CN1063109A

CN1063109A - Improvement to hormone and associated materials

Info

Publication number: CN1063109A
Application number: CN92100141A
Authority: CN
Inventors: R·V·菲什雷利; B·罗布桑
Original assignee: Proteus Molecular Design Ltd
Current assignee: Protherics Medicines Development Ltd
Priority date: 1991-01-09
Filing date: 1992-01-09
Publication date: 1992-07-29
Also published as: GB2267496B; IS3802A; FI933138L; HUT66829A; JPH06504537A; EP0566611A1; CA2100057A1; GB2267496A; AU1161792A; NZ241240A; FI933138A7; GB9314141D0; GB9100468D0; KR930703444A; PT99991A; HU9301979D0; AU652611B2; WO1992012247A1; DK80993A; NO932491L

Abstract

The invention discloses the method for making fish GnRH analogue, said analogue has following sequence:

pGlu—His—Trp—Ser—Tyr—W—Trp—Leu—Arg—Pro—Gly-X-Y-Z

W wherein, X, Y, Z is defined in the specification.Analogue can be connected on the carrier and and start sexual maturity and improve growth rate in order to for example delay.

Description

Improvement to hormone and associated materials

The extension analogue that the present invention relates to be called fish gonadotropin-releasing hormone (pGnRH) and be sometimes referred to as the fish hormone of fish luteinizing hormone-releasing hormone (pLHRH).Specifically, the present invention relates to add an analogue that comprises the formed pGnRH of short amino acid sequence of halfcystine or tyrosine residues at the C-terminal of natural pGnRH aminoacid sequence, under the unaltered basically situation of its conformation in solution, make its polypeptide conjugates be suitable for producing anti-pGnRH antibody, the invention still further relates to the method for immune fish.

The marketable fish aquaculture is the industry that develops rapidly all over the world.West Europe, North America and South America, Canada and East Coast all are the main grown places of fish, particularly Oncorhynchi.For example, the U.S. produced 185,000 tons of Oncorhynchi in 1989, be right after thereafter be catfish market.On the other hand, as the Norway of the Atlantic Ocean, West Europe Oncorhynchi major country of production, the output of 1989 to nineteen ninety is 120,000 tons.It is estimated that to the end of this century, Oncorhynchi market, the world increases by 7.5% every year on average, annual volume of business is 2,000,000,000 pounds.Though at present Europe is the main production area of ocean bream and perch, and culturing carp, also imagining ocean perch and big dace flounder in Eastern Europe and Africa, and in the long term to the business development of Hippoglossoides fish and cod.

In aquaculture, need the sexual maturity of control fish especially, so that stop: the somatocyte growth turns to the sexual gland growth, reduces the quality of fish and shows the commercial secondal sexual character of not expecting.Particularly for salmon fishes, precocious samlet and prematurity samlet can be ended to produce when not reaching the product size samlet and be brought serious economic impact, and reduce its marketable value because be eager to gather in the crops samlet in short duration.In addition, by plant's migration and contribute under the situation in natural gene storehouse, expectation suppresses the reproductivity of having cultured fish fish.

From in logic, problem is associated with the sexually matured fish of results.These can not be sold, and always have that a certain proportion of to have early the male fish of ripe advantage be everlasting ripe before other fishes.Want spended time and funds and isolate sexually matured fish from the prematurity fish, in case fish begins maturation, they more or less all will pass through same time and speed, thereby cause difficulty to results.Therefore, find a kind of sophisticated method of fish of controlling simultaneously to have suitable beyond doubt.

For example, since precocious, the ideal weight that usually can not reach the results fish caused, promptly being forced in not too suitable results in period, the control maturation then can be avoided the amount of missing suitable results period and can make fish long to the ideal size.

Up to now, guarantee to be unlikely that sexually matured effective way takes place is to culture sterile fish.Although carried out a large amount of experimental studies, but still just show that not the colony of two state properties realizes this purpose.

Existing several method is used to control the sexual maturity of fish, as comprises operation castration, triploidization and property control (back two kinds of methods are to belong to genetics technology).Past attempts has attempted with surgical method fish being castrated, but causes the damage of fish to abandon because of their incur great expenses and after carrying out this program eventually.

The triploid method is a kind of method that causes three group chromosomes to exist in the fish individuality, and male and female fish is sterile in order to induced triploid.Yet there are several shortcomings relevant with triploid.At first, this is the time-consuming method of trouble that a kind of needs just can be finished for a long time.For making triploid ovum survival, must carry out high-quality treating processes, all be alive though resulting fish be it seems, this survival rate has comprised the average loss amount of 15-20%, the growth velocity of triploid fish is slower than liploid fish beyond spawning season simultaneously.Near the spawning time time, between the male and female triploid very big-difference is arranged, wherein patrogenesis can be near normal inspection standard and all relevant second features, and female triploid does not then grow tangible ovary, and shows immature outward appearance when normal spawning.

Successfully use the inductive triploid and require to share only female technology (female-only technique), and this commercial be non-remunerative.The shortcoming of other relevant with triploid is, they and diploid compete in same pond and cause growing bad, and the growth of the female fish of triploid does not demonstrate and surpasses the female fish of ripe diploid.

Fish is the height two condition, and promptly they can be in a male and female change, and can controlled other change.For culturing purpose, as the viewpoint from the speed of growth, a kind of sex is normally preferable.Turbot and common eel are female than male example of growing greatlyyer.In samlet, childhood, male and female fish had the similar same speed of growth, but male afterwards fish has grown the feature that does not expect to have.In the ripening stage, male stopping growing, the brown and the love that becomes are picked a quarrel gradually, and its value as food and toy is just very poor.In addition, the rainbow trout (Salmo gairdneri) of raising and Atlantic Ocean Oncorhynchi ripe male in case put into the seawater cultivation pool, just can not be adapted to salt solution and cause death.Can use a kind of generation sex reversing, to obtain the method for all female colonies, but having comprised, it in fish meal, mixes hormone, make fish be not suitable as the food on the dining table, method such as illumination, the chemical sterilization of other control fish fertilizabilitys and to give hormone also all unreliable, uneconomical or be not suitable for the fish of production as food.

Controlled sophisticated immunological method is obtained some success on one's body Mammals.Yet, can not become the solid foundation of studying sashimi (raw fish) Neo-Confucianism to the research of mammalian hormones and immunological response.Fish is a cold blooded animal, and their immunity system can variation to some extent under different condition.Temperature promptly has remarkably influenced to the immune response of fish, shows higher immune response level when consequently in physiological range comparatively high temps being arranged.As if physiological low temperature can postpone the speed of antibody generation and can suppress the foundation of immunological memory, responsive especially to this T lymphocyte.

In addition, the immunity system of fish is subjected to the influence of the hormone that may rationally change.Hormonal readiness can and be arrested equipressure or quality such as influence that oxygen level is low etc. changes because of carrying.Go into sea and sexual maturing period (being two basic hormone change periods) at the troutlet effluent, fish is particularly responsive to disease because body endolymph cell number reduces.

Moreover fish only produces one type antibody molecule, and promptly IgM has five big antibody-likes in definite mammalian body.

The endocrine system that is exactly fish in addition is different in essence in mammiferous endocrine system, and for example the fish gonadotropin release by salmon flying fish such as Oncorhynchi and the generation of fermented red beancurd fish has following sequence:

PGlu-His-Trp-Ser-Tyr-Gly-Trp-Leu-Pro-Gly-NH ₂(Sherwood et al, 1983; " Characterisation of a teleost GnRH ", PNAS USA80:2794-2798), the amino acid on its 7th and 8 is different from Mammals LHRH.

We have now found that, the analogue of a class fish GnRH can be connected on the appropriate carriers molecule and be used for immune fish, so can induce to produce the immunogenic antibody of anti-cross reactivity, the latter to GnRH have high-affinity and can in and endogenous GnRH.

According to an aspect of the present invention, we provide the analogue that the fish of following amino acid sequences GnRH is arranged:

pGlu-His-Trp-Ser-Tyr-W-Trp-Leu-Arg-Pro-Gly-X-Y-Z

Wherein W represents Gly or a D-amino acid

X represents a key, or one or more identical or different amino acid,

Y represents Cys or Tyr or does not have Y, and

Z does not exist or represents one or more identical or different amino acid, or 1 continuous or discrete peptide chain, and each discontinuities exists non-alpha amino acid multifunctional access head part or reverse conversion amino acid, condition be when W be Gly, when X and Y do not exist, have Z.

The residue at W place, position is Gly preferably, and it is naturally occurring residue; The residue at Y place, position is Cys preferably, because it is usually more special, and than being easier to receive on the carrier proteins with the C-terminal Tyr person of obtaining.

X preferably represents relatively small number purpose residue, as 1 to 15, better is 1 to 8, is more preferably 1 to 3, preferably has only one.Though can have any one or a plurality of amino acid on the X position, this amino-acid residue is Gly preferably.Certainly, other amino acid better be to have the amino acid of little side chain such as Ala, Ser, Asn and Val also to may reside on the X position, and this locational any amino-acid residue include within the scope of the invention.Be also to be noted that to comprise more than one identical or different residue on the X position, for example in following sequence:

pGlu-His-Trp-Ser-Tyr-W-Trp-Leu-Arg-Pro-Gly-Gly-Ala-Y-Z。

Should be clear and definite be not have the pGnRH of amino-acid residue to be also included within the scope of the present invention on the X position.

In the preferred embodiment of the pGnRH according to the present invention, Z does not have amino-acid residue on the position, and has following sequence according to the particularly preferred example of analogue of the present invention:

pGlu-His-Trp-Ser-Tyr-Gly-Trp-Leu-Arg-Pro-Gly-Gly-Cys。

Yet we studies show that, comprise on the Z position when adding amino acid generally the conformation of molecule rest part being selected not have a significant effect.If the Z position has comprised such extension, so in preferred version of the present invention, this extension will comprise can be as one or more small segments of the protein sequence of T cell epitope.For example, the small segment of the aminoacid sequence of general formula 1-2-3-4, wherein 1 is that Gly or charged amino acid are (as Lys, His, Arg, Asp or Glu), the 2nd, hydrophobic amino acid is (as Ile, Leu, Val, Met, Tyr, Phe, Trp, Ala), the 3rd, hydrophobic amino acid (being defined as above) or uncharged polare Aminosaeren are (as Asn, Ser, Thr, Pro, Gln, Gly), and the 4th, polare Aminosaeren is (as Lys, Arg, His, Glu, Asp, Asn, Gln, Ser, Thr, Pro), as if can be used as T cell epitope (Rothbard, J.B.﹠amp at least some instances; Taylor, W.R, (1988), A sequence pattern in common to T-cell epitopes, The EMBO Journal7(1): 93-100).Similar small segment can be sequence 1 '-2 '-3 '-4 '-5 ', wherein 1 ' 1,2 ' suitable 2,3 ' and 4 that are equivalent to limit above ' be equivalent to 3,5 ' ibid to be equivalent to 4().Two kinds of forms are included in the general formula that limits below: pGlu-His-Trp-Ser-Tyr-W-Trp-Leu-Arg-Pro-Gly-X-Y-S-T-S ', wherein the T representative contains the peptide sequence small segment (most preferably less than 5 amino acid) of one or more T cell epitopes, it can be type or other structure that limits in the preceding paragraph, and can be separated by any length or the spacer small segment formed, S and S ' (wherein each can suitably and individually be left out) then represent the spacer small segment by any length and the aminoacid sequence formed.The length of S is most preferably less than 5 amino-acid residues, and comprises the residue that for example is selected from Gly, Ala, Pro, Asn, Thr, Ser or multifunction conjunction such as non-alpha amino acid.S ' is most preferably less than 10 amino-acid residues and can comprise multifunction conjunction as non-alpha amino acid; Draw in the amino acid of the S representative that S ' part preferably provides from above.Might represent a whole protein by S ', therefore just not necessarily will be connected on the carrier proteins.

It should be noted, when Z does not exist, can on the X position, provide T cell epitope above-mentioned.

The derivative of basic analogue also is included in the scope of the invention, and wherein Z is or comprises " oppositely reversing " amino acid.The bifunctional amine who promptly has the functional group that is equivalent to certain amino acid.For example one according to the present invention and comprise the amino acid whose analogue of reverse reversing and can have following general formula:

Wherein R is any functional group, is more preferably Gly, and A1 and A2 are more preferably the copy (but not necessarily identical) of one of analogue that this paper of being connected by its C-terminal limits separately.In particularly preferred embodiments, locational Tyr of Y or Cys should be replaced by the amino acid of any kind beyond the residue on the corresponding position of another copy among one of A1 or A2, and perhaps it can be left out.Therefore this dimeric connection is to realize by the side chain of one of residue on the Y position (also can be two residues).In addition, A1 and/or A2 can be natural pGnRH, and oppositely to reverse amino acid whose R can be Cys or Tyr.As previously discussed, the T cell epitope also can be included among the small segment Z.

Analogue can have following general formula:

Wherein B oppositely reverses amino acid, and A1 and A2 are identical or different, and show sequence under having:

pGlu-His-Trp-Ser-Tyr-W-Trp-Leu-Arg-Pro-Gly-X-Y-Z′

Wherein the definition of W, X and Y is the same, and Z ' is identical with aforesaid Z.Just it does not comprise reverse reversing amino acid.

Reverse reversing modification to peptide comprises that the one or more peptide bonds of reversing are to produce the analogue that enzyme liberating is had bigger resistance than initial molecule, and provide a kind of approach easily for producing branch's immunogen, for in wait until big immunogen, this branch's immunogen will contain the epitope of high density.In the reverse solution in enormous quantities that reverses analogue of short chain biologic activity peptide synthesizes, use these compounds to have very big potential value.

Another aspect of the present invention provides the method for making the fish GnRH analogue with following amino acid sequences:

pGlu-His-Trp-Ser-Tyr-W-Trp-Leu-Arg-Pro-Gly-X-Y-Z，

Wherein W represents Gly or D-amino acid,

X represents a key, or one or more identical or different amino acid,

Y represents Cys or Tyr, or does not exist, and

Z does not exist, or represents one or more identical or different amino acid, or continuous or discrete peptide chain, and each discontinuities exists the multifunctional shank of non-alpha amino acid or the amino acid that oppositely reverses, condition be when W be Gly, and there is Z in X and Y when not existing,

This method comprises to be used known chemistry, biology and/or each residue of recombinant technology coupling and separates this analogue.

Can use the peptide synthetic technology of standard, synthesize analogue of the present invention as the solid phase method of peptide synthesis (SPPS) that Merrifield introduces in 1963 (J.Am.Chem, Soc.85,2149).In this method, the peptide chain of growth is covalently bound to insoluble solid phase carrier by its C-terminal.Synthesize by in expected sequence, adding amino acid continuously.In this case, the intermediate steps of all purifying (these steps are for synthetic being absolutely necessary in the solution) all is reduced to simple flushing, because the by product of the Degradation of great majority reaction all has been dissolved in the reaction mixture.The advantage of SPPS is rapidly, relatively easily to finish and is a kind of partly or entirely automated method, uses SPPS might produce the peptide of tens grams.

Briefly, the SPPS by the Merrifield development in 1963 comprises following four-stage:

(A) by in entire synthesis process, all keeping stable covalent linkage, first protected amino acid is connected on the solid phase carrier;

(B) under all stable condition of the blocking group of any side chain functionalities, produce the free amine group acid groups of the residue that is connected again by going to protect;

(C) form amido linkage by condensation, make next protected amino acid and first amino acid coupling.Repeating step B and C finish up to building-up process;

(D) when synthetic finishing, by discharging peptide on the solid phase carrier and removing any amino acid side chain blocking group.

Describe the combination of various amino acid blocking groups in the document, but at present commonly used had only wherein two kinds of combinations.First kind is tert butoxy carbonyl (Boc)/benzyl composition, and it was just used by Merriifield from 1963, and still was widely adopted so far.Be surprisingly found out that recently fluorenyl methoxy phosphinylidyne (the Fmoc)/tertiary butyl system that is developed by people such as Sheppard has also obtained using widely (Sheppard, R.C, 1986, Science Tools, The LKB Instrument Journal33,9).Further example comprises by Arsady; R. wait the Fmoc-polymeric amide method (J.Chem of the use solid-phase resin of people's foundation; Soc.Perkin Trans; 1981,1,529-537); indivedual amino acid (the Atherton that are impregnated in fluorenyl methoxy phosphinylidyne (Fmoc) protection; E.et al, J.Chem, Soc.Perkin Trans; 1983; 1,65-73) and 9-fluorenyl methoxy phosphinylidyne (Fmoc) chemical method (as referring to Atherton, E.et al; 1985; J.Chem.Soc.Chem, Comm, 165).

In addition, the DNA and the RNA molecule of the sequence by genetic code coding each peptide analogs described herein are also included within the scope of the invention.These dna moleculars can be used, in order to produce analogue as herein described in suitable microorganism or eukaryotic cell culture expression system.In such system, synthesized similar molecule and in this document, addressed (as referring to No. 85307311.2, european patent application).

It should be noted, can not use such system directly to make the analogue that mixes reverse reversing amino acid derivative.But can be further purified basic analogue and use the peptide/organic chemistry method of standard to be connected on the reverse reversing amino acid.Describe on polyamides amine type resin solid phase synthesis recently and oppositely reversed the practical of peptide and novel method [Gazerro, H, Pinori, M.﹠amp easily; Verdini, A.S.(1990), A new general Procedure for the solid-phase synthesis of retro-inverso peptides, In " Innovation and Perspective in Solid Phase Synthesis " Ed.Roger Epton.SPC(UK) Ltd.Birmingham, UK].

Preferably analogue is connected on the carrier molecule, to obtain the analogue binding substances.Any small molecules or macromolecular carrier or microorganism can be used, but preferably following carrier commonly used: the key hole

The tuberculin albumen microorganism (PPD) of hemocyanin (KLH), bovine serum albumin (BSA), chicken gamma Globulin (CGG), Toxoid,tetanus (TT), diphtheria toxoid (DPT), purifying, Muramyl dipeptide (MDP), soybean trypsin inhibitor (STT) and Toxins,exo-, cholera or its B subunit.It should be noted that and using under the situation of PPD, with the BCG vaccine cause more favourable (Lachmann, P.et al, 1986, In " Synthetic Peptides as Antigens ", Ciba Foundation Symposium119, pp.25-40).

The carrier of used LHRH analogue during as oneself immunization fish particularly preferably is following microorganism or macromole antigen as vaccine composition: aeromonas salmonicida (Aeromonas salm onici da) (furuncle ulcer disease), Yersinia ruckeri(ERM) and Vibrio anguillarum (Vibrio anguillarium) and Vibrio ordalii(vibriosis) mixture.

Recently, tested the sick effect (latter is caused by the non-bacterium aeromonas salmonicida that moves about of Gram-negative) of anti-furuncle ulcer of following five base type vaccines: whole being killed or destructive cell (vaccine), extracellular products (ECP) and ECP toxoid, whole killed cell ECP share, the antigen of live attenuated vaccine and purifying.Anti-some antigenic hyper-immuneserum that also will produce in fish and mammalian body in addition, is used for passive protection.In addition, successfully produced the vaccine (or vaccine) of anti-redmouth enteritis (enteric redmouth ERM) (this disease is caused by the Gram-negative bacterium Yersinia rukeri that moves about), it comprises the full bacterial cultures of formalin deactivation.The national on the Northern Hemisphere recently commodity vibriosis vaccine that uses comprises the mixture of the most common bacterial classification Vibrio anguillarum and V.ordalii.These vaccines are the cultures of simple deactivation that contain the mixture of full cell and ECP.

Another aspect of the present invention provides the pGnRH analogue that contains with good grounds the present invention or analogue binding substances and the veterinary drug vaccine with vehicle.

These vaccines can be used for oneself immune fish, and one side more of the present invention provides a kind of method that piscinity is grown that suppresses, this method comprises pGnRH analogue or the analogue binding substances that uses significant quantity to said fish, so that the anti-pGnRH antibody titers that is produced is enough to significantly reduce the biological efficiency of endogenous pGnRH.

Can use reagent and the method fully described in the document, make the binding substances that contains the analogue of Cys or Tyr on the Y position at an easy rate.For example, can use N-γ-maleoyl-imino-butyryl acyloxy succinimide (Calbiochem), by thioester bond the fish GnRH analogue that contains the Cys residue on the Y position is connected on the lysine side-chain of carrier protein, simultaneously can with the N-(4-benzene diazonium)-maleimide forms and is connected the diazonium key that the Y position contains the analogue of Tyr residue.

Can use methods known in the art to be easy to peptide and binding substances are purified to necessary degree, these methods are as comprising high performance liquid chromatography, sodium dodecyl sulfate-polyacrylamide gel electrophoresis and FPLC.

Peptide-carrier binding substances of the present invention can be used for following purpose:

(1) immune any animal is used for the anti-fish GnRH antibody of following described specific needs with generation:

(2) fish is carried out oneself immunity of anti-fish GnRH, so that:

-the sexual development of slowing down, stop or disappearing

-control, restriction or elimination fecundity

-treatment GnRH dependency fish tumour

-carry out sashimi (raw fish) Neo-Confucianism/veterinary science research;

(3) as detecting as whether there being the diagnostic reagent of anti-fish GnRH antibody in the serum sample.

Anti-fish GnRH antibody at analogue also constitutes a part of the present invention.For example they can

1) as research sashimi (raw fish) Neo-Confucianism/immunologic research tool

2) as the immunogen that produces anti-alloantibody;

3) carry out passive immunization, so that:

-be implemented in a short time the sexual development of slowing down, stop or disappearing

-reach and control fecundity in a short time

-treatment GnRH dependency fish tumour

-disturb the activity of fish GnRH in addition.

(can carry out passive immunization) by polyclone, mono-clonal or the single area antibody that gives in fish or mammalian body, to produce

4) as the diagnostic reagent that whether has fish GnRH in test example such as the serum sample.

Obviously, above 2) in the anti-alloantibody mentioned also constitute a part of the present invention.

Can according to ordinary method preparation can with the chimeric form of the polyclone of synthetic polypeptide specific combination of the present invention or monoclonal antibody, these antibody or the distinctive form of fish (as referring to Morrison et al, (1984) PNAS(USA) 81,6851-6855; Riech-mann et al, (1988) Nature 332,323-327), and single area antibody (as referring to Ward, E.S, Gussow, D.Griffiths, A.D., Jones, P.and Winter, G.(1989) Nature 341,544-546).As mentioned above, these antibody also are considered to a part of the present invention.

Aspect anti-fish GnRH antibody of detection or fish GnRH, those skilled in the art can use various known immunoassay technologies, as using competition or competitive inhibition detection method or direct and indirect labelling method.The medicine box of the anti-fish GnRH of a kind of preferred detection antibody has comprised analogue of the present invention or analogue binding substances, marking tool and solid phase carrier.Similarly, a kind of medicine box that detects fish GnRH comprised can with antibody or Fab, marking tool and the solid phase carrier of the present invention's analogue specific combination.

Can use fish vaccine through several method, as injection, oral route and by submergence etc.

Injection is the most effectual way of vaccine inoculation in the abdomen control, simultaneously the adjuvant that improves the immune response degree easy to use.Shortcoming is the respective handling that fish need be anaesthetized, and will make the fish withstanding pressure like this and strengthen labour intensity.If but used syringe and the line production system that reuses, 1000 fishes per hour could be injected.But this is unsuitable for the fish less than 15 grams.

The oral vaccine inoculation is suitable for the fish administration to all size in enormous quantities, and the pressure that does not cause because of operation.Shortcoming is to need to use a large amount of vaccines, thereby has improved cost, and the using dosage of every fish of bad control.It is poor that oral vaccine is renderd a service, and causes the level of provide protection low and non-constant, and most of oral administrations are all at the vibriosis vaccine.

Preferred directly immersion method (D.I) is a kind of simple and rapid method, only need contact several seconds with vaccine.This method has now realized automatization and set up the schedule of operation of " bath " and " washing " conversion at vibriosis and ERM vaccine, and only comprises simply vaccine is poured in the accumulator tanks.Also need long duration of contact (about one hour, comprise the oxygenation of water and the pressure that close monitoring fish is subjected to) though this method will consume more vaccine, its labour intensity is littler than injection.

With containing (I) and the known analogue of more than one type of carrier molecule bonded, and/or the mixed vaccine submergence of (II) more than one analogues that combine with carrier molecule equally may be more favourable.In addition, can be with any peptide analogs, their binding substances and composition thereof is added in any suitable adjuvant or the release system and uses, and more than one adjuvant and release system can be lumped together, and forms so-called " super mixture ".Preferred adjuvants and release system comprise aluminium hydroxide (alum), microsphere, liposome, micella, rrna, ISCOMS, Brauns lipoprotein and full cell or microorganism fish vaccine composition.

Further describe the present invention by non-limiting example below.

Embodiment 1

Use the carboxyl terminal of the synthetic pGnRH of the present invention of standard solid-phase Fmoc method to extend form.In the presence of the trifluoroacetic acid by resin on cleavage of peptide, carry out purifying with gel-filtration, ion exchange chromatography and reverse high performance liquid chromatography (HPLC) then.By MBS(m-maleimide benzoyl-N-hydroxy-succinamide phenyl ester) peptide is connected on the various carriers.

Proved that chicken gamma Globulin (CGG) is a kind of good carriers for other vaccines of hardhead trout, and be used in the present embodiment.Also used PPD, because this is a kind of confirmed carrier (although needing to use BCG before immunity) in mammiferous experimental study.

With peptide: protein is 30-40: 1 molar ratio is coupled to the pGnRH analogue on the carrier, and does intraperitoneal injection with three different concentration to rainbow trout (Salmo gairdneri).

Yet the monoclonal anti trout immunoglobulin (Ig) that uses sour jujube root peroxidase (HRP) to connect is monitored serum antibody response with euzymelinked immunosorbent assay (ELISA) (ELISA), surveys once totally 10 weeks weekly.In some test of present embodiment,, or carry out booster shots with pre-immunity of carrier or use adjuvant.To determine to produce the optimum immuning program of anti-pGnRH antibody.

Embodiment 2

Treat sophisticated hardhead trout for one group by the optimum immuning program immunity of determining among the embodiment 1.With the monitoring of method described in the embodiment 1 antibody response, monitor the generation of the speed of growth and sex steroid (as testosterone) simultaneously.When experiment finishes fish is killed, remove gonadectomy and weigh, with determinacy body of gland index (GSI).Fixing sexual gland then is so that carry out Histological research with opticmicroscope.The fish that immunity is crossed has obviously good GSI than untreated control group fish.

Embodiment 3

Extend the rainbow trout (Salmo gairdneri) of analogue (sGnRHa-PPD) immune mature with the salmon gonadin releasing hormone on the protein derivatives that is coupled to the tuberculin purifying.

Method

A) sGnRH-Gly-Cys is connected on the PPD

In the benzoylation cellulose tube, make tuberculin PPD(1.008mg/ml+ phenol sanitas, Evans Limited) to the 0.9%NaCl liquid of under 12 ℃, dialysing, to remove phenol.Use polyoxyethylene glycol (pG20000) to handle then 1.5 hours, PPD is concentrated to 4.032mg/ml.At room temperature make PPD solution (2.5ml, containing 10.08mg PPD mixed 1 hour with 1.08mgN-γ-maleoyl-imino-butyryl acyloxy succinimide (GMBS) in 5.04ul dimethyl formamide (DMF), go up Sephadex G-25M PD-10 post then, and with 3.5ml buffer A (0.05M NaH ₂PO ₄, 0.14M NaCl, pH7.0) wash-out.Be dissolved in sGnRH-Gly-Cys that 4.687ml concentration in the distilled water is 3mg/ml and buffer A (that is, and 14,0616mg SGnRH-Gly-Cys dropwise is added among the PPD.Under nitrogen environment with solution mixed at room temperature 2 hours, and the free thiol content of estimation from start to finish.In the benzoylation cellulose tube, make binding substances to 12 ℃ of liquid of dialysing of distilled water, be stored in-20 ℃ then and descend standby.

B) free thiol analysis

Use reagent 5,5 ' one or two sulphur two (2-nitrobenzoic acid) (DTNS) and go back the free thiol content of ortho states gsh standard substance estimation binding substances.Use the microtiter plate variant of scaled Ell-manShi assay method, wherein in 50 μ l samples, add 2 μ l10mMDTNB and 250 μ l PBS(pH8), after 10 minutes in 405nm place mensuration light absorption ratio.

C) hardhead trout and immune programme for children

Use freezing printing tools difference 130 ripe rainbow trout (Salmo gairdneri)s of mark (the about 30cm of length) of various combinations.Write down length and the weight of every fish and get 600 μ l blood samples mensuration pre-immune serum antibody horizontal.In addition, detect the serum steroid levels, to determine the sex of fish.Fish is divided into two groups, and the sGnRH-Gly-Cys-PPD or the salt solution that are added in respectively in the Freund's complete adjuvant (FCA) are done intraperitoneal injection.Tell treatment group and control group male and female respectively again.Male fish is placed under light and the temperature condition.Femalely further be divided into two groups again: one group is placed under ambient light and the temperature condition (temperature can change) between 14 ℃ or lesser temps, and another group then places under ambient light and the 14 ℃ of conditions.Estimate the influence of temperature antagonism sGnRH-Gly-Cys antibody titers and any difference that in the fish maturation, causes with the one group of fish in back.In the whole experiment, with circumferentially-spaced the fish of each bar stamp is weighed and to detect and bloodletting.With the anti-sGnRH-Gly-Cys antibody (using sGnRH-Gly-Cys-BSA) in the ELISA method detection serum, and use RIA method detection type steroid hormone 17-and/or 11-ketone testosterone (11-ketotestosterone).

D) steroid analysis

Detect the concentration of 17-in the ripe fish serum (E2) and 11-ketone testosterone (11-KT) with radioimmunology.In this analysis, the steroid of radioactivity mark is attached on the limited amount steroid specific antibody, this interaction can partly be suppressed because of adding unlabelled steroid.

E) optimizing of anti-sGnRH-Gly-Cys elisa assay

The salmon class GnRH-Gly-Cys that will be attached on the bovine serum albumin (BSA) is used for the elisa assay method.The 10mgBSA that is added in the 0.5ml buffer A is merged with the 1mgGMBS that is added among the 5 μ lDMF, be coupled on the 6.246mgsGnRH-Gly-Cys at last.ELISA checkerboard detection method can be selected to be suitable for carrying out the sGnRH-Gly-Cys-BSA bag of anti-sGnRH-Gly-CysELISA analysis by concentration in test of maturity.

The result

ⅰ) anti-sGnRH-Gly-Cys antibody titers

(male and female) as shown in table 1 increased anti-sGnRH-Gly-Cys antibody titers with sGnRH-Gly-Cys-PPD immunity (2 μ g sGnRH-Gly-Cys/ fishes are added in intraperitoneal injection among the FCA) 8 weeks of back with respect to control group.

ⅱ) the level of rainbow trout (Salmo gairdneri) ripening period serum 17-β estradiol: with the influence of sGnRH-Gly-Cys-PPD immunity

As shown in table 2, in 4 weeks after immunity, the serum 17-β estradiol level of treatment group and control group adult female trout increases at the same rate.To immunity the 8th week of back, the 17-β estradiol level of control group still continues to increase at the same rate.But in 8 weeks after immunity, the sGnRH-Gly-Cys-PPD group in the ripe raun then comparison has much lower 17-β estradiol level (P＜0.05:t test) according to forming ripe raun.

ⅲ) the mutual relationship between antibody titers and steroid levels

Obviously, after the sGnRH-Gly-Cys-PPD immunity, the increase of antibody titers is that a small amount of increase with ripe rainbow trout (Salmo gairdneri) serum 17-level is mutually related, and data show that pGnRH-Gly-Cys-PPD is active and effective in postponing female rainbow trout (Salmo gairdneri) sexual maturity.

Claims

1. A method for producing a fish GnRH analogue having the following amino acid sequence:

pGIu-His-Trp-Ser-Tyr-W-Trp-Leu-Arg-Pro-Gly-X-Y-Z

wherein W represents Gly or D-amino acid,

X represents a bond, or one or more identical or different amino acids,

Y stands for Cys or Tyr or absent, and

Z does not exist or represents one or more amino acids, which may be the same or different, or a continuous or discontinuous peptide chain, and there is a non-alpha amino acid polyfunctional linker part or an inverted amino acid at each break, provided that when W is Gly, X and Y is absent when Z is present,

The method involves coupling the residues and isolating the analog using known chemical, biological and/or recombinant techniques.

2. The method according to claim 1, wherein W is Gly.

3. A method according to claim 1 or 2, wherein Y is Cys.

4. A method according to any one of the preceding claims, wherein X is one or more glycine residues.

5. A method according to any one of claims 1 to 3, wherein X or Z comprises at least one T cell epitope.

6. A method according to any one of the preceding claims, wherein Z comprises an inverted inversion amino acid.

7. A method according to claim 6, wherein the analog has the general formula:

A ₁ -BA ₂

Wherein B is a reverse inverted amino acid, _A1 and _A2 are the same or different, and have the following sequence:

pGlu-His-Trp-Ser-Tyr-W-Trp-Leu-Arg-Pro-Gly-X-Y-Z'

wherein W, X and Y are as defined in claim 1, and Z' is the same as Z defined in claim 1, but it does not include inverted inversion amino acids.

8. The method according to claim 1, wherein the analogue has the sequence: pGlu-His-Trp-Ser-Tyr-Gly-Trp-Leu-Arg-Pro-Gly-Gly-Cys.

9. A method according to any one of the preceding claims, which comprises linking the analogue to a carrier molecule.

10. A method for the manufacture of a vaccine, wherein an analogue as defined in any one of claims 1 to 8 or a combination of analogues as defined in claim 9 is mixed with a veterinary excipient.

11. A kit for detecting anti-fish GnRH antibody in a sample, said kit comprising an analog as defined in any one of claims 1 to 8, or a combination of analogs as defined in claim 9, a labeling tool and solid phase carrier.

12. Use of an analogue as defined in any one of claims 1 to 9 for the manufacture of a medicament for slowing, stopping or regressing sexual development or controlling fecundity in fish.

13. A method according to any one of claims 1 to 5 or claim 8, wherein the analog is produced as a continuous peptide chain by recombinant techniques.

14. A method of producing an antibody or antigen-binding fragment that specifically binds an analog as defined in any one of claims 1 to 7, the method comprising using an analog as defined in any one of claims 1 to 8 or a claim 9. Immunizing the immunized subject with the defined analog conjugate, and isolating the antibodies formed or antibody-producing cells.

15. A kit for detecting fish GnRH in a sample, said kit comprising the antibody or antigen-binding fragment as defined in claim 14, a labeling tool and a solid phase carrier.

16. Antibody or antigen-binding fragment as defined in claim 15 for use in the manufacture of a medicament for slowing, stopping or regressing sexual development or controlling fecundity in fish.

17. A method for producing an anti-allotype antibody specific for the antibody or antigen-binding fragment defined in claim 14, the method comprising immunizing a subject to be immunized with the antibody or antigen-binding fragment produced by the method of claim 14 and isolating antibodies formed.

18. A method of immunizing fish against fish GnRH to slow down, stop or regress sexual development, or control, limit or eliminate fertility, the method comprising administering to fish an effective amount as defined in any one of claims 1 to 8. or the analogue conjugate as defined in claim 9 or the antibody or antigen-binding fragment as defined in claim 14.

19. A method according to claim 18, wherein the fish is submerged in a mixture containing an analog as defined in any one of claims 1 to 8 or an analog conjugate as defined in claim 9 or an antibody or antigen-binding fragment as defined in claim 14. of water.

20. The method according to claim 18, wherein the analogue as defined in any one of claims 1 to 8 or the analogue conjugate as defined in claim 9 or the antibody or antigen-binding fragment as defined in claim 15 is administered orally .