inexpensively determining whether an animal is an egg-laying animal with high or low levels of hormone pretreatment. More particularly, the invention also relates to methods for determining the amount of inhibin produced by an animal, thereby enabling the determination of the egg production capacity of an animal. Briefly, a method for determining the amount of statin in the blood of an animal comprises the steps of: drawing a blood sample from the animal; contacting the blood sample with an anti-inhibin antibody that is specific for an endogenous inhibin in the animal under conditions that permit the antibody to selectively interact with any inhibin present in said sample; removing any non-interacting antibodies from the interacting antibodies; the amount of antibody that interacts is determined.

Those of ordinary skill in the art will appreciate that immunoassay techniques that can be used in the above-described methods are well known in the art. Thus, any immunoassay technique, labeling and visualization method known in the art may be used in the above methods, including ELISA and Radioimmunoassay (RIA). A preferred immunoassay is an ELISA ("enzyme-linked immunosorbent assay"). The preferred label is horseradish peroxidase. Another preferred marker is a colored latex bead. The colored latex beads can be any color desired for visualization. Preferably, the latex beads are yellow, red, blue or green. The colored latex beads may be empty or solid, but are preferably empty to minimize their weight. The size of the latex beads varies depending on the immunoassay for which they are to be used. One of ordinary skill in the art will be able to determine by routine experimentation the size of the largest bead that is visible but does not interfere spatially with the immunoassay reaction. Preferably, the latex beads are less than 0.5 μ in diameter, and most preferably less than 0.2 μ in diameter.

For example, the concentration of circulating inhibin in the blood of birds can be determined using sandwich ELISA techniques. First, an anti-inhibin antibody directed against a portion of inhibin, or a fragment thereof, is bound to the wells of a microtiter plate. After washing and blocking the plates, a quantity of plasma obtained from the birds to be tested is then added. After allowing any inhibin, if present, in the sample to selectively interact with the immobilized anti-inhibin antibody, the sample is washed out of the plate wells. Next, labeled anti-inhibin antibodies are added to the wells, which antibodies are directed against different portions of inhibin or fragments thereof, rather than the antibodies immobilized in the wells. The antibody can be labeled with any label known in the art, such as horseradish peroxidase. After allowing the labeled anti-inhibin antibody to selectively interact with any immobilized inhibin, any non-interacting labeled anti-inhibin antibody is removed by washing. The amount of inhibin present in the plasma sample is determined by quantifying the amount of labelled anti-inhibin antibody immobilised in the wells using suitable visualisation methods used in the labelling of ELISA. In adjacent plate wells, standard positive and negative controls were performed simultaneously.

It will be appreciated that the reproductive potential of any inhibin-producing animal can be determined by the methods described above. The methods of the invention can be used to determine the amount of statin produced by a female animal of any species that produces statin. Wherein said animal is a bird, a mammal, a fish or a reptile. More specifically, mammals include, but are not limited to: cattle, humans, horses, cats, dogs, sheep, mink, foxes, otters, ferrets, raccoons and pigs. Birds include, but are not limited to: ostrich, pyrone and chicken. Preferably the animal is a bird. More preferably the animal is of the ratite type. A particularly preferred animal is a camel bird. Another preferred animal is a pyrobird. Yet another preferred animal is a chicken.

Animals with high inhibin levels have a lower reproductive potential and, depending on the species concerned and whether they are raised for agricultural purposes, such animals with lower eggs can be slaughtered and not used for reproductive purposes. In contrast, farm animals with lower inhibin levels are higher egg producers and are generally used for breeding purposes without slaughter.

The inhibin levels of an animal vary with its age, species and time of year associated with reproductive stage (if any). Thus, the determination of the egg-laying potential of an animal is related to these factors, and comparing the average statin levels of animals of the same species, about the same age, at the same time of year (if the animal is fertile), a measure of the animal's statin level is most valuable.

Since the amount of inhibin produced by birds varies with the above factors, the relative amounts of inhibin for birds of different ages are shown in the table below. Table 1 shows the variation in production of ratistatin depending on whether the ratites (e.g., avians and ostriches) are poor or good producers of eggs and the age of the ratites.

TABLE 1 age (month) inhibin level reproduction potential 6-12 > 5 poor 6-122-5 moderate 6-120-1.5 good 24+ > 7 poor 24+ 3-7 moderate 24+ 0-2.5 good

In the table above, inhibin levels are associated with a standard pool value of 1 for female ratites, with an average of 50-60 eggs per reproductive period, with good reproductive potential; the pool of female ratites without egg laying function was 7, laying less than 5 eggs per reproductive period, and their reproductive potential was poor.

Another aspect of the invention is a method of producing an animal antibody against another animal class of antibodies (e.g., IgG). Briefly, a method of producing antibodies in an animal against IgG of another animal comprises the steps of: administering to the second animal an effective amount of an antibody from the first animal (e.g., an antibody produced by the method described above), such that an immune response is generated in the second animal against the antibody from the first animal; drawing a blood sample from a second animal; antibodies from the second animal were isolated from serum from the blood sample as described above. Preferably, the second animal is different from the first animal.

The present invention is also directed to a method for quickly, easily, reliably and inexpensively determining whether an animal is immune to challenge with a statin composition. As described above, this method uses antibodies from a second animal that are raised against a class of antibodies from a first animal. Briefly, the method comprises binding a statin or heterologous protein of the invention to a solid phase; contacting the immobilized inhibin with a blood sample of the animal to be tested. Contacting the sample with immobilized inhibin under conditions such that said inhibin selectively interacts with any anti-inhibin antibodies in the sample. After removing the non-interacting antibodies from the sample and washing, an amount of labeled antibodies from the second animal (which are directed against a class of antibodies from the first animal) is added. A labeled antibody directed against this animal antibody will selectively interact with the antibody bound to the immobilized inhibin. After removing the labeled antibodies that have not interacted, the presence or amount of the interacted labeled antibodies is determined by visualizing the markers. Thus, the method detects the presence of antibodies to the inhibin in the animal, thereby determining whether the animal immunoreacts with the administration of the composition comprising inhibin.

It is understood that the method of determining whether an animal has an immune response to administration of a composition comprising a statin can be performed on a female animal of any species. Wherein said animal is a bird, a mammal, a fish or a reptile. More specifically, mammals include, but are not limited to: cattle, humans, horses, cats, dogs, sheep, mink, foxes, otters, ferrets, raccoons and pigs. Birds include, but are not limited to: camel, pyro, rhea, and chicken. Preferably the animal is a bird. More preferably the animal is of the ratite type. A particularly preferred animal is a camel bird. Another preferred animal is a turkey. Yet another preferred animal is rhea.

It will be appreciated by those of ordinary skill in the art that immunoassay techniques for the above-described methods are well known in the art. Thus, any immunoassay technique, labeling and visualization method known in the art may be used in the above-described methods. The preferred immunoassay is ELISA. The preferred label is horseradish peroxidase. Another preferred marker is a colored latex bead. The colored latex beads can be any color desired for visualization. Preferably, the latex beads are yellow, red, blue or green. The colored latex beads may be empty or solid, but are preferably empty to minimize their weight. The size of the latex beads varies depending on the immunoassay for which they are to be used. One of ordinary skill in the art will be able to determine by routine experimentation the size of the largest bead that is visible but does not interfere spatially with the immunoassay reaction. Preferably, the latex beads are less than 0.5 μ in diameter, and most preferably less than 0.2 μ in diameter.

Another embodiment of the present invention is directed to the above method for determining whether an animal has an immune response to administration of a composition comprising a statin, wherein the method is modified as follows. Briefly, the method comprises obtaining a blood sample from an animal; contacting it with a labeled zoostatin or fragment thereof. Contacting the sample with said animal statin under conditions in which the labeled animal statin selectively interacts with any anti-statin antibody in said sample. After removing the marker inhibin from the sample where no interaction has occurred, the presence or amount of the interacting marker inhibin is determined by visualizing the marker. The statins used in this method are selected from, but not limited to, the following: a fusion heterologous inhibin protein of the invention; endogenous inhibin or a fragment thereof; and exogenous inhibin or a fragment thereof. Preferably, the labeled statin is an endogenous statin.

The invention is further illustrated by the following examples which are not to be construed in any way as imposing limitations upon the scope thereof. On the contrary, it is to be clearly understood that resort may be had to various other embodiments, modifications, and equivalents thereof which, after reading the description herein, may suggest themselves to those skilled in the art without departing from the spirit of the present invention and/or the scope of the appended claims.

Example 1 production of a fusion Gene product comprising a Gene encoding expressed Chicken inhibin and a Gene encoding expressed maltose binding protein

The following is a gene (cINA) for the production of a fusion gene product comprising a gene encoding an expressed alpha-subunit chicken inhibin fragment (SEQ ID NO: 2)₅₂₁) And genes encoding maltose binding protein expression). The fusion gene product of the invention is prepared from pMAL^TMthe-C vector kit (from New England Biolabs, Beverly, Massachusetts).

pMAL^TMVectors provide a means for producing a protein that is expressed from a gene cloned in-frame. Inserting the cloned gene downstream of a male gene that encodes maltose binding protein ("MBP") and results in an MBP fusion protein ("MBP-cINA₅₂₁") is used. The method utilizes the affinity of MBP for maltose to generate high-level expression of clone sequences and is used for fusion protein MBP-cINA₅₂₁And (4) purifying by one step.

The following is the inhibin gene cINA₅₂₁Ligation into pMAL^TM-method of the C vector:

1. digestion of 0.5. mu.g of pMAL in 20. mu.l with the restriction endonuclease Pstl^TM-c plasmid DNA.

2. Mu.g of cINA6 plasmid DNA containing the chicken inhibin gene was digested with the same enzyme Pstl.

3. By performing 4. mu.l of pMAL on a 0.8% agarose gel^TMComplete digestion was checked for reactions and 4. mu.l of cINA6 reaction. Then, preparative agarose gels were run and Pstl cINA was purified by prep-A-gene purification kit₅₂₁And (3) fragment.

4. 0.05 units of calf intestinal alkaline phosphatase (NEB #290) was added to the vector DNA digest. The culture was carried out at 37 ℃ for 1 hour.

5. Equal volumes of a 1: 1 phenol/chloroform mixture were added to the vector restriction digest and mixed, after centrifugation, the aqueous (top) phase was removed and placed in fresh test tubes. Repeated with only chloroform.

6. 10 μ g of glycogen or tRNA was added as a carrier to the carrier digest, 1/10 volumes of 3M sodium acetate were added and mixed, and two volumes of ethanol were added. Incubate at-20 ℃ for 30 minutes.

7. Microcentrifuge for 15 minutes. The supernatant was decanted, and the precipitate was washed with 70% ethanol and then dried.

8. Each sample was resuspended in 20. mu.l of water.

9. Mixing 0.2 μ g of the vector digest with 0.5 μ g of the insert digest; add water to 18. mu.l, then add 2. mu.l 10 Xligase buffer; 0.5. mu.l NEB T4 ligase (# 202; -200 units); incubate at 16 ℃ for 2 hours to overnight.

10. Heating at 65 deg.C for 5 min; cooled on ice.

11. Mu.l of the ligation mixture was mixed with 100. mu.l of competent DH5 alpha (or any lacZ alpha-complement strain) and incubated on ice for 15-30 minutes. Heat to 42 ℃ and hold for 2 minutes.

12. 1ml of LB was added and incubated at 37 ℃ for 60 minutes. Spread on LB plates containing 100. mu.g/ml ampicillin. The culture was carried out overnight at 37 ℃. Colonies were picked with sterile toothpicks on LB amp master plates and LB amp plates containing 80. mu.g/ml Xgal and 0.1mM IPTG. The culture was carried out at 37 ℃ for 8 to 16 hours. Lac phenotypes were scored on Xgal plates and "white" clones were recovered from the master plate.

13. One or both of the following methods were used to screen for the presence of the insert:

A. the DNA of the micro-formulation is prepared. Digestion with appropriate restriction endonucleases to determine the presence and orientation of the insert.

B.i) culture of 5ml of culture in LB amp broth to approximately 2X 10⁸/ml。

ii) 1ml of sample was taken. Microcentrifuge for 2 minutes, the supernatant was discarded and the cells were resuspended in 50. mu.l protein gel SDS-PAGE sample buffer.

iii) IPTG is added to the remaining culture to 0.3mM, e.g.15. mu.l of 0.1M stock solution. The culture was carried out at 37 ℃ for 2 hours under good aeration.

iv) 0.5ml of sample was taken. Microcentrifuge for 2 minutes, the supernatant was discarded and the cells were resuspended in 100. mu.l SDS-PAGE sample buffer.

v) boil the sample for 5 minutes. Mu.l of each sample was electrophoresed on a 10% SDS-PAGE gel together with a panel of protein MW standards and MBP in SDS-PAGE sample buffer as provided. The gel was stained with Coomassie Brilliant blue. The induction band is readily visible at a position corresponding to the molecular weight of the fusion protein. The molecular weight of MBP alone is 42,000 daltons.

Example 2

Production of the fusion heterologous protein "MBP-cINA" comprising Chicken inhibin and maltose binding protein₅₂₁”

The following is the production of the fusion heterologous protein "MBP-cINA" comprising chicken inhibin and maltose binding protein₅₂₁"to (1). Example 1 expression of the fusion Gene product fused heterologous maltose binding protein-inhibin protein "MBP-cINA₅₂₁", as follows:

1. 80ml of concentrated broth + glucose and ampicillin (see media and solutions below) were inoculated with 0.8ml of overnight cultured cells containing the fusion plasmid of example 1.

2. Culturing at 37 deg.C under good aeration to 2 × 10⁸Individual cell/ml (A)₆₀₀0.5). 1ml samples were taken and microcentrifuged for 2 minutes (non-induced cells), the supernatant was discarded and the cells were resuspended in 50. mu.l SDS-PAGE sample buffer. Vortexed and placed on ice.

3. IPTG (isopropyl thiogalactoside) is added to the remaining culture to a final concentration of 0.3mM, for example at H₂0.24ml of 0.1M mother liquor in O (see media and solutions). At 37 deg.CThe culture was continued for 2 hours. A0.5 ml sample was taken and microcentrifuged for 2 minutes (to induce cells), the supernatant was discarded and the cells were resuspended in 100. mu.l SDS-PAGE sample buffer. Vortex to resuspend the cells and place on ice.

4. The culture was divided into two aliquots. Cells were harvested by centrifugation at 4000 Xg for 10 minutes. The supernatant was discarded and 1 pellet (sample a) was resuspended in 5ml lysis buffer (see media and solutions). Another 1 pellet (sample B) was resuspended in 10ml 30mM Tris-Cl, 20% sucrose, pH8.0 (8 ml per 0.1g wet cell weight).

5. The samples were frozen in a dry ice-ethanol bath (or overnight at-28 ℃). Thawing in cold water (20 ℃ is more efficient than 70 ℃ but takes longer).

6. Sonicated and measured by using Bradford or at A₂₆₀Release of nucleic acid assay protein release monitors cell rupture until it reaches a maximum. 0.6ml of 5M NaCl was added.

7. Centrifuge at 9,000 Xg for 20 minutes. The supernatant (crude extract 1) was decanted and stored on ice. The pellet was resuspended in 5ml lysis buffer. This is a suspension of insoluble material (crude extract 2).

The heterologous fusion maltose binding protein-inhibin protein "MBP-cINA" produced as described above₅₂₁"column purification as follows:

1. amylose resin (1.5g) was swelled in 50ml column buffer (see media and solution) in a 250ml filtration flask for 30 min. The gas was removed with a getter. Poured into a 2.5X 10cm column. The column was washed with 3 column volumes of the same buffer + 0.25% Tween 20.

The amount of resin required depends on the amount of fusion protein produced. The resin binds to a bed volume of about 3mg/ml, so a column volume of about 15ml is sufficient for yields of up to 45mg of fusion protein per liter of culture. A50 ml syringe stoppered with silanized glass wool can be used in place of the 2.5cm column. The ratio of the column height to the diameter should be less than or equal to 4.

2. The crude extract was diluted 1: 5 with column buffer + 0.25% Tween 20. By [10 × (column diameter cm)²]The diluted crude extract was loaded in ml/hour. This is about 1 ml/min for a 2.5cm column.

The aim of the dilution of the crude extract was to reduce the protein concentration to about 2.5 mg/ml. If the crude extract is of low concentration, it is not over-diluted. One empirically good method is to produce about 120mg protein in 1g of wet-weight cells.

3. Wash with 2 column volumes column buffer + 0.25% Tween 20.

4. Wash with 3 column volumes column buffer without Tween 20.

5. The fusion protein "MBP-cINA" was eluted with column buffer +10mM maltose + 0.1% SDS (optionally 10 mM. beta. -mercaptoethanol, 1mM EGTA)₅₂₁". Fractions of 10-20 parts and 3ml were collected. By, for example, Bradford assay or A₂₆₀Protein in the fractions is measured; the fusion protein containing fraction has easily detectable proteins. The fusion protein eluted soon after the empty volume of the column. Culture media and solutions

Concentrated medium + glucose and ampicillin per liter: 10g of tryptone, 5g of yeast extract, 5g of NaCl and 2g of glucose. And (3) high-pressure sterilization: sterile ampicillin was added to 100. mu.g/ml.

0.1M IPTG mother liquor 1.41g IPTG (isopropyl- β -o-thiogalactoside); adding H₂O to 50 ml. Filtering and sterilizing.

0.5M sodium phosphate buffer, pH7.2 (mother liquor)

(A)69.0g NaH₂PO₄H₂O to 1 liter of H₂And (4) in O.

(B)70.9g Na₂HPO₄To 1 liter of H₂And (4) in O.

117ml of (A) were mixed with 383ml of (B). The pH of this mother liquor should be 7.2. Diluted to 10mM, p with column bufferH should be 7.0. Lysis buffer 20ml 0.5M Na/l final concentration₂HPO₄10mM phosphate 1.75g NaCl 30mM NaCl10 ml% Tween 200.25% Tween j200.7ml β -mercaptoethanol 10mM β -ME ("β -ME") (optional) 20ml 0.5M EDTA (pH 8) 10mM EDTA10 3871M EGTA (pH 7) 10mM EGTA with HCl or NaOH to a final concentration of 7.0 column buffer per liter 20ml 0.5M sodium phosphate pH 7.210 mM phosphate 29.2g NaCl 0.5M NaCl1ml 1mM azide 1mM 0.7mM β -ME (optional) 10mM β -ME1ml M EGTA (pH 7.0) 1mM EGTA (optional) if necessary to a final concentration of 20ml 0.5M sodium phosphate pH 7.210 g NaCl 30mM NaCl1ml M mM NaCl 1mM Na phosphate 1mM β -ME (7 mM β -ME) 1mM azide 1mM ethanol (optional) pH7.0 mM 1mM β -mercaptoethanol 1mM β -5 mM EDTA (optional) pH 7.7 mM EDTA 1mM β -1 mM EDTA 1mM 1M EGTA (optional) pH 78 pH9 mM β -EDTA (optional) 1mM EGTA (optional) pH7.0 mM EGTA (optional) 1mM ethanol) If necessary, adjusted to pH7.0

Fused chicken inhibin-MBP heterologous protein' MBP-cINA₅₂₁The purity after "passing through the column" is illustrated in column "E" of FIG. 1. The column labeled "F" is the eluate from the column when no heterologous protein is loaded on the column (negative control). The column labeled "B" represents the plasmid pMAL^TM-C-vector standard. The column labeled "C" is a molecular weight standard. The column labeled "D" is the actual pMAL^TMThe C vector (this vector was used to prepare the fused chicken-inhibin-MBP heterologous protein "MBP-cINA" before insertion of the inhibin gene as described in example 2₅₂₁". The above proteins were electrophoresed on SDS-PAGE gels in SDS-PAGE sample buffer and stained with Coomassie Brilliant blue.

Example 3

Immune anti-inhibin ostrich

The following is a method for immunizing camels against inhibin. The ostrich is primarily immunized about six months before the first breeding season and then boosted six months at one-month intervals. Therefore, it is preferred that the ostrich is administered with the primary immunization when it is between about 18 and 24 months of age. The primary immunization comprises between about 1.5 and 3.0mg of a fusion heterologous protein comprising a myostatin (alpha subunit fragment) and a maltose binding protein produced by the methods of examples 1 and 2. The boost immunization comprises between about 0.75 to 1.5mg of the fusion heterologous protein. In the primary immunization, the fusion heterologous protein was emulsified in Freund's complete adjuvant (Sigma chemical Co., St. Louis, Mo.), and in the booster immunization, the fusion heterologous protein was emulsified in Freund's incomplete adjuvant (Sigma). The fusion heterologous protein composition was injected subcutaneously at three sites along the upper leg region of camelids.

Example 4

Production of selectively anti-inhibin camel antibodies

The following are methods of producing camel antibodies ("camel anti-chicken inhibin antibodies") that are selective against heterologous proteins of the invention comprising chicken inhibin and maltose binding protein. More specifically, the camel antibody is an IgG antibody. The heterologous protein utilized was a fusion protein comprising chicken statin and maltose binding protein produced by the methods described in examples 1 and 2. To produce this antibody, camel birds were immunized with the allo-protein gallistatin-MBP as described in example 3. The amount administered to the ostrich must be sufficient to elicit an immune response from the ostrich against the heterologous protein. Approximately 5ml of blood was drawn from the ostrich, and then the anti-inhibin antibody was isolated from the remaining blood sample. Any separation method known in the art may be used to isolate the antibody. Preferably, standard ELISA techniques are used to bind the affinity to the HPLC column.

Example 5

Production of goat antibody selectively against ostrich antibody

The following is a method of producing goat antibodies selectively directed against a panel of camelid antibodies, including the antibodies produced in example 4, more specifically IgG camelid antibodies. In this way, the goat is immunized with 0.5 to 3.0mg of camel IgG, so that the immune response occurs in the goat against the camel IgG. Ostrich IgG was obtained from a serum bank obtained from different ostriches. Blood was obtained from goats. Goat anti-camel IgG is then isolated from the sample using standard techniques known in the art.

Serum pools were purified using standard methods including precipitation with 50% ammonium sulfate followed by fractionation using a protein sepharose column. Preferably, IgG precipitation is performed as follows. 12ml serum containing IgG was diluted 1: 1 with 50mM-Tris (pH 8.0). Next, 24ml of saturated ammonium sulfate (all at 4 ℃ C.) was added slowly with stirring, and the mixture was stirred for about 2 hours. The mixture was centrifuged at 10,000rpm for 10 minutes to collect the precipitate. The pellet was resuspended in 50mM-Tris saline (to 12ml) and dialyzed against 2L of 50mM-Tris overnight at 4 ℃ in a final volume of 20 ml.

Preferably, subsequent fractionation using a protein-A sepharose column is as follows. The column contained protein A-Sepharose CL-4B (Pharmacia Biotech, Piscataway, NJ). The column was loaded with approximately 5ml of ammonium sulfate/serum precipitate. The sample was allowed to bind to protein A-Sepharose for approximately 30 minutes. Next, the column was washed with 0.1M phosphate buffer (pH7.5), and the adsorbed IgG was eluted with 0.1M glycine (pH 2.8). Finally, the eluted fractions were neutralized by adding a few drops of 1M Tris-HCl (pH 9). The quality of the purified camel IgG was visually tested after SDS-polyacrylamide gel electrophoresis prior to administration to goats.

The immunization method and adjuvant utilized are not critical to the present invention, and as such, any method known in the art may be utilized, and any adjuvant system known in the art may be used. Preferably, the purified camelid IgG is injected subcutaneously into the goat. Booster injections with incomplete freund's adjuvant at four week intervals are also preferred. Preferably, the purified IgG is administered with freund's complete adjuvant or Hunter's titermax (Sigma chemical company, st. After three to four injections, the goat developed a satisfactory immune response.

Next, 5 to 10ml of blood was taken from the goat, and goat antibodies against ostrich IgG were isolated from the blood sample. Any method known in the art may be used to isolate goat antibodies from a blood sample. Separation of goat antibodies from the sample the preferred method is to pass the blood sample through a camelid IgG column. Goat antibody was then collected from the column by washing the column with glycine buffer-pH 8.0.

Example 6

Monitoring the immune response of ostriches after vaccination with the fusion heterologous protein

The following is a method of monitoring the immune response of the ostrich after inoculating it with the fusion heterologous protein consisting of the chicken inhibin and maltose binding protein produced by the methods of examples 1 and 2, wherein the immune response is monitored by using the goat antibody produced in example 5. To determine whether a ostrich should immunoreact with the fused inhibin-MBP heterologous protein, the heterologous protein is first bound to a solid phase. Next, 5 to 10ml of blood was extracted from the ostrich immunized with the heterologous protein and serum was separated from the blood. Contacting the immobilized heterologous protein with serum under conditions wherein the heterologous protein selectively interacts with any anti-inhibin antibodies in the serum. After washing, the goat antibody labeled with HRP, produced in example 5, was added. The labeled goat antibodies then selectively interact with the camel antibodies bound to the immobilized heterologous protein. After removing the non-interacting labeled antibody, the presence or amount of the interacting HRP-labeled goat antibody was determined by visualization of the label. Preferably, the label is visualized by the addition of nitro blue tetrazolium ("NBT") (a substrate for HRP).

Those of ordinary skill in the art will understand that: immunoassay techniques for the above methods are well known in the art. Thus, any immunoassay technique, labeling and visualization method may be used for the above method. The preferred immunoassay is an ELISA and the preferred label is horseradish peroxidase. Another preferred marker is colored latex beads.

Example 7

Determination of the reproductive potential of camels

The following is a method for determining the reproductive potential of camelids by quantitatively determining the amount of inhibin in the blood of the camelids. The circulating inhibin concentration in camel bird blood can be measured using Radioimmunoassay (RIA) or standard sandwich ELISA techniques. First, the anti-inhibin antibody produced in example 4 is bound to the wells of a microtiter plate. After washing and blocking the plate, a certain amount of plasma or serum obtained from the ostrich is then added to the well of the microtiter plate. After any inhibin in the sample (if present) is allowed to selectively interact with the immobilized anti-inhibin antibody, the sample is washed from the wells of the plate. Second, a different anti-inhibin antibody (instead of the antibody produced in example 4, which is conjugated to horseradish peroxidase) was added to the wells. HRP-conjugated anti-inhibin antibodies are distinct from immobilized anti-inhibin antibodies in that they are selective against different portions of inhibin. After the labeled anti-inhibin antibody is allowed to selectively interact with any immobilized inhibin, any non-interacting labeled anti-inhibin antibody is removed by washing. The amount of inhibin present in the plasma sample is determined by adding NBT to the wells and visualizing the amount of immobilized labeled anti-inhibin antibody in the wells. Standard positive and negative controls were performed simultaneously in adjacent plate wells. Many immunoassay techniques, labeling and visualization methods are known in the art. Thus, any immunoassay, labeling and visualization technique may be used in the present invention.

Example 8

Increasing egg production in quails

As described above, the chicken inhibin alpha-subunit cDNA clone (cINA6) inserted into the EcoR 1 site of Bluescript was P.A. Johnson (Corne)ll university) is given. Excision of a DNA fragment from the cINA6 clone ("cINA") using PstI digestion₅₂₁”)。cINA₅₂₁The DNA fragment includes most of the mature chicken inhibin alpha-subunit. The fragment (cINA)₅₂₁) Cloning in-frame into the maltose binding protein ("MBP") vector p-MAL^TMin-C, appropriately sized fusion proteins (lane E; FIG. 1) were detected after IPTG (isopropyl. beta. -D-thiogalactopyranoside) induction and SDS-PAGE. Formed protein conjugates ("MBP-cINA)₅₂₁") used as an antigen to immunize against circulating inhibin levels in prepubertal female Japanese quails, as described below.

Hatched quails were hatched in a modified 2S-D Petersime type incubator battery for quails. The initial incubation temperature was about 37.8 ℃, decreasing by about 2.8 ℃ per week until room temperature was reached. During the growth period (i.e. until about 6 weeks of age), quail dosing of food (28% CP, 2,800Kcal ME/kg feed) was started, providing sufficient water. Use of continuous dim light (221x), 14 hours light (280 to 3001 x): dark for 10 hours. At 25 days of age, 50 quails were randomly divided into two equal injection groups (25 birds per group) as follows: (1) MBP-cINA in Freund's adjuvant₅₂₁(＂MBP-cINA₅₂₁FRN "), or (2) freund (adjuvant control; "FRN"). Birds immunized against inhibin (group 1) were administered approximately 0.75mg of MBP-cINA per bird in the appropriate control vehicle₅₂₁. An equivalent injection volume (0.2ml) of FRN was administered to group 2 as a vehicle. All injections were injected subcutaneously using a tuberculin syringe with a 25 gauge needle. After the initial injection, before closing into the egg laying cage (single), the wings of quail were fixed to identify them by treatment. Perweekly subcutaneous administration of about 0.375mg of MBP-cINA to each bird₅₂₁The booster inhibin immunization of (a), or appropriate control immunostimulation, is continued for five consecutive weeks (i.e., at 32, 39, 46, 53 and 60 days of age) followed by three additional immunostimulations every 35 days (i.e., at 95, 130 and 165 days of age). Quail feeding rationed diet (21% CP, 2,750Kcal ME/kg feed) provided sufficient water.

Daily female quail daily egg production ("HDEP") and mortality ("MORT") measurements were recorded for 20 consecutive weeks, beginning at age 41 days (day one of the egg laying cycle considered). In addition, the average day age of the FIRST eggs ("FIRST") and the day age of the female quails to reach 50% egg production ("FIFTY") or the maximum egg production as described above were calculated for each treatment group.

Female quail daily egg production data were subjected to analysis of variance (ANOVA) using a fully randomized design with treatment split placement. The major region consisted of two injection treatments (MBP-cINA)₅₂₁FRN, or FRN), 20 laying periods of 7 days, each including split.

In female quails, neutralization of inhibin immunity significantly accelerates puberty. Mean day age of eggs produced by FIRST was reduced (P <. 0088) in inhibin-treated female quails for approximately six days (Table 2). Day-old to reach FIFTY production was also significantly reduced in inhibin-treated female quails (12 days: P <.01) (Table 3).

The effect of statin treatment on daily egg production (HDEP) in female quails also existed, most notably at the beginning and end of the laying cycle (fig. 2). For example, MBP-cINA is being used when compared to PRN controls₅₂₁Significantly large (P <.05) average HDEP rates were observed in FRN-treated female quails, with week 1 (16.5 vs. 2.6%), week 2 (50.0 vs. 28.6%), week 4 (96.6 vs. 79.7%), again at week 15 (98.8 vs. 86.9%), week 16 (96.9 vs. 86.3%), week 18 (85.7 vs. 66.1%), and week 20 (96.8% vs. 73.8%). The total HDEP rate (including all 20 weeks of egg laying) for inhibin-treated female quails was 83.5%, compared to 75.4% for the control group.

In addition to accelerating puberty, prolonging egg production and increasing overall egg production intensity, statin treatment reduced the time required to reach peak egg production (approximately 3 weeks) (FIG. 2; compare MBP-cINA at week 4)₅₂₁FRN 96.6% HDEP versus 96.6% HDEP at week 7). Although the difference in peak HDEP values was not statistically evaluated, the difference in treatment at average day age, when female quails reached 50% HDEP levels (FIFTY), reflected the peak capacity.

Mortality was not a factor in this study, as only eight birds died (three controls, five treatments). Such MORT (16%) is within expected limits for quails up to 180 days of age.

TABLE 2

Effect of inhibin immune neutralization on average (. + -. SE) day-old in first egg production of Japanese quails

Treatment of the first egg-laying day age

FRN¹ 56.15＝1.82^a

MBP-cINA₅₂₁/FRN² 50.38＝1.08^b

¹Control in freund adjuvant.

²＝MBP-cINA₅₂₁V FRN-maltose binding protein in freund's adjuvant-chicken α 515-inhibin fusion protein.

^a，b(P＜.0088)。

TABLE 3

Effect of inhibin immune neutralization on average (. + -. SE) day-old in 50% egg production of Japanese quails

Treatment of 50% egg laying day age

FRN¹ 73.04＝3.78^aMBP-cINA₅₂₁/FRN² 61.00＝2.70^b

¹Control in freund adjuvant.

²＝MBP-cINA₅₂₁V FRN-maltose binding protein in freund's adjuvant-chicken α 515-inhibin fusion protein.

^a，b(P＜.01)。

The incidence of non-shell and thin-shell eggs occurs more frequently in control birds (rather than inhibin-immunized birds), particularly in the late stages of the egg-laying cycle. This indicates that: a greater number of defective eggs (i.e., eggs that are not suitable for consumption or are likely to break before consumption, or fail to serve as hatching eggs) are associated with the control treatment.

Example 9

Improving egg laying capacity of camel birds

Protein conjugates (MBP-cINA)₅₂₁) Used as an antigen to immunize pre-pubertal female camels against circulating inhibin levels, thereby accelerating the onset of egg production in the treated camels. The procedure described in example 8 was followed with the following exceptions: the average age at puberty for untreated camel birds is between about 28 to 32 months. The following is a treatment schedule for treating camel birds having an approximate weight range of about 150 to 300 pounds: primary (first) injection of 5.0mg of the heterologous protein of the invention at its age of 26 months; 2.5mg booster was injected at the age of months 27, 28, 30, 32, 34, 36.

Example 10

Improving egg laying capacity of pyrotechnics

Protein conjugates (MBP-cINA)₅₂₁) Used as an antigen to immunize pre-pubertal mother turkeys against circulating inhibin levels, thereby accelerating egg production in the treated turkeys. The procedure described in example 8 was followed with the following exceptions: the average age at puberty for untreated birds is about 20 months. The following is a treatment schedule for treating a bird having an approximate weight range of about 50 to 90 pounds: primary (first) injection of 3.0mg of the heterologous protein of the invention at its age of 18 months; 2.5mg booster was injected at age 19, 20, 22, 24, 26, 30 months.

Example 10

Improving egg laying ability of chicken

Protein conjugates (MBP-cINA)₅₂₁) The pre-pubertal hens were immunized with the antigen against circulating inhibin levels, thereby accelerating egg production in the treated hens. The procedure described in example 8 was followed with the following exceptions: the average age at puberty for untreated chickens was about 20 weeks. The following is a treatment schedule for treating chickens having an approximate weight range of about 2.0 to 3.5 pounds: the initial (first) injection of 1.5mg of the heterologous protein of the invention at its age of week 15; 0.75mg boost was injected at 17, 20, 24, 30, 40 and 50 weeks of age.

Example 11

Improving the egg laying capacity of turkeys

Protein conjugates (MBP-cINA)₅₂₁) The pre-pubertal hen was immunized against circulating inhibin levels using as an antigen, thereby accelerating egg production in the treated turkeys. The procedure described in example 8 was followed with the following exceptions: the average age at puberty for untreated turkeys was about 30 weeks. The following is a treatment schedule for treating turkeys having an approximate weight range of about 9.0 to 12 pounds: primary (first) injection of 2.0mg of the heterologous protein of the invention at its age at week 28; 1.0mg boost was injected at 29, 30, 34, 38, 46 and 54 weeks of age.

Example 13

Improving egg laying ability of parrots

Protein conjugates (MBP-cINA)₅₂₁) Used as an antigen to immunize prenatal mother parrots against circulating inhibin levels, thereby accelerating the onset of egg laying in the treated parrots. The procedure described in example 8 was followed with the following exceptions: the average age at puberty for untreated parrots was about 30 weeks. The following is a treatment schedule for treating parrots having an approximate weight range of about 0.5 to 1.25 pounds: age at its 28 th monthA primary (first) injection of 0.75mg of a heterologous protein of the invention; 0.375mg booster was injected at age 29, 30, 32, 34, 36 and 38 months.

Of course, it should be understood that: the foregoing relates only to the preferred embodiments of the present invention and numerous modifications or variations may be made therein without departing from the spirit and scope of the invention as set forth in the following claims.

Sequence listing (1) general information (i) applicants:

(A) name: agricultural technology Co Ltd

(B) Street: jones and Askew, Peachtree street 191, Ste.3700

(C) City: artalagin (R)

(D) State: root of Hodgkin's disease

(E) The state is as follows: united states of america and america

(F) ZIP code (ZIP): 30303

(G) Telephone: (404)818-3700

(H) Faxing: (404)818-3799

(A) Name: louisiana State university A & M management board

(B) Street: jones and Askew, Peachtree street 191, Ste.3700

(C) City: artalagin (R)

(D) State: root of Hodgkin's disease

(E) The state is as follows: united states of america and america

(F) ZIP code (ZIP): 30303

(G) Telephone: (404)818-3700

(H) Faxing: (404)818-3799(ii) invention name: inhibin composition and method of enhancing productivity (iii) number of sequences: 2(iv) computer readable form:

(A) type of medium: flexible disk

(B) A computer: IBM PC compatible machine

(C) Operating the system: PC-DOS/MS-DOS

(D) Software: patent Release #1.0, version #1.30(EPO) (2) SEQ ID NO: 1, information: (i) sequence characteristics:

(A) length: 521 base pairs

(B) Type (2): nucleic acids

(C) Chain type: single strand

(D) Topological structure: linear (ii) molecular type: DNA (genome) (iii) hypothetical structure: or (iv) antisense strand: no (ix) feature:

(A) name/keyword: CDS

(B) Position: the 303(xi) sequence description: SEQ ID NO: 1CTG CAG CGC CCA TCG GAG GAC GTG GCC GCC CAC ACC AAC TGC CGC CGG 48 LeuGln Arg Pro Ser Glu Asp Val Ala Ala His Thr Asn Cys Arg Arg 51015 GCG TCC CTC AAC ATC TCT TTC GAG GAG CTG GGC TGG GAC AAT TGG ATC 95 AlaSier Leu Asn Ile Ser Phe Glu Glu Leu Gly Trp Asp Asn Trp Ile 202530 GTG CAC CCC AGC AGC TTC GTT TTC CAC TAC TGC CAC GGG AAC TGT GCC 144 ValHis Pro Ser Ser Phe Val Phe His Tyr Cys His Gly Asn Cys Ala 354045 GAA GGC CAC GGG CTG AGC CAC CGG CTG GGG GTG CAG CTG TGC TGC GCC 192 GluGly His Gly Leu Ser His Arg Leu Gly Val Gln Leu Cys Cys Ala 505560 GCG CTG CCC GGC ACC ATG CGC TCA CTG CGT GTC CGC ACC ACC TCT GAT 240 AlaLeu Pro Gly Thr Met Arg Ser Leu Arg Val Arg Thr Thr Ser Asp 65707580 GGT GGC TAC TCC TTC AAG TAC GAG ACG GTG CCC AAC ATC CTG GCG CAG 288 GlyGly Tyr Ser Phe Lys Tyr Glu Thr Val Pro Asn Ile Leu Ala Gln 859095 GAC TGC ACC TGT GTC TAGCAGCTGG CATGCACGGC CAGACCCGCG TGGATCTCCC 343 AspCys Thr cysval 100CGTTGCCTCT GGACTGCCCC AGTGCCAGAT GATGAGCCCA TCCCAGGGAT GGAGGAGTCA 403CTCACACGGG CACTGCGCAG CCCGGAGCAG GGAGAGGGAC CCAGGTGGAA GTTTTGGTGG 463TGCCACCCTC CCTTTGACTG CCAGGGTTTC ATGGTTTCAG GTTGCGTGGG TGCTGCAG 521(2) SEQ ID NO: 2, information: (i) sequence characteristics:

(A) length: 101 amino acids

(B) Type (2): amino acids

(D) Topological structure: linear (ii) molecular type: protein (xi) sequence description: SEQ ID NO: 2: leu Gln Arg Pro Ser Glu Asp Val Ala Ala His Thr Asn Cys Arg Arg 51015 Ala Ser Leu Asn Ile Ser Phe Glu Glu Leu Gly Trp Asp Asn Trp Ile 202530 Val His Pro Ser Ser Phe Val Phe His Tyr Cys His Gly Asn Cys Ala 354045 Glu Gly His Gly Leu Ser His Arg Leu Gly Val Gln Leu Cys Cys Ala 505560 Ala Leu Pro Gly Thr Met Arg Ser Leu Arg Val Arg Thr Thr Ser Asp 65707580 Gly Gly Tyr Ser Phe Lys Tyr Glu Thr Val Pro Asn Ile Leu Ala Gln 859095 Asp Cys Thr Cys Val

100

Claims (26)

1. A method of increasing the production capacity of an avian, the method comprising administering to the avian an effective amount of a heterologous protein comprising an alpha-subunit avian inhibin protein, or a fragment thereof, and a carrier protein to increase the production capacity of the avian.

2. The method of claim 1, wherein said bird is selected from the group consisting of ratite, psittaciform, falconoid, picoforme, strigiformes, passeriformes, coraciformes, falliformes, cuuliformes, columbiformes, galliformes, anseriformes, and heroodies.

3. The method of claim 2 wherein said ratite is a camel, pyro, rhea, wingless, or pyro.

4. The method of claim 2, wherein said bird is a chicken, turkey, parrot (parrot), parrot (parakeet), makaw, falcon, eagle (eagle), quail, eagle (hawk), pigeon, cockatoo (cockatoo), whooping bird, saloon bird, or the like,Bird, twitter, goose, duck, canary, blackcock, giant beak bird or sparrow.

5. The method of claim 1, wherein said inhibin protein has or comprises an amino acid sequence substantially identical to SEQ ID NO: 1, and (b) 1 identical sequences.

6. The method of claim 1 wherein said carrier protein is maltose binding protein, bovine serum albumin, ovalbumin, flagellin, keyhole limpet hemocyanin, thyroglobulin, serum albumin, gamma-globulin, a sexual germ cell containing antigen Ia, or a polymer of amino acids.

7. The method of claim 1, wherein said bird is a female bird.

8. The method of claim l, wherein said bird is a male bird.

9. A fused heterologous protein comprising an α -subunit inhibin protein, or a fragment thereof, fused to a carrier protein.

10. The fused heterologous protein of claim 9, wherein said inhibin protein has or comprises a sequence substantially identical to the sequence set forth in SEQ ID NO: 2, and 2, the same sequence.

11. The fused heterologous protein of claim 9, wherein said carrier protein is maltose binding protein, bovine serum albumin, ovalbumin, flagellin, keyhole limpet hemocyanin, serum albumin, gamma globulin, thyroglobulin, a sexual germ cell containing antigen Ia, or a polymer of amino acids.

12. A method of producing a fused heterologous protein, the method comprising the steps of:

(a) providing a double-stranded cDNA encoding an alpha-subunit inhibin, or a fragment thereof;

(b) providing a vector having encoded information for producing a carrier protein;

(c) ligating said inhibin cDNA to said vector;

(d) inserting the ligated vector into an expression system;

(e) expressing a fusion heterologous protein comprising a inhibin protein or a fragment thereof and the carrier protein.

13. The method of claim 12 wherein said inhibin cDNA has or comprises an amino acid sequence substantially identical to SEQ ID NO: 1, and (b) 1 identical sequences.

14. The method of claim 12, wherein the inhibin protein, or a fragment thereof, of said expressed fusion heterologous protein has, or comprises, a sequence substantially identical to the sequence set forth in SEQ ID NO: 2, and 2, the same sequence.

15. The method of claim 12 wherein said carrier protein is maltose binding protein, bovine serum albumin, ovalbumin, flagellin, keyhole limpet hemocyanin, thyroglobulin, serum albumin, gamma-globulin, a sexual germ cell containing the Ia antigen, or a polymer of amino acids.

16. A fused gene product comprising a gene encoding the expression of alpha-subunit avian inhibin protein, or a fragment thereof, fused to a gene encoding the expression of a carrier protein.

17. The fusion gene product of claim 16, wherein said gene encoding the expression of α -subunit avian inhibin protein encodes a polypeptide having or comprising an amino acid sequence substantially identical to the amino acid sequence of SEQ ID NO: 2 expression of proteins of the same sequence.

18. The fusion gene product of claim 16, wherein said gene encoding the expression of α -subunit avian inhibin protein is substantially identical to the gene set forth in SEQ ID NO: 1 are identical.

19. The fusion gene product of claim 16 wherein said gene encoding for expression of a carrier protein encodes maltose binding protein, bovine serum albumin, ovalbumin, flagellin, keyhole limpet hemocyanin, serum albumin, thyroglobulin, gamma-globulin, a sexual germ cell containing antigen Ia, or a polymer of amino acids.

20. A composition comprising a cell containing a vector, wherein said vector comprises a DNA sequence encoding a statin or fragment thereof and a carrier protein, and wherein said vector is capable of expressing the statin or fragment thereof fused to the carrier protein when present in the cell.

21. The composition of claim 20, wherein said DNA sequence encoding inhibin, or a fragment thereof, is SEQ ID NO: 1.

22. a composition comprising a vector, wherein said vector comprises a DNA sequence encoding a statin or fragment thereof and a carrier protein, and wherein said vector is capable of expressing the statin or fragment thereof fused to the carrier protein when present in a cell.

23. The composition of claim 22, wherein said DNA sequence encoding inhibin, or a fragment thereof, is SEQ ID NO: 1.

24. a method of increasing the production capacity of an avian, the method comprising administering to the avian an effective amount of a fusion gene product comprising a gene encoding the expression of an alpha-subunit avian inhibin protein, or a fragment thereof, and a gene encoding the expression of a carrier protein, to increase the production capacity of the avian.

25. A method comprising implanting into an animal cell comprising a vector, wherein said vector comprises a DNA sequence encoding a statin or fragment thereof and a carrier protein, and wherein said vector is capable of expressing the statin or fragment thereof fused to the carrier protein when present in the cell.

26. The method of claim 25, wherein said DNA sequence encoding inhibin, or a fragment thereof, is SEQ ID NO: 1.