CN1189777A - Tissue Adhesives Utilizing Synthetic Crosslinkers - Google Patents

Abstract

The present invention provides adhesives and sealants capable of firmly bonding tissues by using a protein-based polymer having a repeating unit derived from a natural structural protein as a backbone of crosslinking functionality. In particular, block copolymers of elastin and silk fibroin are used which have lysine substitutions in spaced apart units, wherein the amino groups can be crosslinked with a bifunctional crosslinking agent.

Description

Tissue Adhesives Utilizing Synthetic Crosslinkers

This application is a continuation-in-part of Application Serial No. 08/435,641 filed May 5, 1995, the disclosure of which is incorporated herein by reference.

The field of the invention is that of physiologically acceptable compositions for use as tissue adhesives and sealants.

Bonding of separated tissues is required in many situations. Suture and fixation are effective and well-established methods of wound closure. However, there are some cases in which surgical procedures are limited to highly trained specialists (eg, microsurgery) or inaccessible due to tissue or organ fragility (eg, endoscopic procedures) when traditional repair procedures are not satisfactory , or body fluid loss (including capillary "seepage") and cannot be used. Tissue adhesives or sealants have been developed to meet these needs. They are used to seal or reinforce wounds that have been sutured or stapled, as well as exploring free-standing uses. The main commercial products are fibrin adhesives and cyanoacrylates. However, both products have obvious limitations which limit their wide application.

Nitrile acrylates are mainly used for closure of skin wounds in facial and plastic surgery. The advantages of cyanoacrylates are their almost instant bonding speed and strong bond strength. However, the speed of bonding can be a disadvantage since the bonded tissue must be re-cut to shape it into the desired shape. Furthermore, because it works by mechanical interlocking, it can only be used on dry substrates, limiting its use as a sealant, and it is relatively stiff relative to the surrounding tissue. Cytoacrylates are known to be toxic to certain tissues and although their biodegradability need not be considered, possible degradation products are also suspected of being carcinogenic.

Fibrin adhesives containing blood-derived fibrinogen, factor XIII and thrombin were first used as sealants and hemostats and have been used in many different surgical procedures in vivo. They have been shown to be non-toxic, biocompatible and biodegradable. They inhibit excessive bleeding and reduce fibrosis. However, even mild stretching of tissues bonded with fibrin breaks the bond. Three to ten minutes are required for initial bonding, but 30 minutes to several hours are required to fully develop strength. Depending on the application, this product may also reabsorb too quickly. The use of recombinantly produced fibrinogen, factor XIII, thrombin and related components (eg fibrin, activated factor XIII) has not been shown to improve the onset time or strength of fibrin adhesives. Fibrin adhesives derived from heterologous human and animal sera may provoke immune side effects and put patients at risk of viral infection. It is not realistic to obtain and apply an intrinsic fibrin adhesive that can take into account patient safety.

Therefore, there is a considerable need to develop products that have the biocompatibility of fibrin but have a faster onset of action and increased strength. The product should be readily available, more desirable than natural sources, easy to administer and reabsorbable over time.

Tissue Adhesives In: Tissue Adhesives in Surgery, Matsumoto, T., Medical Laboratory Publishing Co., 1972 and Sierra, D.H., Journal of Applied Biomaterials, 7:309-352, 1993 Blocks with repeating units Methods for preparing protein polymers are described in US Patent 5,243,038 and EPA 89.913054.3.

The present invention provides polymeric compositions and methods of use in which polymeric tissue can be chemically crosslinked in situ to provide novel crosslinked polymeric products with favorable mechanical and biological properties, such as strong adhesion to tissue. The composition can be used in a variety of applications involving its physical, chemical and biological properties in bonding separated tissues to provide at least one bond including stable, flexible and resorbable properties.

The tissue of interest contains high molecular weight recombinant polymers with one or a combination of repeating units associated with naturally occurring structural proteins. Of particular importance are the repeating units of fibroin, elastin, collagen, and keratin, especially collagen and the structures of fibroin and elastin. The polymer has functional groups that can be chemically cross-linked with physiologically acceptable cross-linking agents under physiological conditions, so it can form a bond with strong adhesive properties to various substrates, which is strong enough to maintain the bond between the substrates. Mechanical properties, and can be formulated into compositions with good reabsorption properties.

Of particular importance, the compositions of the present invention provide strong adhesion to tissue and maintain separated tissue in contacting spatial relationship. The subject composition may also be used as a sealant, where the composition may be used to fill voids in tissue, reinforce tissue masses or bond synthetic materials to tissue. The composition of interest may also be used in vivo as a depot by mixing with a pharmaceutical composition, or as an adhesive for bonding tissues, or for other adhesions or simply as a sustained release source of drugs.

The functional groups used for cross-linking may be all the same or a combination of functional groups and may contain functional groups of naturally occurring amino acids such as amino groups such as lysine, carboxyl groups such as aspartic acid and glutamic acid, guanidine, Such as arginine, hydroxyl groups such as serine and threonine, and sulfhydryl groups such as cysteine. Preferably, the functional group is amino (including guanidino).

The molecular weight of the polymer is at least about 15 KD, generally at least about 30 KD, preferably at least about 50 KD and usually not more than 250 KD, more usually not more than about 150 KD. The polymer has at least two functionalities, and more usually about at least four functionalities, each generally having an equivalent weight in the range of about 1KD to 40KD, more usually in the range of about 3KD to 20KD, preferably in the range of 3-10KD, at least 3, usually 6 functionalities for crosslinking. If desired, mixtures of polymers may be used wherein the polymers have a combination of functionalities or different functionalities such as carboxyl and amino, mercapto or aldehyde, hydroxyl and amino, and the like. Thus, based on the functionality and crosslinking agent, amides, imines, ureas, esters, ethers, urethanes, thioethers, disulfides and the like can be formed.

The monomer units in the polymer can be selected from fibroin, GAGAGS (sequence 01); elastin, GVGVP (sequence 02); collagen GXX, wherein X'S can be the same or different, and at least 10 % but not more than 60% of X'S is proline, and keratin, AKLK/ELAE (SEQ ID NO: 3). The desired functional group may replace an amino acid in the monomer unit or as a single amino acid or part of an intervening group of not more than about 30 amino acids, generally not more than 16 amino acids. In the previous examples it was provided that one or more of the repeats in the repeat block were modified and introduced into a cross-linking functionality which is not normally present. Thus, lysine may be substituted for valine, arginine for glycine, serine for alanine, and other similar substitutions. In more recent examples, there may be intervening functionality in the blocks of repeating units, the number of intervening functionalities being based on the aforementioned ranges.

Of particular importance are copolymers, either block copolymers or random copolymers, preferably block copolymers, in which, in the case of elastin and fibroin, the ratio of elastin units to fibroin units is between 16 - 1:1; preferably in the range of 8-1:1, where the blocks have different ratios. Generally, in block copolymers, each block contains at least two units and not more than about 32 units, usually not more than about 24 units. The appropriate number of functionalities present in the polymer can be provided by substituting amino acids in units with amino acids bearing appropriate functional groups or by using intervening groups between blocks.

The repeat unit of a single amino acid contains 3 to 30 amino acids, usually 3 to 25 amino acids, more usually 3 to 15 amino acids, especially 3-12 amino acids, more especially 3-9 amino acids. At least 40%, usually at least 50%, more usually at least 70% by weight of the protein polymer is composed of fragments of repeat units comprising at least two identical consecutive repeat units. The repeating blocks in the polymers used typically contain at least 2, 4, 7 or 8 units, and combinations thereof, wherein the unit modified by the crosslinking functionality is counted as one unit.

In most cases, the polymers of the present invention carry within the polymer chain reactive functional groups of naturally occurring amino acids and, if desired, side groups to provide the desired functionality. For example, carboxyl groups can be reacted with polyamines to exchange 1 or n amino groups with one carboxyl functional group. Amino groups can be substituted with polycarboxylic acids to replace amino groups with several carboxyl groups. The mercapto group can be replaced with an aldehyde by reaction with an aldehyde alkene such as acrolein to provide an aldehyde functionality. Other functionalities such as phosphate esters, activated alkenes such as maleimide, thioisocyanate, etc. can also be introduced if desired. This functionality can vary widely from natural products in order to provide the possibility of crosslinking. In some cases, this may entail increasing the number of functionalities per molecular weight without increasing the functionality of the entire chain. This is a greater variation in the substitution of one functionality for another, such as aldehydes for thiols, thus facilitating the choice of crosslinker.

The crosslinking agent is generally bifunctional in that the functionalities may be the same or different (although higher functionalities may be possible) and are not more than four functional. Various crosslinkers can be used based on the specific functionality available on the polymer. The crosslinking agent is usually at least about 3 carbon atoms and not more than 50 carbon atoms, generally between about 3-30 carbon atoms, more usually between 3-16 carbon atoms. The chain linking two functionalities is at least one atom and not more than about 100 atoms, usually not more than about 60 atoms, preferably not more than 40 atoms, especially not more than about 20 atoms, wherein the atoms may be carbon , oxygen, nitrogen, sulfur, phosphorous, or similar atoms. The linker may be aliphatic saturated or unsaturated, preferably aliphatic, and may contain functionalities such as hydroxyl, ester, amide, thioether, amino, and phosphite. Crosslinking groups can be thiohydric or hydrophilic.

Various reactive functional groups can be used, such as aldehyde groups, isocyanates, mixed carboxylic anhydrides such as ethoxyformic anhydride, activated olefins, activated halogens, amino groups, and the like. The degree of crosslinking and reactivity can be controlled by selecting the appropriate functionality of the protein polymer and the crosslinking agent.

A variety of crosslinking agents can be used, especially those that have been used previously and have been found to be physiologically acceptable. Crosslinkers that can be used include dialdehydes such as glutaraldehyde, activated dienes, diisocyanates such as tetramethylene diisocyanate, hexamethylene diisocyanate, octamethylene diisocyanate, anhydrides such as succinic acid Dianhydride, ethylenediaminetetraacetic dianhydride, diamines such as hexamethylenediamine, cyclo(L-lysyl-L-lysine), etc. The crosslinking agent may also contain asymmetric functionality, for example, activated alkenals such as acrolein and quinoids, activated halocarboxylic anhydrides, and the like. The crosslinking agent is usually commercially available or can also be conveniently synthesized according to a conventional method or before the adhesive is used or in situ.

In some cases, it may also be desirable to react a second physiologically acceptable compound as a modifying unit with a multifunctional, usually bifunctional, compound to alter the crosslinking properties. The addition of the second compound may increase the rate of cross-linking, alter the solubility characteristics of the cross-linking agent, increase or decrease the strength of the cross-linked polymer, increase or decrease the rate of reabsorption, or provide other interesting physical, chemical or biological properties. characteristic. The multifunctional second compound can be reacted with the crosslinking compound before or simultaneously with the protein. If the reaction is performed first, then the resulting crosslinked product will be physiologically acceptable and when reacted simultaneously, with the multifunctional second compound in situ, the crosslinking compound and the resulting crosslinked product will be physiologically acceptable. The ratio of the multifunctional second compound to the crosslinking compound is generally in the range of about 0.1-2:1, more usually in the range of about 0.1-1:1, depending on when the multifunctional second compound Reactivity with cross-linking agents, the number of cross-links desired in the final protein composition, the size of bridges between protein molecules, and other similar factors.

The properties of the multifunctional second compound can vary widely. The functional group may be the same as or different from the functional group in the polymer, but reactive with the functionality of the crosslinking compound. For example, the second multifunctional compound may bear amino and/or hydroxyl groups while the protein has amino or hydroxyl functionality. Diurethanes can be produced by using diisocyanates and diols. Thus, the chains of the cross-linked protein will contain 2 or more urethanes.

In many cases, the multifunctional second compound includes internal functionality that does not participate in the reaction but can provide other properties to the cross-linking agent or to the cross-linked protein product. Properties of interest include hydrophilicity, hydrolytic instability, susceptibility to enzymatic degradation, biocompatibility, shear strength, and the like. Most of the internal functionalities will contain oxygen, sulfur and nitrogen atoms, such as ethers, carboxylates including urethanes, amino groups, amides, ketones, dithiols and the like. To enhance the reabsorption rate, ester groups are important, while to enhance hydrophilicity, the same groups and ethers, such as polyoxyalkylene groups, can be used.

The multifunctional second compound generally has at least 2 carbon atoms and no more than 50 carbon atoms, usually no more than about 30 carbon atoms, desirably no more than about 16 carbon atoms per heteroatom. Both natural and synthetic bifunctional compounds can be used. Typical compounds include lysine, arginine, bis-(2'-aminoethyl)malonate, citrate, lysyllysine, 2'-aminoethylglycinate, O, N - diglycyl ethanolamine, divinyl glycol diglycinate, cystine, and the like. To provide the terminal amino group, various low molecular weight amino acids can be used, especially glycine and alanine incorporated into the inserted bifunctional compound, such as ethylene glycol, diethylene glycol, and tetraethylene glycol, propylene glycol, 1- 4-Butylcaine-2-diol, ascorbic acid, etc.

The desired composition can be prepared by combining the protein polymer and crosslinking agent, either or both, with a bulking agent, before the adhesive is used. The two compositions are conveniently mixed according to conventional methods, for example, by injecting the components into a central reactor with a syringe and mixing by drawing them into the syringe and pushing them back and forth. Alternatively, the two compositions are dispersed at the application site simultaneously. In some cases, it may be desirable to allow the crosslinker to partially react with the protein prior to the addition of the multifunctional second compound. Alternatively, the multifunctional second compound can be mixed with the protein prior to mixing with the cross-linking agent.

Typically, the polymer is obtained as a dispersion or solution, especially an aqueous solution, with a concentration of protein polymer generally in the range of about 50 mg-18/ml, more usually in the range of 100-800 mg/ml. The solution is buffered at a pH that either increases or slows the rate of crosslinking. The pH is usually in the range of about 2-12, more usually 8-11. Various buffers can be used, such as phosphate, borate, carbonate, etc. The cations can affect the properties of the product and in this sense the alkali metals potassium and sodium are preferred. Usually between about 5-40%, more usually between about 5-20%, preferably between 10-20% by weight of the protein composition, and the like are prepared within the desired time frame in order to provide a composition that is easy to handle. things. The concentration of the buffer is generally in the range of about 50-500 mM. Other reagents such as stabilizers, surfactants, and the like may also be present in the protein solution. If present, the concentration of the second multifunctional compound is determined according to the ratio of the crosslinker to the polymer.

The ratio of crosslinker to polymer will vary widely depending on the crosslinker, the number of functionalities in the polymer, the rate of hardening desired, and the like. Generally, the weight ratio of polymer to crosslinker is at least about 1:1 and not more than 100:1, usually not more than about 50:1, usually between 2 and 50:1, but may in some cases No more than 30:1. The equivalent ratio of protein to cross-linker is usually between about 0.1-1:3, more usually about 0.5-2:2. The basis for selecting the equivalent ratio of the protein cross-linker is the rate of preparation, the reactivity of the cross-linker, the relative solubility of the cross-linker in the mixture, the physiological properties of the cross-linker, the desired stability of the cross-linked product degree, and the like.

Various bulking agents, especially natural proteins, may be used if necessary. The bulking agent generally does not comprise more than 50%, usually not more than about 20%, more usually not more than about 10%, by weight of the composition. Extenders that can be used include, but are not limited to: synthetic polymers, addition and condensation polymers, proteinaceous and non-proteinaceous, such as polylactides, polyglycolides, polyanhydrides, polyorthoesters, polyvinyl compounds , polyenes, polyacrylates, polyethylene glycols, polyesters, polyvinyl alcohols, polyethers, copolymers and their derivatives; and natural polymers, e.g., proteins and non-proteins, including collagen, fibrinogen, Fibronectin, laminin, keratin, chitosan, heparin, dextran, alginate, cellulose, glycosaminoglycans, hyaluronic acid, poly(polysaccharide), derivatives thereof, and the like. Extenders can adjust overcrossing time and provide desired physical or physiological properties to the adhesive.

Based on an overlap shear strength test performed on experimental slices, the overlap shear strength is at least 100, preferably at least About 250, more preferably at least about 300, usually no more than about 4000, more usually no more than 3000 g/ ^cm2 .

The composition of interest may be applied to the tissue in any convenient manner, for example, using a syringe, catheter, cannula, manually applying the composition, spraying or other similar methods. The composition of interest may be applied to the tissue while or before the tissue incision is maintained in contacting relationship. The composition of interest rapidly develops a substantial shear stress strength in order to maintain tissue contact. In some cases, it is necessary that the composition degrades after some necessary period of time, usually after a period of at least one week and generally not more than about four weeks.

Applicable tissues include blood vessels such as arteries, veins or capillaries, muscles, nerves, organs such as liver, spleen, etc., lungs, dura mater, colon, and other similar tissues.

In addition to their use as adhesives, the subject compositions can also be used to seal or fill defects, such as voids or holes in tissue, and thus act as sealants. Thus, the composition can be used to stop the flow of fluid, such as blood, from ruptured blood vessels, such as arterial veins, capillaries and the like. For the intended composition to be used as a sealant, it may be applied as described above where the defect is to be sealed. The composition can be injected into normal or abnormal tissue to augment tissue mass, such as skin.

The subject composition can also be used in the preparation of articles by itself or in combination with other materials. In one application, an article is prepared for in vivo use in a mammal, wherein the advantages are biocompatibility, reabsorbability, vascularization, tissue adhesion and/or cohesive ability, and the like. Various articles can be manufactured, such as gels, films, fibers, coatings, preparations such as needles and screws, or flowable injectable compositions that act and bind or seal tissue, Form a drug depot, augment tissue or act as a filler, coating or the like. The resulting product can be prepared according to a conventional method such as molding, extrusion, precipitation from a solvent, solvent evaporation, and the like. Flowable sustained-release formulations can be obtained by using molecular dispersions, fine particles in a medium saturated with polymers; using melts, wherein the melt temperature is achieved by adding physiologically acceptable additives; and the like.

The article may be used in connection with implanting the article into a mammalian host or applying it to a surface of a mammalian host, such as wound treatment, burn dressing, and the like. In these cases, the effect of the item may last for a predetermined period of time and may be replaced by a natural material produced by natural processes, thus requiring that the material be reabsorbed after it has fulfilled its purpose until the natural process has re-established the natural structure. Thus, the composition can be used to maintain tissue contact, cover tissue, microencapsulate cells of organs, provide a coating that cells can invade and replace the composition with a natural composition, such as bone, soft tissue and the like.

In order to enhance the hardness of the polymerized composition, the composition may be partially prepolymerized. During prepolymerization, the polymer generally has at least about 3% and no more than 75% of the total number of crosslinked chains compared to the completion of crosslinking. The number of cross-linking chains should allow easy processing of the resulting product and provide sufficient time before onset of action. Alternatively, the functional group can be reacted with an excess of cross-linking agent such that the degree of cross-linking of the protein is replaced by the functionality of the cross-linking agent. The protein with the polymerized functionality can then be used to cross-link the protein with the original degree of cross-linking or a second compound with a multifunctionality.

The subject composition may also be used as a sustained release formulation to provide a relatively uniform release of a physiologically active product, such as a drug. The drug can be mixed in an appropriate concentration with the target composition before cross-linking. As the cross-linked polymer degrades, the drug can be released due to diffusion and erosion of the outer surface of the sustained release formulation. By controlling the dosage form of the slow-release agent, the degree of cross-linking, the concentration of the drug and the like, the physiological therapeutic level of the drug can be continuously maintained. Depending on the particular composition and application, the period required for absorption can be as short as 0.5 day and as long as 4, 6 or 8 weeks or longer.

The protein polymer composition can be prepared by conventional methods. See, for example, US Patent 5,243,038, the disclosure of which is incorporated herein by reference. Briefly, sequences containing many repeating units can be synthesized, the complements of which lead to dsDNA with overhangs. A series of dsDNA can be prepared and constructed as a cloning vector for the protein gene by stepwise transfer. In this way monomers are obtained which can be verified by sequencing that their sequence has not been altered, which are subsequently multimerized, cloned and expressed. Further details are found in the patents provided above.

The following examples are provided for purposes of illustration and not limitation.

Example 1

method

The construction of synthetic DNA and its large-scale use for polypeptide synthesis is described in US Patent 5,243,038; PCT/US89/05016 and PCT/US 92/09485, the disclosures of which are incorporated herein by reference. Modifications to these methods and others are as follows.

1 Purification of DNA Using Filters and Columns

A. Ultrafree-Probind filter device ("Probind", Millipore): The DNA solution was added to the filter device and centrifuged in a Sorvall Microspin 24s at 12,000 RPM for 30 seconds.

B. Microcon-30 filter membrane (Amicon): The DNA-containing solution was washed by adding to the filter and changing the water twice followed by centrifugation (12,000 RPM) in a microcentrifuge tube for 6 minutes.

C. Bio-Spin6 column ("Bio-Spin", BioRad): The DNA solution was equilibrated in TEAB (triethylammonium bicarbonate, pH 7.0) before passing through the column, and centrifuged in a Sorvall RC5B centrifuge tube (with HB4 rotor, 2,500RPM, 4 minutes).

2. Phosphatase treatment of DNA

Phosphatase treatment of DNA was performed by resuspending ethanol-precipitated restricted DNA in 20 mM Tris-HCl pH 8.0, 10 mM MgCl ₂ to a final DNA concentration of 20 μg/ml. Shrimp alkaline phosphatase (SAP) was added at a concentration of 2μ/μg DNA and the mixture was incubated at 37°C for 1 hour, heat-inactivated at 65°C for 20 minutes, then passed through a Probind filter membrane and then passed through a Bio-Spin column.

3. Preparative Agarose Gel Electrophoresis

For agarose ligation, the buffer used was 1×TAE (50 mM Tris-acetate, pH 7.8).

4. Agarose DNA Ligation

Melt the agarose at 65°C, then lower it to 37°C and add ligation buffer (5X=100mM Tris-HCl, pH7.5, 50mM MgCl ₂ , 50mM DTT, 1mM ATP); then place the reaction tube at room temperature and Ligase (1000 units T4 DNA ligase (NEB)) was added. The reaction volume is typically 50 μl. The reaction solution was incubated at 15°C for 16-18 hours.

5. Purification of agarose DNA with Ultrafree® ^- MC filter unit

This method can be used to cut agarose to a size of 400 μl. After agarose gel electrophoresis, stain with ethidium bromide to visualize and cut out the agarose block with the desired DNA band. Then, the agarose was frozen at -20°C for one hour and then quickly thawed at 37°C for 5 minutes. Then, the agarose is thoroughly impregnated. It was then transferred to the sample cup of the filter unit and centrifuged at 5,000 xg for 20 minutes in a conventional microcentrifuge. The agarose was then resuspended in 200 μl of Tris-EDTA, or other buffer, and incubated at room temperature for 30 minutes to elute other DNA from the gel. The mixture was centrifuged at 10,000 RPM for 20 minutes. At this point, the DNA is present in the filter tube and separated from the agarose fragments, making it available for subsequent DNA manipulations.

6. Preparation of antibodies against synthetic peptides

This is done using methods as described in US Patent 5,243,038, PCT/US89/05016 and PCT/US92/09485.

7. Immunoblotting of Proteins in Gels

Another method of detecting ¹²⁵ I-protein A can also be used. The method is based on a chemiluminescent signal emitted by horseradish peroxidase (HRP). The chemiluminescent reagents are conveniently commercially available from n suppliers such as Amersham and DnPont NEN. Western blots were prepared and blocked with BLOTTO. Some methods for introducing the HRP-tagged enzyme include, for example, hapten/anti-hapten-HRP, biotinylated antibody/streptavidin-HRP, or goat or mouse-anti-rabbit TgG-HRP. These reagents were purchased from various manufacturers such as BioRad or Amersham, and occasionally biotinylated antibodies were used in our laboratory with biotin NHS from Vector Laboratories (Burlinggame, CA. (CaT. #SP-1200)) according to the manufacturer's instructions. Preparation of product brochures. The following is a method for detecting the expression of protein aggregates.

The blot was placed in 15 ml of BLOTTO solution containing biotinylated goat anti-rabbit TgG (BioRad) (1:7500) dissolved therein and shaken gently at room temperature for 2 hours. Then, the filters were washed for 30 minutes (3 changes of TSA (50 mM Ths-HCl pH 7.4, 0.9% NaCl, 0.2% sodium azide)) and then washed in TBS containing 0.1% TWEEN 20 for 5 minutes. The blot was then incubated with gentle rotation for 20 minutes at room temperature (20 ml of TBS with 0.1% Tween20 (100 mM Ths base, 150 mM NaCl, pH 7.5) HRP-streptavidin (Amersham) (1:1000 dilution) in). Then, the blot was washed 3 times in TBS containing 0.3% Tween20, 5 minutes each time, and then washed 3 times in TBS containing 0.1% Tween20, 5 minutes each time. The blot was then incubated in 12 ml of equal volumes of #1 and #2 (Amersham) developing solution mixed in equal amounts for 1 minute with swirling. Blots were removed and autoradiographed.

8. Protein expression detection

In a 250 ml flask, the culture solution was incubated overnight at 30° C. with 50 ml of LB medium. Add kanamycin at a final concentration of 50 μg/ml and culture with stirring at 30° C. (200 rPm). When the OD ₆₀₀ of the culture solution reached 0.8, 40 ml was removed into a new flask preheated at 42° C. and incubated at the same temperature for about 2 hours. The culture solution (30°C and 42°C) was frozen and OD ₆₀₀ was measured. Cells were harvested by centrifugation and aliquoted at 1.0 _OD600 for Western detection using appropriate antibodies.

9. Amino Acid Detection

Amino acid derivatives were detected on a reversed-phase HPLC using a Waters 600E system.

10. Peptide Synthesis

Synthetic peptides were prepared on a Ramin/Protein Technologies PS3 FMOC peptide synthesizer. Both synthesis and cleavage were done using the methods provided in the instrument manual.

11. In vitro DNA Synthesis

β-cyanoethylphosphoramidite, control-well glass columns, and all synthesis reagents were purchased from Applied Biosystems, Foster City, California. Synthetic oligonucleotides were prepared using the phosphite triester method on an Applied Biosystems Model 381A DNA synthesizer with a 10-fold excess of protected phosphoramidites and 0.2 μmol of nucleotides bound to a synthesis support column. The chemistry used for the synthesis was conventional as recommended in the instruction manual for the synthesizer and found in (Matteucci et al., J. Am. Chemical Society, 103:3185-3319 (1981)). Deprotection and cleavage of oligomers on solid support were performed according to routine protocols provided by Applied Biosystems. The synthesis recovery obtained by measuring the OD value of the deprotected group with the method recommended by Applied Biosystems is greater than 97.5%.

The crude oligonucleotide mixture was purified by preparative gel electrophoresis using Applied Biosystems, 1992, Method for the Evaluation and Isolation of Synthetic Oligonucleotides. The concentration of the acrylamide gel is between 10-20% depending on the oligomer concentration. If necessary, the purified oligomers were identified by UV projection, excised from the gel and extracted by pressing and leaching (Smith, Enzymology, 65:371-379 (1980)).

For the synthesis of oligonucleotides longer than 100 bases, the synthesis cycle is replaced by the Applied Biosystems recommended protocol with the 381A DNA Synthesizer. All reagents used are fresh. All reagents were provided by Applied Biosystems except acetonitrile (Burdick and Jackson Cat #017-4 with water content less than 0.001%) and 2000A pore size column (Glen Rosearch). Due to the length of the oligomers, it is necessary to have pauses during the synthesis to replace reagent bottles that have run out of reagents. The pause is at the beginning of the cycle and the pause time should be as short as possible. At the beginning of each synthesis cycle, wash with TCA after detritylation 2 to 3 times more than recommended. The capping reaction time was also extended to control truncated failure sequences. The synthesized DNA was desalted and subjected to PCR amplification.

12. DNA sequence analysis

Data were stored and analyzed using DNA Strider, DNA Inspectim IIe or DNA id's Apple Macintosh personal computer software.

13. Dideoxy DNA sequence analysis of double-stranded plasmid DNA

Minipreps of plasmid DNA were made as described in US Patent No. 5,243,038. Primers were synthesized on a DNA synthesizer and plasmid DNA was annealed following the steps of MB sequencing. Sequencing reactions were performed using Sequenase (United States Biochemicals) and its recommended conditions. All sequences were electrophoresed on polyacrylamide gels.

14. PCR amplification

The PCR reactions were performed in Perkin Elmer thin-walled Gene Amp ^™ reaction tubes in a volume of 100 μl. About 1 μm of each primer was added to 1×PCR buffer (supplied by Perkin Elmer, 10× solution), 200 μM dNT, 5 μTaq enzyme, and a certain concentration of target DNA. Amplification was carried out on a Perkin Elner DNA thermal cycler (Model 480) with 30 cycles of 12 minutes each, with cycle parameters of 95°C, 62°C and 72°C. Various reaction samples were detected by electrophoresis on 1.5% low-melting point agarose gel in 0.5×TA buffer. The reaction mixture giving the desired band was collected and centrifuged through a Probind filter to remove Taq enzyme, then passed through a Microcon-30 filter and a Bio-Spin column. The DNA solution was concentrated in vacuo.

15. Synthesis of glycine 2-aminoethyl ester from diamine:

Concentrated sulfuric acid (9.90 g, 0.101 mol) was diluted in 10 ml of water. Glycine (7.50 g, 0.100 mole), 2-aminoethanol (6.10 g, 0.100 mole) and dilute sulfuric acid were placed in a 250 ml three-necked round bottom flask, equipped with a stopper, mechanical stirrer, heating mantle, and Dean-Stark Moisture valve to protect the contents of the unit from moisture with a nitrogen flow. Toluene (100ml) was added and the contents of the apparatus were refluxed until no more water evaporated. The apparatus was dismantled and the toluene was decanted, then a vacuum line was attached to remove residual toluene from the reaction. The product was used without further purification. The Fourier transform infrared spectrum of the reaction product showed strong carboxyl absorption at 1736 cm ¹ and 1672 cm ¹ . Compared with the spectra of glycine ethyl ester hydrochloride and glycylglycine hydrochloride, the product of this reaction was estimated to be an approximately 4:1 mixture of glycine 2-aminoethyl ester and N,O-diglycyl ethanolamine. Lysine Choline Ester:

Concentrated sulfuric acid (11.40 g, 0.120 mole) was diluted into water (10 ml). Lysine monohydrochloride (13.69g, 0.075mole), choline chloride (10.47g, 0.075mole) and dilute sulfuric acid were placed in a 250ml single-neck vessel equipped with a magnetic stirring bar, heated in a hot oil bath, and a vacuum tube. in a round bottom flask. Vacuum was applied gradually and heat was applied gradually to remove volatile components entering the valve cooled with liquid nitrogen. The reaction was terminated when the oil bath temperature reached 110°C and the pressure dropped to 0.024 mm-Hg. The product was shown to be homogeneous by thin layer chromatography (cellulose, acetic acid/acetonitrile/water 5:65:30 v/v/V, visualized in nebulized ninhydrin, Rf = 0.25). It can be used directly. Diglycine 1,3-propanediester:

Concentrated sulfuric acid (10.78 g, 0.110 mole) was diluted into water (10 ml). 1,3-Propanediol (7.61 g, 0.100 mole), glycine (15.0 g, 0.200 mole), and dilute sulfuric acid were placed in a 250 m three-neck circle equipped with a stopper, a mechanical stirrer, an oil bath, and a Dean-Stark water valve in the bottom flask. The contents of the device were protected from moisture with a stream of nitrogen. Add toluene (100ml), 130°C oil bath, and reflux the contents until no further water volatilization occurs (about 9 hours). Disassemble the apparatus and dump the toluene. The reaction was dissolved in water (29ml) with stirring at room temperature. On cooling to -20°C for 18 hours, fine white crystals precipitated from the solution which could be removed by filtration. The filtrate was poured into methanol (250ml) pre-cooled to 3°C to precipitate a semi-solid slurry. The supernatant was decanted and the slurry was triturated in batches and triturated with methanol (50ml) to give a granular solid (12.55g). A solid sample (9.19g) was boiled with methanol (18.4ml) and water (7.9ml), filtered while hot and crystallized at 4°C for 18 hours. The precipitate was filtered while cold, deposited on a funnel, rinsed with methanol, acetone and air dried to give a white crystalline solid (6.89g). Using a pH rise, a sample of the product was titrated with aqueous KOH. The apparent equivalent weight of each amine was 201 g/mol; an acidic contaminant was also present with an apparent equivalent weight of 601 g/mole. The Fourier transform chromatogram showed that the monocarboxyl absorption peak was at 1744cm ^-1 .

16. Fermentation conditions

The fermentors used to express the protein polymer are usually 15LMBR, 10L working volume, or 13L Braun Bostat E, 8.5L working volume. The choice of fermenter and its size is not very critical. Any medium suitable for the growth of EColi can be used. Nitrogen sources can be used ranging from NZ amines to organic salts and carbon sources are generally glycerol or glucose. All fermentations are carried out under conditions selectively suitable for the quality control (such as kanamycin, ampicillin, etc.). The fermentation method used to express protein polymers in Ecoli was fed-batch. Fed-batch is the preferred method for fermenting recombinant organisms, although other methods may also be used.

The fed-batch method is performed using a growth phase in which cells transition from exponential to stationary phase. This transition is usually the result of depletion of essential nutrients or accumulation of metabolic by-products. When the former is the cause, adding nutrients to the system allows cell division to continue. One or more essential nutrients can be gradually added to the reaction tank during the fermentation so that the net volume increases. The result is a controlled growth rate to optimize biomass and expression levels. The production of protein aggregates can be induced by providing appropriate physical or chemical signals based on the expression system used when the number of cells in the culture medium is at or near a maximum. Production then continues until the accumulated product reaches a maximum level (Fiestchko, J., and Riteh, T, Chemical Engineering Communications. 1986, 45: 229-240; Seo, J.H.; Bailey, J.E., Biotechnology and Bioengineering 1986, 28:1590-1594).

Embodiment 2 SELP8K, the construction of SELP8E and CLP6

Polymers designated SELP8K and SELP8E were prepared, characterized by functional groups for crosslinking. The construction of these polymers is described below (starting from the aforementioned gene monomer, SELPO) (see US Patent No. 5,243,038, PSY1298). SELP8K and SELP8E amino acid monomer sequence design: SELP8K MONOMER (GAGAGS) ₄ (GVGVP) ₄ GKGVP (GVGVP) ₃ (Sequence 04) SELP8K MONOMER (GAGAGS) ₄ (GVGVP) ₄ GEGVP (GVGVP) ₃ (Sequence 05) SELP8 construction:

Plasmid PSY1378 (see U.S. Pat. No. 5,243,038) was digested with Ban I REN, purified by agarose electrophoresis, and passed through a NACS column. Then, the DNA was precipitated with ethanol in 2.5M ammonium acetate and digested with Fok I REN in advance. _PPT0134 (see PCT /US92/09485) were connected, extracted with phenol/chloroform, and precipitated with ethanol.

The product of this ligation mixture was transformed into E. coli strain HB101. Quality control DNA extracted from transformants was purified and analyzed by Nra I and Xmn I RENs digestion. Check _P PT0255 with the required restriction sites was obtained and used for subsequent constructions.

Quality control DNA _P PT0255 was treated with Cfr10I REN followed by RNAse treatment. The digested fragments were separated by agarose gel electrophoresis, and the DNA was excised and self-ligated. The ligation product was transformed into E. coli strain HB101. Plasmid DNA from transformants was purified and digested with Nae I and Stu I RENs for analysis. Plasmid _PPT0267 with the desired deletion was used for subsequent constructions.

The two oligonucleotides synthesized are shown in Table 1 and purified as described in Example 1.

Table 1

5-CTGGAGCGGGTGCCTGCATGTACATCCGAGT-3' (SEQ ID NO: 06)

3-CCGAGACCTCGCCCACGGACGTACATGTAGGCTCA-5' (sequence

07)

The two oligonucleotide chains were annealed and ligated with the plasmid _P PT026 DNA digested with Ban II and Sca I RENs, purified by agarose gel electrophoresis, and passed through a NACS column.

The product of this ligation reaction was transformed into E. coli strain HB101. Plasmid DNA extracted from transformants was purified and digested with Dra I.

Plasmid DNA with the correct digestion sites from both clones was sequenced. The plasmid DNA designated _PPT0287 was found to be correct and selected for further construction.

Plasmid DNA pSY1298 (see US Pat. No. 5,243,038) was digested with Ban II REN, and the SELPO gene fragment was purified by agarose gel electrophoresis, passed through NACs column, and finally connected to _P PT0287 digested with Ban II. The enzyme was removed by extraction with phenol/chloroform and precipitated with ethanol.

The ligation product was transformed into E. coli strain HB101. Plasmid DNA extracted from transformants was purified and analyzed by Dral REN digestion. Plasmid DNA from this clone showing the correct restriction sites was further digested with Ban II, Aha II and Stn I RENs. Plasmid _PPT0289 carries the desired SELP8 monomer sequence (see Table 2).

Table 2: SELP8 gene monomer sequence

Bani BaniigGT GCC TCT GGA GCT GCGC GCG GGC TCT GGA GGA GGT GGT GGTCCA CCA AGA CCGCGC CGC CCG AGA CCA CAC GGT CCAG A G A G S G V p gga GTT CCG GGT GGC GGC GTT CGA GGA GGT GGT GGT GGA GGA GGA GGCAT CAA GGC CAA CAA GGC CAA CAA CAA CAA CAT GGA CACV G V p g V p g V p g v

                           SmaIGGT GTT CCA GGC GTA  GGT GTG CCC GGG  GTA GGA GTA  CCA GGG  GTA GGCCCA CAA GGT CCG CAT  CCA CAC GGG CCC  CAT CCT CAT  GGT CCC  CAT CCGG   V   P   G   V    G   V   P   G    V   G   V    P   G    V   G

Baniigtc CCT GGA GGT GCT GGT AGC GCA GCA GCGC GCGC TCGCAG GGCAG GGCAG GGC CGC CCA CCA CCG CCG CGC CGC CGC CGC CGC CGC CGC CGC CGC CGC CGC CCG CCG :08 & 09) Construction of SELP8K and SELP8E gene monomers

An oligonucleotide chain capable of cloning a SELP8 gene monomer with a single base polymorphism at position 90 was synthesized. Cloned lysine and glutamic acid oligonucleotides were generated from single synthesis using both adenine and guanine at this position (see Table 3). The synthesis was performed on an Applied Biosystems DNA synthesizer (model 381A) with a 2000A synthesis column provided by Glen Research. The time needed to change bottles in this synthesis is reduced. After synthesis, the 202 base DNA fragment was deprotected and excised from the column by treatment with 30% ammonium hydroxide at 55°C for 6 hours.

表35′-ATGGCAGCGAAAGGGGACCGGGCTCTGGTGTTGGAGTGCCAGGTGTCGGTGTTCCGGGTGTAGGCGTTCCGGGAGTTGGTGTACCTGGA(A/G)AAGGTGTTCCGGGGGTAGGTGTGCCGGGCGTTGGAGTACCAGGTGTAGGCGTCCCGGGAGCGGGTGCTGGTAGCGGCGCAGGCGCGGGCTCTTTCCGCTAAAGTCCTGCCGT-3′(SEQ ID NO：10)

The other two DNA strands are used as primers for PCR amplification. The two chains are:

1.5'-AAGAAGGAGATATCATATGGCAGCGAAAGGGGACC-3' (SEQ ID No: 11)

2.5'CGCAGATCTTTAATTACGGCAGGACTTTAGCGGAAA-3' (SEQ ID No: 12)

PCR amplification and product purification were performed as described in Example 1.

DNA was resuspended and Ban II REN digested as described in Example 1. Then, the digested DNA was separated by low-melting point agarose gel electrophoresis and connected with _PPT0289 digested with Ban II RENs, and then purified by NACs column. The product of the ligation reaction was transformed into E. coli strain HB101. Plasmid DNA extracted from isolated transformants was purified and analyzed by digestion with ApaL I and EcoN I RENs. Cloned plasmid DNA with the correct restriction site was analyzed by Asp700REN digestion to distinguish clones encoding lysine or glutamic acid at this polymorphic site. Cloned plasmid DNA carrying each polymorphism was purified and analyzed by DNA sequencing. Plasmid _PPT0340 contains the desired SELP8K monomer sequence (see Table 4) and _PPT0350 contains the desired SELP8E monomer sequence.

Table 4: SFLP8K gene monomer sequence

BanI BanII

GGT GCC GGT TCT GGA GCT GGC GCG GGC TCT GGT GTT GGA GTG CCA GGT

CCA CGG CCA AGA CCT CGA CCG CGC CCG AGA CCA CAA CCT CAC GGT CCA

G A G s G A G A G S G V G V P G

Econigtc GGT GTT CCG GGT GGC GGC GGC GGA GGA GGA GGT GGT GGT GGA AAACAG CCA CAA CAA CAA CAA GGC CAA CAA CAA CAA CAT G V P G V V p Gkggg GGG GGG GGT GTG CCG GGC GTT GGA GTA CCA GGT GTA GGCCCA CAA GGC CCC CAT CCA CAC GGC CCG CAA CCT CAT GGT CCA CAT CCGG V p G V G G V P G V P V G V G

Smai Baniigtc CCG GCG GCG GCT GCT GCT AGC GCA GCA GCGC GCGC TCGCAG GGC CGC CGC CGC CCA CCG CCG CGC CGC CGC CCG CGCV P G A G A G A G S g s G (SEQ ID NO: 13 & 14) SELP8K polymer construction

Plasmid DNA derived from _PPT0340 was digested with Ban I REN and the digested fragments were separated by agarose gel electrophoresis. A 192bp SELP8K gene fragment was excised and purified with a NACS column. The purified fragment was connected with the plasmid _PPT0317 digested with Ban I REN, and passed through Millipore Probind and Bio-Spin6 columns. The DNA was then treated with Shrimp alkaline phosphatase (SAP) as described in Example 1.

The product of this ligation reaction was transformed into E. coli strain HB101. Transformants resistant to kanamycin were selected. Plasmid DNA of each transformant was purified and examined for its increased size due to the SELP8K monomer multiple DNA insertion. Several clones in which bases ranging from 200 bp to about 7 kb were inserted were obtained. Clones with 6-32 repeats were used to express SELP8K protein polymers ( _PPT0341 , _PPT0343 , _PPT0344 , _PPT0345 and _PPT0347 ). SELP8K expression analysis

The culture solution that had been kept overnight at 30°C was inoculated into 50 ml of LB medium placed in a 250 ml flask. Kanamycin was added at a final concentration of 50 μg/ml and cultured at 30° C. with shaking (200 rpm). When the _OD600 of the culture solution reached 0.8, 40 ml was transferred to a new flask preheated at 42°C and incubated at the same temperature for about 2 hours. The cultures (30°C and 42°C) were frozen and the _OD600 was measured. Cells were harvested by centrifugation and split at 1.0 _OD600 for Western analysis using anti-SLP antibody.

E. coh strain HB101 containing plasmids _P PT0341, _P PT0343, _P PT0344, _P PT0345 and _P PT0347 were grown as described above. Proteins produced by these cells were detected by Western blot to detect the response of the cells in this chamber to the SLP antibody. Each clone produced a strongly reactive band. The apparent molecular weight of the product ranges from about 35KD to over 250KD. _{The PPT0345} strain produced a SLP antibody-reactive band with an apparent molecular weight of 80,000. The amino acid sequence of the expected SELP8K polymer encoded by QP _P PT0345 is as follows. pPT0345 SELP8K 884 AA MW 69,772

MDPVVLQRRDWENPGVTQLNRLAAHPPFASDPMGAGSGAGAGS

[(GVGVP) ₄ GKGVP(GVGVP) ₃ (GAGAGS) ₄ ] ₁₂

(GVGVP) ₄ GKGVP(GVGVP) ₃ (GAGAGS) ₂

GAGAMDPGRYQDLRSHHHHHH (SEQ ID No: 15) SELP8K purification

SELP8K was produced fermentatively in E. coli strain _P PT0345. The product was purified from cell biomass by cell lysis, centrifugation to remove debris, and affinity chromatography. The purified product was detected by SDS-polyacrylamide gel electrophoresis, immunoreactivity of polyclonal antiserum reactive with silk-like peptide moiety (SLP antibody), and amino acid analysis. A protein band with an apparent molecular weight of 80,000 can be seen on the SDS-PAGE gel stained with amide black and transferred to the membrane, and the same band can be seen on the Western blotting with SLP antibody. As expected, amino acid analysis (shown in Table 5) indicated that the product was rich in glycine (43.7%), alanine (12.3%), serine (5.3%), proline (11.7%), and valine (21.2%). This product also contains 1.5% lysine. The amino acid composition in the table below shows the relationship between the composition of the purified product and the theoretically expected composition encoded from the synthetic gene sequence.

Table 5: Amino acid analysis of purified SELP8K

Amino Acids pmoles Actual % Composition Theoretical % Composition

Ala 1623.14 12.3 12.2

Asx 122.20 0.9 0.8

Glx nd nd nd 0.4

Phe 58.16 0.4 0.1

Gly 5759.31 43.7 41.5

His 46.75 0.4 0.8

He 43.87 0.3 0

Lys 198.21 1.5 1.5

Leu 39.54 0.3 0.5

Met 36.01 0.3 0.3

Pro 1534.21 11.7 12.4

Arg 70.84 0.5 0.6

Ser 703.83 5.3 6.1

Thr nd nd nd 0.1

Val 2797.47 21.2 22.4

Tvr 140.87 1.1 0.1

nd = CLP6 production not detected

CLP6 (CLP6 refers to DCP6) was prepared using _{the PPT0246} strain as described in PCT/US92/09485. Multigram quantities of protein aggregates are purified using conventional protein purification, extraction, and isolation methods. The crystallized product is a white, spongy substance and is very soluble in water.

CLP6 pPT0246 1,065 AA MW 85,386

MDPVVLQRRDWENPGVTQLNRLAAHPPFASDPM

[(GAHGPAGPK) ₂ (GAQGPAGPG) ₂₄ (GAHGPAGPK) ₂ ] ₄

GAMDPGRYQLSAGRYHYQLVWCCK

(SEQ ID NO: 16)

The construction polymer design element of embodiment 3 SELPOK polymer

The structure of the SELP8K interpolymer composed of a silk-like block (SLP module) and an elastin-like module (ELP module) is as follows; [(SLP module) ₄ (ELP block) ₈ ]. In addition, it is also possible to design polymers with different reabsorption and dissolution characteristics without changing their adhesive properties by tuning the length of the SLP block to that of the ELP block. Compared to SELP8K, SELPOK contains half-length crystallizable SLP blocks while maintaining the dispersed frequency of its ELP fragments.

Polymers were also designed with intervening sequences to facilitate reabsorption in vivo by proteolytic cleavage with collagenase (92kd) and cathepsin. SELPOK was used as the backbone for these designs, but these sites are available for many different polymer backbone sequences. Suitable insertion sites are chosen so that they facilitate access to the catalytic groove of the protease. Most proteases bind four amino acids upstream of this cleavage site. Therefore, the insertion sequence should be free of hydrogen bonding and crystallization that can be induced by blocks such as SLP.

The beta structure of SELPOK is broken after the proline of the first ELP block. SELPOK-CS1 contains two consecutive collagenase cleavage sites (PLGP) in the inserted six amino acids (SEQ ID NO: 17). The insertion site was chosen so that at least one proline was removed from the SLP block (GAGAGS GVGVP LGPLGP GVGVP (SEQ ID NO: 18). SELPOK-CS2 contains multiple cathepsin B (ARR), L (FF), restriction sites for S and H (FVR) and plasmin. Choose a suitable insertion site so that at least one proline is removed in the SLP module (GAGAGS GVGVP GFFVRARR GVGVP) (SEQ ID NO: 19). Plasmid _P Construction of PT0317 plasmid

Plasmid DNA _P SY1262 (see US Patent No. 5,243,038) was linearized with Pvu II REN, and then passed through a Probind filter membrane and a Bio-Spin6 column. Then, the DNA was treated with Shrimp alkaline phosphatase (SAP). Then, the linearized _P SY1262 DNA was ligated with DNA derived from _P QE-17 (QLAGEN Catalog #33173) prepared as follows. Plasmid DNA _P QE-17 was digested with Bg III and Hind III RENs and the 366P fragments shown in Table 6 were purified with Probind filter membrane and Biospin column. The DNA was further purified using a Microcon-30 filter and the filtrate containing the 36 bp fragment was retained. Then, the DNA was treated with DNA polymerase I and purified with Probind filter and Biospin column (see Example 1).

Table 6 5'-GATCTTCGATCTCATCACCATCACCATCACTA (SEQ ID NO: 20) 3'-AAGCTAGAGTAGTGGTAGTGGTAGTGATTCGT (SEQ ID NO: 21)

The product of this ligation reaction was transformed into E. coli HB101 strain. Plasmid DNA of this transformant was purified and analyzed by digestion with Bstl107I and EcoRVRENs enzymes. Clones containing the desired DNA fragment were further digested with Bstl 1071 and BstYI RENs to detect the orientation of the insertion. Cloned plasmid DNA with the correct restriction sites was purified and checked by DNA sequencing. Check _P PT0317 contained the desired DNA insert and was used for further DNA construction. SELPOK Polymer Construction

An oligonucleotide shown in Table 7 was synthesized with an Applied Biosystems DNA synthesizer (model 381A) and a 2000A synthesis column provided by Glen Research. After synthesis, the 93 base DNA fragment was deprotected and cleaved from the support column by treatment in ammonium hydroxide at 55°C for 6 hours.

Table 75'-ATGGCAGCGAAAGGGGACCGGTGCCGGCGCAGGTAGCGGAGCCGGTGCGGGCTCAAAAAGGGCTCTGGTGCCTTTCCGCTAAAGTCCTGCCGT-3' (SEQ ID NO: 22)

A PCR reaction was performed using the same two DNA primers as described for the construction of the SELP8K gene monomer and the reaction product was purified. DNA was resuspended and digested with Ban I REN. The digested DNA was then separated on a low melting point agarose gel and ligated with _PPT0285 (see PCT/US92/094850) previously digested with Ban I REN and purified by NACS column. The product of the ligation reaction was transformed into E. coli HB101 strain. The transformant plasmid DNA was purified and digested with EcoRI and Ban II RENs for analysis. Then, cloned plasmid DNA with the correct restriction sites was purified and analyzed by DNA sequencing. Plasmid _PPT0358 contained the desired sequence and was used for subsequent DNA construction.

Plasmid DNA derived from _PPT0340 was digested with Ban II REN and the digested fragments were separated by agarose gel electrophoresis. The SELPOK gene fragment of 156bp (see Table 8) was cut out and purified with an Ultrafree-Mc filter membrane, and then passed through a Bio-Spin6 column.

Table 8BANIIG GGC TCT GGT GGA GGA GGA GGT GGT GGT GGT GGT GGT CCG GGT GGC GTTC GTTC CCGA CAA CAA CCT CCA CAA CAA CAA CAAG CAAG CAAG CAAG S G V V p g V g V VCCGGa GTT GGT GGT GTA CCT GGT GGT GGT CCG GGG GGG GGT GGT GGT GTG CCGGC CCA CAA CAA CAT GGA CCA CCA GGC CCC CCC GGCP G V g V p g V pg V pg V pGA GGA's GGC GTC CCG GGA GCG GGT GCT GGT AGCCCG CAA CCT CAT GGT CCA CAT CCG CAG GGC CCT CGC CCA CGA CCA TCGG V G V P G V G V P G S A G A

BanIIGGC GCA GGC GCG GGC TCCCG CGT CCG CGC CCG AGG A G A G S (SEQ ID NOS: 23&24)

The purified fragment was connected with the plasmid _P PT0358 digested with Ban II REN, and passed through a Probind filter membrane and a Microcon-30 filter membrane. Then, the digested fragments were separated by agarose gel electrophoresis. Then the plasmid DNA was excised and purified with Ultrafree-MC filter membrane and Bio-Spin6 column (see Example 1) successively.

The product of this ligation reaction was transformed into E. coli HB101 strain. Transformants resistant to chloramphenicol were screened. Plasmid DNA of each transformant was purified and examined for increased size due to insertion of SELPOK multiplex DNA. Several clones were obtained with inserts of different sizes. Plasmids _P PT0359, _P PT3060 and _P PT0374 containing repeats of 18, 2 or 6 SELPOK gene monomers, respectively, were used for subsequent constructions.

Plasmid DNA of _P PT0359 and _P PT0374 was digested with Ban I REN and the digested fragments were separated by agarose gel electrophoresis. About 2800bp and 1000bp SELPOK gene fragments were cut out and purified by NACS column. Then, the purified fragment was connected with the plasmid _PPT0317 digested with Ban I REN, and passed through a Probind filter membrane and a Bio-Spin column. Then, the DNA was treated with Shrimp alkaline phosphatase (SAP), and passed through a Probind filter membrane and a Bio-Spin6 column successively (see Example 1).

The product of this ligation reaction was transformed into E. coli HB101 strain. Transformants resistant to kanamycin were screened. Plasmid DNA of each transformant was purified and tested for increased size due to SELPOK multiple DNA insertions. Several clones were obtained. Plasmids _P PT0364 and _P PT0375 were used for expression of SELPOK. SELPOK expression analysis

E. coli HB101 strain (containing plasmids _P PT0364 and _P PT0375) was cultured and grown as described in Example 1. The reactivity of proteins produced by these cells to ELP antibody was examined by SDS-PAGE. It can be found that each tested sample has a strong reaction band, and its apparent molecular weight is about 95KD and 35KD respectively.

pPT0364 SELPOK 1000 AA MW 80,684

MDPVVLQRRDWENPGVTQLNRLAAHPPFASDPM

[(GAGAGS) ₂ (GVGVP) ₄ GKGVP(GVGVP) ₃ ] ₁₆

(GAGAGS) _{2GAGAMDPGRYQDLRSHHHHHH}

(SEQ ID NO: 25)

pPT0375 SELPOK 376 AA MW 31,445

MDPVVLQRRDWENPGVTQLNRLAAHPPFASDPM

[(GAGAGS) ₂ (GVGVP) ₄ GKGVP(GVGVP) ₃ ] ₆

(GAGAGS) _{2GAGAMDPGRYQDLRSHHHHHH}

(SEQ ID NO: 26) SELPOK-CS1 polymer construction

Plasmid _pPT0360 was digested with Bn I REN and the digested fragment was separated by agarose gel electrophoresis. The SELPOK gene fragment of about 300bp was cut out and purified through Ultrafreee-M1 filter membrane and Bio-Spin6 column successively. This purified fragment was ligated with Fok I REN digested plasmid _PPT0134 (see PCT/US92/09485). The enzyme was inactivated by heating at 65°C for 20 minutes and the ligation mixture was passed through a Probind filter. Then, the DNA was treated with Shrimp alkaline phosphatase (SAP), and passed through Probind filter membrane and Bio-Spin6 column respectively.

The product of this ligation reaction was transformed into E. coli HB101 strain. Transformants resistant to chloramphenicol were screened. Plasmid DNA from each transformant was purified and analyzed by Dral REN digestion. _PPT0363 showed the correct restriction map and was used for subsequent DNA construction.

The oligonucleotide chains shown in Table 9 were synthesized with an Applied Biosystems DNA synthesizer (model381A) and a 2000A synthesis column (provided by GlenResearch). After synthesis, the 141 base DNA fragment was deprotected and excised from the support column by treatment in ammonium hydroxide at 55°C for 6 hours.

Table 95' - ATGGCAGCGAAAGGGGACCGCCGGTGCGGGCTCTGGTGTTGGAGTGCCGCTGGGTCCTCTTGGCCCAGGTGTCGGTGTTCCGGGTGTAGGCGTTCCGGGAGTTGGTGTACCTGGAAAAGGTTTCCGCTAAAGTCCTGCCGT-3'

(SEQ ID NO: 27)

A PCR reaction was performed using the same two DNA primer strands as described in Construction of the SELP8K gene monomer and the reaction product was purified. Then, the DNA was resuspended and digested with BsrFI and EcoNI RENs. The digested DNA was treated with Probind and Microcon-30 filter, ligated with _PPT0363 digested with BsrFIREN, treated with ProBind filter and Bio-Spin6 column and further digested with EcoNIREN. The digested fragments were separated by agarose gel electrophoresis. The larger DNA band of about 2000 bp was cut out and purified with Ultrafree-M1 filter membrane and Bio-Spin6 column (see Example 1) successively.

The product of this ligation reaction was transformed into E. coli HB101 strain. Plasmid DNA from each transformant was purified and assayed by Asp 700I and Eco 0109 IRENS digestion. Plasmid DNA of clones exhibiting the correct restriction pattern was purified and analyzed by DNA sequencing. Plasmid _P PT0368 (see Table 10) contained the desired sequence and was used for subsequent DNA construction.

Table 10BANIIG GGC TCT GGT GGA GGA GGA GGA CCG CTG GGT CCT CTT GGC CCA GGT GTCC CCG AGA CCA CAA CCT CAC GGC CCA CCA CCG GGT CCA GGC GTT CCG GGA GGT GGT GGT GGA CCT GGA AAACCA CAA GGC CCA CAA GGC CAA CAA CAA CAA CAA CAT GGA CCT GGA CCT TTG V P g V g V p g k GGG GGG GGG ’ss GGT GTACCA CAA GGC CCC CAT CCA CAC GGC CCG CAA CCT CAT GGT CCA CATG V P G V G V P G V G V P G V

BaniigGC GTC CCG GGA GCG GCG GCT GCT GCC GCA GCA GCGC GCGC GGC TCTCG CAG GGC CCA CCA CCA CCG CCG CCG CGC CGC CGC CGC CGC CGC CCG CGC CCG CGC CGC CGC CGC CGC CGC CCG V p g A G A G s (SEQ ID NOS: 28 & 29)

Plasmid DNA _PPT0368 was digested with Ban II REN, and the digested fragment was separated by agarose gel electrophoresis. The 174bp SELPOK-CS1 gene fragment was cut out and purified through Ultrafree-M1 filter membrane and Bio-Spin6 column successively. The purified fragment was ligated with the plasmid _P PT0358 digested with Ban II REN, and then passed through a Probind filter membrane and a Microcon-30 filter membrane. Subsequently, the digested fragments were separated by agarose gel electrophoresis. The plasmid DNA was cut out again and purified with Ultrafree-M1 filter membrane and Bio-Spin6 column (see Example 1) successively.

The product of the ligation reaction was transformed into E. coli HB101 strain. Transformants resistant to chloramphenicol were screened. Plasmid DNA of each transformant was purified and examined for increased size due to SELPOK-CS1 multiple DNA insertions. Several clones were obtained with insert sizes ranging from 1000 bp to approximately 3000 bp. Plasmid _PPT0369 containing 16 repeats of the SELPOK-CS1 gene monomer was used for subsequent construction.

Plasmid DNA of _PPT0369 was digested with BanI REN, followed by passage through Probind filters and separation of the digested fragments by agarose gel electrophoresis. The SELPOK-CS1 gene fragment of approximately 2800bp was cut out and purified through an Ultrafkee-M1 filter membrane, and desalted through a Bio-Spin6 column. Then, the purified fragment was connected with the plasmid _P PT0317 digested with BanIREN, and passed through Probind filter membrane and Bio-Spin6 column successively. Subsequently, the DNA was treated with Shrimp alkaline phosphatase (SAP), and passed through a Probind filter membrane and a Bio-Spin6 column (see Example 1) successively.

These ligation products were transformed into E. coli HB101 strain. Transformants resistant to kanamycin were screened. Plasmid DNA of each transformant was purified and examined for increased size due to SELPOK-CS1 multiple DNA insertions. Several clones were obtained. Plasmid _PPT0370 was selected for the expression of SELPOK-CS1. SELPOK-CS1 expression analysis

E. coli HB101 strain (containing plasmid _P PT0370) was cultured and grown as described in Example 1. The reactivity of proteins produced by these cells to ELP antibody was examined by SDS-PAGE. A strong reaction band with an apparent molecular weight of approximately 90KD was observed in each assay. pPT0370 SELPOK-CSl 934 AA MW 76,389

MDPVVLQRRDWENPGVTQLNRLAAHPPFASDPM

((GAGAGS) ₂ (GVGVP) ₁ LGPLGP(GVGVP) ₃ GKGVP(GVGVP) ₃ ] ₁₅

(GAGAGS) _{2GAGAMDPGRYQDLRSHHHHHH}

(SEQ ID NO: 30) SELPOK-CS2 polymer construction

An oligonucleotide chain as shown in Table 11 was synthesized using an Applied Biosystems DNA synthesizer (model 381A) and a 2000A synthesis column provided by Glen Research. After synthesis, the 147 base DNA fragment was deprotected and cleaved from the support column by treatment in ammonium hydroxide at 55°C for 6 hours.

Table 11 5' - ATGGCAGCGAAAGGGGACCGCCGGTGCGGGCTCTGGTGTTGGAGTGCCAGGCTTCTTTGTACGTGCACGCCGTGGTGTCGGTGTTCCGGGTGTAGGCGTTCCGGGAGTTGGTGTACCTGGAAAAGGTTTCCGCTAAAGTCCTGCCGT-3' (SEQ ID NO: 31)

A PCR reaction was performed using the same two DNA primers as described in Construction of the SELPSK gene monomer and the reaction product was purified. Then, the DNA was resuspended and digested with BsrFI and Eco NI RENs. The digested DNA was processed with ProBind and Microcon-30 filter, Bio-Spm6 column and ligated with _PPT0363 digested with BsrFIREN, processed with ProBind filter and Bio-Spin6 column and further digested with Eco NI REN. The digested fragments were separated by agarose gel electrophoresis. The larger DNA band of about 2000bp was cut out and purified with Ultra M1 filter membrane and Bio-Spin6 column successively.

The ligation reaction product was transformed into E. coli HB101 strain. Plasmid DNA from each transformant was purified and analyzed by digestion with Asp 700I and Dra III RENs. Plasmid DNA of those clones showing the correct restriction pattern was purified and analyzed by DNA sequencing. Plasmid _P PT0367 (see Table 12) contained the desired sequence and was used for subsequent DNA construction.

Table 12BANIIG GGC TCT GGT GGA GGA GGA GGC TTC TTC TTT GCA CGC CGTC CCG AGA CCA CAA CCT CCT CCG AAG AAA CAT GCAG GCG GCAG S G V P G F V R RGGT GGT GGT GGT GGT GGT CCG GGT GGC GTT CGA GGA GGT GGT GGT GGT GGT GGACCA CAG CAA CAA CAA CAA GGC CAA CAA CAA CAA CAA CAA CAT GGA CAA CAT GGA CAA CAA CAA CAA CAA CAA GTT GGA GTA CCA GGT GTATTT CCA CAA GGC CCC CAT CCA CAC GGC CCG CAA CCT CAT GGT CCA CATK G V P G V G V P G V G V P G V

BaniigGC GTC CCG GGA GCG GCT GCT GCT GGC GCA GCA GCGC GCGC GGC GGC CAG GGC CGC CGC CCA CCA CCA CCG CCG CGC CGC CGC CGC CGC V P g A G A G (SEQ ID NOS: 32 & 33)

Plasmid DNA _P PT0367 was digested with Ban II REN, treated with a Probind filter and Bio-Spin6 column and the digested fragment was separated by agarose gel electrophoresis. The 180bp SELPOK-CS2 gene fragment was cut out and purified with Ultrafree-M1 filter membrane and Bio-Spin6 column successively. The purified fragment was ligated with Ban II REN digested plasmid _PPT0358 and passed through Probind and Microcon-30 filters. The digested fragments were then separated by agarose gel electrophoresis. Plasmid DNA was excised and purified successively with Ultrafree-M1 filter membrane and Bio-Spin6 column (see Example 1).

The product of this ligation reaction was transformed into E. coli HB101 strain. Transformants resistant to chloramphenicol were screened. Plasmid DNA of each transformant was purified and examined for increased size due to insertion of SELPOK-CS2 multiplex DNA. Several clones with insert sizes ranging from 200b0 to about 3000bp were obtained. Plasmids _P PT0371 and _P PT0372 containing 18 and 15 repeats of the SELPOK-CS2 gene monomer, respectively, were used for subsequent constructions.

Plasmid DNA of _PPT0372 was digested with Ban I PEN, then passed through a Probind filter, and the digested fragment was separated by agarose gel electrophoresis. The SELPOK-CS2 gene fragment of about 2800bp was cut out and purified through Ultrafree-M1 filter membrane, and desalted with Bio-Spin6 column. Then, the purified fragment was connected with the quality inspection _P PT0317 digested with Ban I REN, and passed through a Probind filter membrane and a Bio-Spin6 column. The DNA was treated with Shrinp alkaline phosphatase (SAP), passed through a Probind filter membrane and then a Bio-Spin6 column (see Example 1).

The product of this ligation reaction was transformed into E. coli HB101 strain. Kanamycin-resistant transformants were screened out. Plasmid DNA in each transformant was purified and examined for increased size due to multiple DNA insertions of SELPOK-CS2. Several clones were obtained. Plasmid _PPT0373 was selected for expression of SELPOK-CS2. SELPOK-CS2 expression analysis

The Ecoli HB101 strain containing the plasmid _P PT0373 was cultured and grown as shown in Example 1. The reactivity of proteins produced by these cells to ELP antibody was examined by SDS-PAGE. A strong reaction band representing a molecular weight of approximately 90 KD was observed in each assay. pPT0373 SELPOK-cS2 964 AA MW 83,218

MDPVVLQRRDWENPGVTQLNRLAAHPPFASDPM

[(GAGAGS) ₂ (GVGVP) ₁ GFFVRARR(GVGVP) ₃ GKGVP(GVGVP) ₃ ] ₁₅

Purification of (GAGAGS) ₂ GAGAMDPGRYQDLRSHHHHHH (SEQ ID NO: 34) SELPOK and SELPOK-CS1

SELPOK and SELPOK-CS1 were produced in E. coli _P PT0364 and _P PT0370 strains, respectively. The product was obtained by lysing the cell biomass, precipitation by polyethyleneimine and ammonium sulfate centrifugation, and ion exchange chromatography to remove insoluble debris. The purified product was checked by SDS-PAGE, immunoreactivity with polyclonal antiserum reactive with elastin-like peptide block (ELP antibody), and amino acid analysis.

For SELPOK, a protein band with an apparent molecular weight of 95,000 can be seen separated by SDS-PAGE stained with amide black, transferred to the membrane, and the same band reacts with the ELP antibody on the Western blot. As expected, amino acid analysis (see Table 13) showed that the product was enriched in glycine (41.0%), alanine (8.0%), serine (4.5%), proline (14.1%), and valine (26.8%) %). The product also contains 1.9% lysine. The amino acid composition shown in the table below shows the correlation between the amino acid composition of the purified product and the theoretically expected theoretical composition induced by the synthetic gene sequence.

Table 13

Amino acid analysis of purified SELPOK

pMoles Mole% Theoretical Mole%

ASX 28.10 0.6 0.7

GLX 26.90 0.6 0.4

SER 199.84 4.5 4.0

GLy 1812.07 41.0 40.5

HIS 28.45 0.6 0.7

ARG 20.49 0.5 0.5

THR 0 0.0 0.1

ALA 355.29 8.0 8.0

PRO 623.22 14.1 15.0

TYR 8.47 0.2 0.1

VAL 1183.63 26.8 27.3

MET 17.21 0.4 0.3

ILE 4.83 0.1 0.0

LEU 20.66 0.5 0.4

PHE 7.57 0.2 0.1

LYS 84.02 1.9 1.8

Total 4420.75

For SELPOK-CS1, a protein band with an apparent molecular weight of 90,000 can be seen on the SDS-PAGE gel stained with amide black, transferred to a membrane, and the same band can be seen on Western blotting with ELP antibody. As expected, amino acid analysis (see Table 14) showed that the product oligomerized glycine (40.0%), alanine (7.6%), serine (5.2%), proline (16.3%), and valine (23.3%) %). This product also contains 1.5% lysine. The amino acid composition table shown below shows the correlation of the composition of the purified product with the theoretical composition induced by the synthetic gene sequence as expected.

Table 14

Amino acid analysis of purified SELPOK-CS1

pMoles Mole% Theoretical Mole%

ASX 16.43 0.7 0.7

GLX 10.59 0.5 0.4

SER 119.96 5.2 3.6

GLY 924.51 40.0 39.6

HIS 13.85 0.6 0.7

ARG 11.26 0.5 0.5

THR 0 0.0 0.1

ALA 175.07 7.6 7.3

PRO 376.40 16.3 16.7

TYR 2.49 0.1 0.1

VAL 537.96 23.3 24.5

MET 5.19 0.2 0.3

ILE 0 0.0 0.0

LEU 76.62 33 0.4

PHE 2.58 0.1 0.1

LYS 35.68 1.5 1.6

Total 2308.59

Embodiment 4. The detection test method of CLP6 and SELP8K characteristic

Tiseel adhesive system. The rat skin was washed with water, blotted dry and cut into 1 cm x 4 cm strips. The adhesion was tested using Tiseel kit VH (Osterreiches Institute for Fur Blood Derivatives, GmbH, A-1220, Vienna, Austria) according to the manufacturer's instructions. Shear Tensile Strength Test of Overlapping Part of Rat Skin

The ability of the adhesive composition to adhere to skin was determined using the in vitro rat skin overlap shear strength test. Adhesive was applied to the underside of strips of skin harvested from mouse skin. The second skin is folded so as to create an adhesive surface of approximately 1 cm ² . A 100 gram weight is applied to the lamination and the bond is usually allowed to cure at room temperature for 2 hours, wrapped in plastic to prevent drying. The laminated connection is mounted on an Instron tensile tester or similar device and tension is applied. Detected overlapping regions that failed to load were recorded and normalized. Adhesive system with glutaraldehyde. Rat skin was washed with water, blotted dry, and cut into approximately 1 cm x 4 cm strips. Distill glutaraldehyde, store frozen and thaw quickly before use. Fetal bovine serum albumin was solubilized as described in Godman's instructions (Goldman, WO94/01508). CLP6 was dissolved in 150 mM HEPES + 30 mM NaCl at a concentration of 600 mg/ml and the pH was adjusted to 7.5. SELP8K was dissolved in 150 mM HEPES + 30 mM NaCl at the concentrations shown in Table 15 and the pH was adjusted to 7.5. SELP8K was dissolved in 150 mM HEPES + 45 mM NaCl at the concentrations shown in Table 15 and adjusted to pH8. Aliquots of the indicated proteins were spread on two skins prior to the addition of the glutaraldehyde solution. Put the second piece of skin on top and rub the lower skin to disperse the component, and adjust the overlapping area to about 1 cm ² , cover with plastic paper to prevent drying, and cure at 25°C under a pressure of 100 g/cm ² Hour. Adhesive system with 1,6-(diisocyanato)hexane. Rat skin was washed with water, blotted dry, and cut into approximately 1 cm x 4 cm strips. SELP8K was made into solution at a concentration of approximately 50% w/w in the specified buffer. A 1:1 v/v mixture of hexamethylene diisocyanate and Pluronic L-61 surfactant was prepared. 20 μl of SELP8K solution followed by 2 μl of dilute HMDI was applied to one piece of skin. Put the second piece of skin on top of the lower skin and rub to mix the components, adjust the overlapping area to about 1 cm ² , cover with plastic paper to prevent drying, and place at 25°C for 2 hours under a pressure of 100 g/cm ² solidified. result

To provide a baseline for subsequent adhesion tests, ethyl cyanoacrylate and Tisseel fibrin glue were tested. The results are shown in the table below.

Table 15: Basic examples of lamination shear tension Reagent dose Tensile Strength normal saline not applicable 13±4 Tiseel fibrin glue -25mg 261±51 ethyl cyanoacrylate 25mg 385±119

All data are based on at least three test specimens. All test results are based on a two hour cure time.

This target composition was compared to the protein binding system described by Goldman (WO94/01508). 10 microliters of glutaraldehyde (concentration as above) was added to all experiments. The results are shown in the table below.

Table 16: Lamination Shear Tension of Glutaraldehyde Cured Adhesive Systems Reagent dose Tensile strength Ovalbumin + glutaraldehyde (30μ) 200mg/mL 10μL 2.5N 6mg/2.5mg 50±10 Hypoplastic collagen (denatured) + glutaraldehyde (25μL) 125mg/ml 10μL 2.5N 3mg/2.5mg 148±47 CLP6 + glutaraldehyde (40 μL) 600 mg/mL 10 μL 2.5N 24mg/2.5mg 306±98 CLP6 + Glutaraldehyde (20μL) 600mg/mL 10μL 2.5N 12mg/2.5mg 171±42 SELP8K(30μL) +glutaraldehyde600mg/ML1.0N300mg/mL 1.0N300mg/mL(impure) 1.0N300mg/mL(impure) 0.1N287mg/mL 2.5N100mg/L 1.0N100mg/mL 2.5N 18mg/1mg9mg/1mg9mg/1mg9mg/0.1mg7mg/2.5mg3mg/1mg3mg/2.5mg 545±153452±54234±51 ^* 210±57 ^* 374±90361±47274±17 *This SELP8K preparation is known to be impure and was detected to have approximately half the adhesion of the more thoroughly pure material.

The data in the table above demonstrate that the target polymer provides superior adhesion when used in glutaraldehyde-cured systems under conditions comparable to collagen and ovalbumin. Despite the lower number of amino acids used for cross-linking, the SELP8K polymer provided the highest tensile strength among the rat skin superimposed shear results. The above results demonstrate that even the dose of glutaraldehyde as low as 100 μg/ ^cm2 can obtain a significant adhesive effect. The quality and purity of this glutaryl is known to be important for good crosslinking (Rujigork, Dewijin, Boon, Journal of Materials Science and Medicine 5:80-87 (1994)); Whipple, Rata, J. Org. Chem., 39:1666-1668 (1974). Glutaraldehyde used in these experiments was distilled and diluted to 2.5N and stored at -20°C until use.

In the following studies, hexamethylene diisocyanate was used. It has been found that it is necessary to add the same volume of diluent to obtain good adhesion, otherwise the cure will be too fast. The results are shown in the table below, where n=12.

Table 17: Lamination Shear Tensions of Adhesive Systems Related to HMDI Reagent dose Tensile strength SELP8K 20 μL×50% w/w HMDI/L-61 1:1 v/v 2 μL×50% v/v buffer: (100 μL water + 10 μL 1M KHCO ₃ ) 10mg1mg 585±203 SELP8K 20μL×50%w/w HMDI/L-61 1:1v/v 2μL×50%v/v Buffer: (100μL 50mM PO ₄ (pH6.8)+5μL 1M KHCO ₃ ) 10mg1mg 503±21 SELP8K 20μL×50%w/w HMDI/L-61 1:1v/v 2μL×50%v/v Buffer: (100μL 50mMPO ₄ (pH68)+10μL 1MKHCO ₃ ) 10mg1mg 451±67 SELP8K 20μL×50%w/wHMDI/L-61 1:1v/v 2μL×50%v/v Buffer: (100μL 50mMPO ₄ (pH6.8)) 10mg1mg 362±71

Example 5 Evaluation of the properties of SELPOK (SEOK) and SELPOK-CS1

A series of formulations were prepared with different formulation compositions and their laminated shear strengths were measured. In addition, a series of protocols were used to prepare protein gels that provide adhesion. These schemes are as follows: Scheme A SEOK 17% w/w

                                                                                                           

Potassium Carbonate 1:1

Isocyanate:amine 131:1 to prepare protein jelly

The 1:2 refers to the nominal ratio of amino groups derived from lysine to amino groups derived from SEDK. The 1:1 refers to the nominal ratio of carbonate ions per amino group derived from SEOK and lysine. The 131:1 refers to the nominal ratio of isocyanate groups to amine groups derived from SEOK and lysine.

A stock buffer solution was prepared by dissolving 0.0157 g of lysine hydrochloride, 0.0710 g of potassium carbonate, and 0.00371 g of Evans blue dye in 7.526 ml of deionized water. Add 620.6 μl of stock buffer to 127.1 mg of SEOK in an Eppendorf tube. The mixture was vortexed until a homogeneous solution was obtained. The solution was centrifuged at 5000 rpm for 30-60 seconds to remove air bubbles. The solution was then drawn into a 1 ml syringe for application to the test skin. Preferably, the dye is encapsulated in the protein jelly for easier visualization of the distribution of the jelly on the test skin. Preparation of HMDI coagulant

The HMDI coagulant was prepared by dissolving 1.75 mg of Sudan red dye in pure 1.00 g of 1,6-hexanediisocyanate. The purpose of selectively embedding dyes in the protein jelly is to more conveniently observe the distribution of the coagulant on the test skin. Preparation of rat skin

Freshly collected rat skins were frozen and stored at -20°C. Thaw before use and cut into 1em x 3em strips. Remove all fascia from the strip with a razor. Choose a leather strip with the same width and thickness. The prepared mouse skin samples were placed in a sterilized pad soaked in PBS and wrapped in an anti-drying plastic bag, and temporarily stored at 37°C. Adhesive application

In a greenhouse at -37°C, 15 µl of the protein jelly was applied to each of two strips of mouse skin, 30 µl in total, and cut into approximately 1 cm ² /piece of skin with a stainless steel spatula. A total of 1.8 μl of HMDI coagulant was applied evenly to the skin such that 3 parallel strips of HMDI were applied to the first skin and two X-shaped strips were applied to the second skin. The skins were immediately laminated, covered with a plastic film to prevent drying, and pressed with a weight of 100 grams. The junction skin was cured at 37°C for 15 minutes. Before carrying out the tensile test with an Instron Model 55 tester, measure the length and width of the laminated connecting skin with a ruler to an accuracy of 1 mm. Set the speed of the crosshead to 25 mm/min. The results of the composite shear resistance test are reported in units of g/ ^cm2 . Means and standard deviations were calculated for at least triplicate tests. Option B SEOK 17% w/w

       Lysine Hydrochloride 1:2

Potassium Carbonate 1:1

Isocyanate:amine 14.5:1

The procedure was as in Protocol A, except that a two-stage process was used to remove air bubbles in the protein jelly. After centrifugation, the jelly was placed in a reduced pressure (26 Hg) apparatus for 30 minutes. The volume of HMDI coagulant applied to the superimposed junctional skin was 2.0 μl. Option C SEOK 17% w/w

      Lysine Hydrochloride 1:2

Potassium Carbonate 1:1

Isocyanate:amine 7.3:1

The procedure was as in protocol B except that the HMDI coagulant conjugate was changed. 5.2 mg of Sudan dye was dissolved in 10.735 g of neat Pluronic Surfactant L-31 by heating at 100°C for 10 minutes. After cooling to room temperature, the same weight of 1,6-dicyanoethane was added to the mixture. This mixture is prepared before use. Option D1 SEOK 17% w/w

          Lysine Hydrochloride 1:2

Potassium Carbonate 1:1

Isocyanate:amine 14.5:1

The procedure was as in Scheme B except that 4.75 mg of Pluronic Surfactant L-31 was added to 74.6 mg of SEOK. The SEOK to lysine buffer ratio was also as described in Protocol B. Option D2 SEOK 17% w/w

         Lysine Hydrochloride   1:2

Potassium Carbonate 1:1

Isocyanate:amine 14.5:1

Except that 1.07 mg of Pluronic surfactant L-31 was added to 74.7 mg of SEOK, the rest of the steps were the same as in protocol B. The ratio of SEOK to lysine buffer is as described in Protocol B. Option E SEOK 17% w/w

                                                                                 

Potassium Carbonate 1:1

Isocyanate: Amine 7.3:1

Except that 5.1mg of Pluronic L-31 was added to 85.0mg of SEOK, the rest of the steps were as in Scheme B. The HMDI coagulant composition was also varied. 5.2 mg of Sudan Red was dissolved in 10.735 g of neat Pluronis Surfactant L-31 by heating at 100°C for 10 minutes. After cooling to room temperature, an equal amount of 1,6-dicyanohexane was added to the mixture. This mixture is prepared before use. Option F SEOK 17% w/w

         Lysine Hydrochloride   1:2

Sodium borate pH9.5

Isocyanate:amine 14.5:1

Follow protocol B except that the stock buffer is prepared by dissolving 0.46 g of lysine hydrochloride, 1.24 g of boric acid in 92.2 ml of deionized water. The pH of the solution was adjusted to 9.52 with 7.8 ml of 2N sodium hydroxide solution. Evans blue dye was dissolved in the buffer at a concentration of 0.50 mg/ml, and the solution was filtered through a 0.45 μm syringe filter. 333.5 μl of storage buffer was added to 68.3 mg of SEOK in an Eppendorf tube. The mixture was vortexed until it was completely solubilized. Option G SEOK 17% w/w

                                                                 

Potassium borate pH9.5

Isocyanate:amine 14.5:1

The procedure was the same as in Protocol B, except that 0.46 g of lysine hydrochloride and 1.24 g of boric acid were dissolved in 99.1 ml of deionized water to prepare a stock buffer. The pH of the solution was adjusted to 9.52 by adding 0.9 ml of 10N potassium hydroxide solution. Evans blue dye was dissolved in this buffer at a concentration of 0.50 mg/ml and filtered through a 0.45 micron syringe filter. 367.2 μl of stock buffer was added to 75.2 mg of SEOK in an Eppendorf tube. The mixture was shaken on a vortex until it was completely solubilized. Option H SEOK 17% w/w

                                                                 

Lithium Carbonate pH9.5

Isocyanate:amine 14.5:1

Except that 42.7 mg of lysine hydrochloride was dissolved in 10.0 ml of deionized water, and 14.3 mg of lithium carbonate was added to make the pH 9.55, the rest of the steps were as in Scheme B. Evans blue dye was dissolved in this buffer at a concentration of 0.50 mg/ml and filtered through a 0.45 μm syringe filter. Option I SEOK 17% w/w

                                                                 

Sodium carbonate pH9.5

Isocyanate:amine 14.5:1

Follow protocol B except that 1.06 g of sodium carbonate and 0.46 g of lysine hydrochloride are dissolved in 99.2 ml of deionized water. The pH of the solution was adjusted to 9.54 with 0.8 ml of concentrated hydrochloric acid. Evans blue was dissolved in this buffer solution at a concentration of 0.50 mg/ml and filtered through a 0.45 micron syringe filter. Scheme J SEOK 17% w/w

                                                                               

Potassium Carbonate

Isocyanate: Amine 14.5:1

Follow protocol B except that 1.38 g of potassium carbonate and 0.46 g of lysine hydrochloride are dissolved in 99.1 ml of deionized water to prepare the stock buffer. The pH of the solution was adjusted to 9.53 with 0.9 ml of concentrated hydrochloric acid. Evans blue dye was dissolved in the buffer at a concentration of 0.50 mg/ml and filtered through a 0.45 μm syringe filter. Scheme K SEOK 17% w/w

         Lysine Hydrochloride   1:2

                                                                       

Isocyanate:amine 14.5:1

The procedure was the same as in Protocol B, except that 42.7 mg of lysine hydrochloride was dissolved in 10.0 ml of deionized water and 55.2 mg of cesium carbonate was added to make the pH 9.52 to prepare a stock buffer. Evans blue dye was dissolved in the buffer at a concentration of 0.50 mg/ml and filtered through a 0.45 μm syringe filter. Scheme L SEOK 17% w/w

          Lysine Hydrochloride 1:2

Calcium carbonate 1: 1

Isocyanate:amine 14.5:1

The procedure was as in Protocol B, except that 20.8 mg of lysine hydrochloride was dissolved in 10.0 ml of deionized water and 68.6 mg of calcium carbonate was added to prepare the stock buffer. Evans blue dye was dissolved in this buffer at a concentration of 0.50 mg/ml and filtered through a 0.45 μm syringe filter. Plan M SEOK 17% w/w

          Lysine Hydrochloride 1:2

Potassium Carbonate pH9.0

Isocyanate:amine 14.5:1

The procedure was as in Protocol B except that 103.9 mg of lysine hydrochloride was dissolved in 50.0 ml of deionized water and 473.3 mg of potassium carbonate was added to prepare the stock buffer. The pH of the buffer was adjusted to 9.00 with concentrated hydrochloric acid. Scheme N1 SEOK 17% w/w

        Lysine Hydrochloride   1:2

Potassium Carbonate 1:1

Isocyanate:amine 14.5:1

Iodide:carbonate 1:2

The procedure was the same as protocol B except that potassium iodide was added to the stock buffer at a concentration of 5.70 mg/ml. The gum has a nominal pH of about 11. Option N2 SEOK 17% w/w

          Lysine Hydrochloride 1:2

Potassium Carbonate pH9.0

Isocyanate:amine 14.5:1

Iodide:carbonate 1:2

The procedure was as in Protocol B, except that potassium iodide was added to the stock buffer at a concentration of 5.70 mg/ml. Scheme O1 SEOK 17% w/w

        Lysine Hydrochloride   1:2

Potassium Carbonate 1:1

Isocyanate:amine 14.5:1

Glucose 1.09M

Follow protocol B except that 1.2755 grams of glucose is added to 6.495 ml of this stock buffer. The pH of the solution was 10.5. Scheme O2 SEOK 17% w/w

        Lysine Hydrochloride   1:2

Potassium Carbonate pH9.0

Isocyanate:amine 14.5:1

Glucose 1.09M

Follow protocol B except that 1.2755 grams of glucose is added to 6.495 ml of this stock buffer. The pH of the buffer was adjusted to 9.00 with concentrated hydrochloric acid. Scheme P1 SEOK 17% w/w

                                                                               

Potassium Carbonate 1:1

Isocyanate: Amine 14.5:1

                                                             

The procedure was the same as Protocol B except that 0.5226 grams of urea was added to 5.087 ml of stock buffer (nominal 1.5M). Scheme P2 SEOK 17% w/w

      Lysine hydrochloride   1:2

Potassium carbonate pH9.00

Isocyanate:amine 14.5:1

                                                             

Follow protocol B except that 0.5226 g of urea is added to 5.807 ml of stock buffer. The pH of the buffer was adjusted to 9.00 with concentrated hydrochloric acid. Scheme Q SEOK 17% w/w

      Lysine hydrochloride   1:2

Potassium Carbonate 1:1

                                                                                           

Except that the volume of the HMDI coagulant was reduced to 1.0 μl, other steps were as in protocol B. Scheme R SEOK 17% w/w

      Lysine hydrochloride   1:2

Potassium Carbonate 1:1

Isocyanate:amine 6.7; 6.5; 6.1:1

Except that the HMDI coagulant was diluted with toluene at 1:1 w/w, 1:3 w/w, or 1:5 w/w, the rest of the steps were as in Scheme B. The volume of concentrated HMDI coagulant applied to the superimposed junctions was 2 μl, 4 μl, or 6 μl, respectively. Scheme S SEOK 17% w/w

      Lysine Hydrochloride 1:2

Potassium Carbonate 1:1

Isocyanate:amine 6.1; 5.7; 5.6:1

Except that the HMDI coagulant is diluted with methylcyclohexane at 1:1w/w, 1:3w/w, or 1:5w/w, the rest of the steps are the same as in plan B, and applied to the dilute HMDI at the superimposed junction Coagulant volumes were 2 μl, 4 μl, or 6 μl, respectively. Scheme T SEOK 17% w/w

        Lysine Hydrochloride 1:2

Potassium Carbonate 1:1

Isocyanate:amine 8.5; 9.4; 9.7:1

Except diluting the HMDI coagulant with chloroform at 1:1 w/w, 1:3 w/w, or 1:5 w/w, the rest of the steps were the same as protocol B. The volume of dilute HMDI coagulant applied to the superimposed junction was 2 μl, 4 μl, or 6 μl, respectively. Scheme U SEOK 17% w/w

      Lysine Hydrochloride   1:2

Potassium Carbonate 1:1

Isocyanate:amine 8.0; 8.7; 7.8:1

Except that dichloromethane was used to dilute the HMDI coagulant at 1:1 w/w, 1:3 w/w, or 1:5 w/w, the remaining steps were as in Scheme B. The volume of dilute MDI coagulant applied to the superimposed junctions was 2 μl, 4 μl, or 6 μl, respectively. Scheme V1 SEOK 33%w/w

      Lysine Hydrochloride   1:1

Potassium Carbonate 3:2

Preparation of isocyanate:amine 5.0:1 protein jelly

A stock buffer solution was prepared by dissolving lysine hydrochloride (9.14 mg/ml), potassium carbonate (41.6 mg/ml), and Evans dye (0.50 mg/ml) in deionized water. The mixture was filtered through a glass fiber plug before use. 113.9 mg SE8K, and 227.8 mg of stock buffer were placed in an Eppenborf tube and vortexed until dissolved. Centrifuge at 5000 rpm for 30 seconds to remove air bubbles in the solution. The protein jelly was then drawn into a 1.00 ml syringe and allowed to stand at room temperature for 20 minutes before spreading over the superimposed junctions of the test specimens. Preparation of HMDI coagulant

The HMDI coagulant was prepared by dissolving 3.75 mg of Sudan dye in Pluronic Surfactant L-61 and adding an equal amount of 1,6-diisocyanohexane. Preparation of rat skin

Freshly harvested rat skins were frozen at -20°C. Thaw and cut into 1 cm x 3 cm strips before use. Choose those strips of the same width and thickness and remove the fascia and musculature of the thiopine. Before use, these mouse skin specimens were placed between sterile liners soaked in PBS, wrapped in plastic bags to prevent drying, and temporarily stored at 37°C. Adhesive Application

Apply 35 μl of protein jelly to one end of the mouse skin and cut into small pieces of about 1 cm ² (5-10 times with a stainless steel spatula). Excess protein jelly was transferred to a second piece of rat skin with a stainless steel spatula and handled similarly. A total of 3.8 μl of HMDI coagulant was applied to the skin in proportion to three parallel strips of HMDI on the first skin. The skins were immediately used to form a lap joint, rubbed against each other to disperse the HMDI coagulant, covered with a piece of plastic film to prevent drying, and pressed with a 100 gram weight. Allow the connection to cure for 15 minutes at 37°C. The length and width (accurate to 1 mm) of the laminated connection were measured with a ruler before performing the tensile test on an Instron Model 55 detector. Set the crosshead speed to 25mm/min. Laminate shear tension is reported in units of g/ ^cm2 . Means and standard deviations were calculated for at least triplicate assay results. Solution V2 SE8K 33% w/w

                                                                               

Potassium Carbonate 3:2

Isocyanate: Amine 5.0:1

The protocol was followed except that lysine hydrochloride (4.59 mg/ml), potassium carbonate (31.2 mg/ml), and Evans blue dye (0.50 mg/ml) were dissolved in deionized water to prepare the stock buffer V1 method. Solution V3 SE8K 33%w/w

                                                                                               

Potassium Carbonate 3:2

Isocyanate: Amine 5.0:1

The method of Protocol V1 was followed except that potassium carbonate (13.83 mg/ml) and Evans blue dye (0.50 mg/ml) were dissolved in deionized water to prepare stock buffer. Option W1 SEOK 17% w/w

Arginine 1:4

Potassium Carbonate 1.2:1

Isocyanate: Amine 13.1:1

Follow the procedure of Protocol A except that the buffer is prepared by dissolving arginine (2.4 mg/ml), potassium carbonate (9.46 mg/ml), and Evans blue dye (0.50 mg/ml) in deionized water . The volume of HMDI coagulant used for superimposed junctions was 2.0 [mu]l. It is assumed that only the α-amino group of arginine participates stoichiometrically in the coagulation reaction. Option W2 SEOK 17% w/w

Cysteine 1:2

Potassium Carbonate 1:1

Isocyanate:amine 13.1:1

Follow Protocol A except that cysteine (1.38 mg/ml), potassium carbonate (9.46 mg/ml), and Evans blue dye (0.50 mg/ml) are dissolved in deionized water to prepare the buffer Methods. The volume of HMDI coagulant used for superposition ligation was 2.0 μl. Scheme W3 SEOK 17% w/w

Tyrosine 1:2

Potassium Carbonate 1:1

Isocyanate:amine 13.1:1

In addition to dissolving tyrosine (2.07mg/ml), potassium carbonate (9.46mg/ml), and Evans blue dye (0.50mg/ml) in deionized water to prepare the buffer, the other protocol A method. The volume of HMDI coagulant used for superposition ligation was 2.0 [mu]l. Scheme W4 SEOK 17% w/w

1,3-BDSA 1:2

Potassium Carbonate 1:1

Isocyanate:amine 13.1:1

In addition to 1,3-benzenedisulfonic acid disodium salt-hydrate (1,3-BDSA) (3.79mg (mg/ml), and potassium carbonate (9.46mg/ml), and Evans blue beam material (0.50 mg/ml) was dissolved in deionized water to prepare the buffer, and the other followed the method of scheme A. The volume of HMDI coagulant applied to the superimposed junction was 2.0 μ l. Scheme X SEOK 17% w/w

Peptide RGRGRGKGKGK 1:2 (SEQ ID NO: 35)

Potassium Carbonate 1:1

Isocyanate:amine 14.5:1

Except that the synthetic peptide RGRGRGKGKGK (sequence 35) (4.4 mg/ml), potassium carbonate (9.46 mg/ml), and Evans blue dye (0.50 mg/ml) were dissolved in deionized water to prepare the buffer, other The method of scheme A. Option Y SEOK 17% w/w

    Lysine Choline Ester   1:4

Potassium Carbonate 1.12:1

Isocyanate:amine 14.5:1

Protocol B was followed except that the buffer was prepared by dissolving lysine choline ester (29.2 mg), potassium carbonate (123.9 mg), and Evans blue dye in 13.09 ml of deionized water. The buffer was filtered through a 0.45 μm syringe filter before use. The protein gel was prepared by dissolving SEOK (63.5 mg) in 310.0 μl of buffer. Plan Z SEOK 17% w/w

AEGly 1:4

Potassium Carbonate 1:1

Isocyanate:amine 14.5:1

Protocol B was followed except that glycine 2-aminoethyl ester, AEGly (see Example 1, Diamine Synthesis) was used to prepare the buffer. An aliquot of glycine 2-aminoethyl ester (28.9 mg, 236 mg/ml), and potassium carbonate (48.6 mg) was dissolved in 5.127 ml of water. 2.20 mg of Evans blue dye was added and the solution was filtered through a 0.45 μm syringe filter. Scheme AA SE8K 17% w/w

 Lysine Hydrochloride 1:1

Potassium Carbonate 1:1

Isocyanate:amine 12.0:1

The protocol was followed except that the buffer was prepared by dissolving lysine hydrochloride (3.73 mg/ml), potassium carbonate (11.33 mg/ml), and Evans blue dye (0.50 mg/ml) in deionized water VI method. Pluronic Surfactant L-61 cannot be added to this HMDI coagulant. The protein polymer used was SE8K. The volume of coagulant used for this junction was 2.0 μl. Scheme AB SEOK-CS1 17% w/w

    Lysine Hydrochloride 0.94∶2

Potassium Carbonate 0.96:1

Isocyanate:amine 16.4:1

The protocol was followed except that the buffer was prepared by dissolving lysine hydrochloride (1.84 mg/ml), potassium carbonate (8.37 mg/ml), and Evans blue dye (0.50 mg/ml) in deionized water A method. The protein polymer used was SEOK-CS1. The volume of coagulant used for this connection was 2.0 μl. Scheme AC SEOK 17% w/w

    Lysine Hydrochloride 1:2

Sodium borate pH9.5

  Isocyanate: Amine 7.3:1

Procedure F was followed except that the volume of HMDI coagulant applied to the superimposed junction was 1.0 μl. Scheme AD SEOK 17% w/w

                                                                                                         

Sodium borate pH9.5

Isocyanate:amine 7.3:1

Except for diluting the HMDI coagulant with cyclohexane at 1:1 v/v, 1:3 v/v, or 1:5 v/v, the steps in Scheme F were followed. The volume of dilute HMDI coagulant applied to the superimposed junction was 2 μl, 4 μl, or 6 μl, respectively. Scheme AE SEOK 17% w/w

                                                                                                                                 

Sodium borate pH9.5

Isocyanate:amine 7.3:1

Except for diluting the HMDI coagulant with 1,1,1-trichloroethane at 1:1 v/v, 1:3 v/v, or 1:5 v/v, the steps of Scheme F were followed. The volume of dilute HMDI coagulant applied to the superimposed junction was 2 μl, 4 μl, or 6 μl, respectively. Scheme AF SEOK 17% w/w

                                                                                                         

Sodium borate pH9.5

Isocyanate:amine 7.3:1

Except diluting the HMDI coagulant with ethyl acetate at 1:1 v/v, 1:3 v/v, 1:5 v/v, or 1:9 v/v, the steps of Method F were followed. The volume of dilute HMDI coagulant applied to the superimposed junction was 2 μl, 4 μl, 6 μl, or 10 μl, respectively. Scheme AG SEOK-CS1 17% w/w

1,3-PG 0.39:2

Potassium Carbonate 0.496:1

Potassium bicarbonate 0.496:1

Isocyanate:amine 2.5:1

Continue to use the method of plan B, with the following changes. Preparation of protein jelly

Add 81.6 mg of protein polymer SEOK-CS1 to 331 μl of deionized water in an Eppendorf tube and shake on a vortex mixer until dissolved. To this solution was added 11.43 μl of an aqueous solution (10% w/w) of diglycine 1,3-propylene glycol ester (1,3-PG); 14.73 μl of an aqueous solution of potassium carbonate (10% w/w); and 9.43 [mu]l of aqueous potassium bicarbonate (10%, w/w). The contents were shaken on a vortex mixer until homogeneous. The solution was centrifuged at approximately 5000 rpm for 30-60 seconds to expel air bubbles, and the jelly was then placed under reduced pressure (26 inches Hg) for 30 minutes. Preparation of HMDI coagulant

Neat 1,6-diisocyanatohexane (100 mg), and Sudan red dye (2.4 mg) were dissolved in 1-chloro-2,2,2-trifluoroethyl difluoromethyl ether (2.342 g) . The volume of dilute HMDI coagulant applied to the superimposed junction was 4 μl. Scheme AH SEOK 17% w/w

                                                                                                                                               

Potassium Carbonate 1:1

Preparation of isocyanate:amine 5.0:1 protein jelly

A protein jelly was prepared by dissolving the protein polymer SEOK at 17% w/w in 10 millimolar lactic acid aqueous solution (0.90 mg/ml). The pH of the protein jelly was about 3.5 using wide range pH paper. An accelerated curing solution was prepared by dissolving 1.66 grams of potassium carbonate in 10 ml of deionized water. Preparation of HMDI coagulant

1,6-Hexane diisocyanate (5.04 g), Pluronic surfactant F-127 (0.0033 g), and Sudan red dye (0.0020 g) were dissolved in chloroform (2.24 g) to prepare HMDI coagulant. Preparation of rat skin

Freshly collected rat skins were frozen and stored at -20°C. Thaw and cut into 1 cm x 3 cm strips before use. Remove the fascia on the thong with a razor. Choose leather strips that are the same width and thickness. The prepared mouse skin specimens were placed between sterile liners soaked in PBS and wrapped in anti-drying plastic bags for temporary storage at 37°C. Adhesive use

The strips were placed on glass petri dishes in a greenhouse at 37°C. Use a stainless steel spatula to apply 1.0 μl of HMDI coagulant to the surface of approximately ¹ cm at the end of each two skins, for a total of 2.0 μl. 2.0 µl of the potassium carbonate solidified solution was divided into six drops, and the drops were added to one of every two skins and superimposed connections were immediately performed. A 100 gram weight was applied to the joint and then placed at 37°C for 15 minutes to allow the adhesive to cure.

The stack was connected to an Instron proficiency tester and tested for reliability as described below.

The first test used the surfactant Pluronic L-31.

Table 18: Effect of Surfactant Pluronic L-31 SEOK jelly K ₂ CO ₃ L-Lys HMDI+Pluronic Laminated shear strength g/cm ² plan 1 SEOK17%w/w 1:1 1:2 none 2143±328 A 2 SEOK17%w/w (repelling gas) 1:1 1:2 none 2901±685 B 3 SEOK17%w/w (repelling gas) 1:1 1:2 HMDI/L-31 1:1 (3.3% w/w relative to total jelly) 1248±370 C 4 SEOK 17% w/w (repellent gas) + L-31 (1.04% w/w relative to total jelly) 1:1 1:2 none 745±209 D1 5 SEOK 17% w/w (repellent gas) + L-31 (0.24% w/w relative to total jelly) (1.4% w/w wrt SEOK) 1:1 1:2 none 1499±159 D2 6 SEOK 17% w/w (repellent gas) + L-31 (0.17% w/w relative to total jelly) (1.0% w/w wrt SEOK) 1:1 1:2 HMDI/L-31 1:1 (3.3% relative to total jelly) 677±284 E.

In the next experiment, various buffers were used in conjunction with lysine as a polyfunctional group.

Table 19

Adhesion effect using 17% SELPOK (SEOK) in various pH9.5 buffers buffer Shear strength g/cm2 CV% plan 1 Na ₃ BO ₃ /L-Lys (1:2) ^* 2360±475 20% f 2 K ₃ BO ₃ /L-Lys (1:2) 2037±338 17% G 3 Li ₂ CO ₃ /L-Lys (1:2) 188±38 20% h 4 Na ₂ CO ₃ /L-Lys (1:2) 1168±274 twenty three% I 5 K ₂ CO ₃ /L-Lys (1:2) 2393±631 26% J 6 Cs ₂ CO ₃ /L-Lys (1:2) 233±56 twenty four% K 7 CaCO ₃ /L-Lys (1:2) 88±2 2% L

*Molar ratio of amino groups derived from lysine relative to amino groups derived from SEDK SELPOK

In the next experiment, various non-reactive and chemically reactive additives were added to the formulation to determine the effect of the additive on shear strength.

Table 20 Adhesive effect using 17% SELPOK dissolved in carbonate buffer with additives buffer pH Shear strength g/cm ² cv plan 1 K ₂ CO ₃ (2:2)/L-Lys (1:2) without additives 10.59.0 2901±6851618±293 24% 18% BM 2 K ₂ CO ₃ (2:2)/L-Lys (1:2) plus KI (0.043Mole/L) 119.0 2538±441826±211 17% 26% N1N2 3 K ₂ CO ₃ (2:2)/L-Lys (1:2) plus glucose (1.09 Mole/L) 10.59.0 1896±5571398±358 29% 26% O1O2 4 K ₂ CO ₃ (2:2)/L-Lys (1:2 plus urea (1.50Mole/L) 119.0 2674±846239±38 32% 16% P1P2

In the following experiments, various organic solvents were used in which the cross-linking agent was dissolved prior to mixing with the buffered aqueous solution of the protein.

Table 21 uses HMDI to add volatile diluent and is dissolved in the binding effect of lysine-boric acid buffer (pH9.5) Thinner boiling point Dilution ratio [v/v] coagulant volume Shear strength g/cm ² CV% plan 1 none Untested 1:0 1μL 852±173 20% AC 2 Cyclohexane 81° 1:11:31:5 2 μL 4 μL 6 μl 9009531053 Untested AD 3 1,1,1-Trichloroethane 75° 1:11:31:5 2 μL 4 μL 6 μL 781±13943±119816±51 2% 13% 6% AE 4 ethyl acetate 77° 1:11:31:51:9 2 μL 4 μL 6 μL 10 μL 869±41741±296685±147658±131 5% 40% 21% 20% AF

Table 22 uses HMDI to add volatile diluent and is dissolved in the binding effect of lysine-carbonic acid buffer (pH10) Thinner boiling point Ratio [w/w] coagulant volume Shear strength g/cm ² CV% plan 1 none Untested 1:0 1μL 2713±234 6% Q 2 toluene 110° 1:11:31:5 2 μL 4 μL 6 μL 2303±5022295±2101178±282 22% 9% 24% R 3 Methylcyclohexane 101° 1:11:31:5 2 μL 4 μL 6 μL 2508±2342160±1111364±395 9% 5% 29% S 4 Chloroform 61° 1:11:31:5 2 μL 4 μL 6 μL 2075±3702836±6201493±223 18% 22% 15% T 5 Dichloromethane 40° 1:11:31:5 2 μL 4 μL 6 μL 2389±5422636±5042511±493 23% 19% 20% u

In the following experiments, different multifunctional agents were used to crosslink polymers with various functionalities, either symmetrical or asymmetrical, and the intervening chains with side chains of different functional groups were aliphatic family or aromatic. Sometimes, the multifunctional used for crosslinking is hydrolytically labile with a hydrolysis sensitive bond in the linkage.

Table 23 Adhesive effect of SELP8K, SELPOK or SELPOK-CS1 with different polyfunctional agents protein buffer Ratio ^* multifunctional agent Shear strength g/cm ² plan SE8K 33% w/w _{K2CO3 3} :2 2:21:20:2 Lysine 1328±2031212±2411161±383 V1V2V3 SE0K 17% w/w _{K2CO3 1} :1 1:2 Lysine Arginine Cysteine Tyrosine 3,5-Disulfocatechol Tyrosine 2143±3281176±748741±66622±339399 AW1W2W3W4 SE0K 17% w/w _{K2CO3 1} :1 1:2 Peptide RGRGRGKGKGK 850±184 x SE0K 17% w/w _{K2CO3 1} :1 1:2 Lysine 2901±685 B SE0K 17% w/w _{K2CO3 1} :1 1:4 Lysine Choline Ester 2385±502 Y SE0K 17% w/w _K2CO3 1.12 _: 1 1:4 Glycine 2-Aminoethyl Ester N,O-Diglycyl Ethanolamine Sulfate Salt in a 4:1 Mixture 3269±422 Z SE0K-CS1 17% w/w _{K2CO3 1} :1 1:2 Lysine 2196±275 AB SE0K-CS1 17% w/w _{K2CO3 1} :1 0.77:2 Diglycine 1,3-propanediester 3028±392 AG

^* The ratio of the added nucleophile to the available amino groups of the protein backbone.

In Table 24, various formulations containing SELP8K, SELPOK, SELPOK-CS1 were compared.

Table 24 SELP8K, SELPOK know the adhesion effect of SELPOK-CS1 polymer Ratio _{of K2CO3} to amine Amine Ratio ^* Shear strength g/cm ² CV% plan SE8K 17% w/w 1:1 2:2 2854±1027 36% AAA SE0K 17% w/w 1:1 1:2 2143±328 15% A SE0K 17% w/w 10mM lactic acid in water 1:1 0:2 532±207 39% AH SE0K-CS1 17% w/w 1:1 1:2 2196±275 13% AB

^* Ratio of lysine-derived amine groups to protein-derived amine groups.

It is evident from the above results that the present invention provides a composition that rapidly produces components having a broad spectrum of characteristics. The object composition provides a strong adhesive component with good shear strength which develops within a short time. The present invention can also provide compositions for filling tissue defects or air conditioning or for expanding tissue. Thus, the protein polymers of the present invention can be used as tissue adhesives, which result in resorbable, physiologically compliant compositions that maintain long-term strength, and as sealants, among other uses.

All publications and patent applications mentioned in this specification are hereby incorporated by reference to the same extent as if they were individually cited.

Now that the invention has been fully described, it will be apparent to those skilled in the art that many changes and modifications can be made therein without departing from the spirit or scope of the appended claims.

Sequence Listing (1) General Information (i) Applicant: STEDRONSKY, Erwin R

      CAPPELLO, Joseph(ii) Title of Invention: Tissue Adhesive Utilizing Synthetic Crosslinking Agent(iii) Serial Number: 35(iv) Mailing Address:

(A) Recipients: FLEHR, HOHBACH, TEST, ALBRITTON and HERBERT

(B) Street: Four Embarcadero Center, Suite200

(C) City: San Francisco

(D) Continent: CA

(E) Country: United States

(F) Zip code: 94111 (v) Computer readable form

(A) Media type: floppy disk

(B) Computer: IBM PC compatible

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

(D) Software: PatenIn Release, #1.0, Version#1.30(vi) Current application materials:

(A) Application number:

(B) Filing date:

(C) Classification number: (viii) Lawyer / agent information

(A) Name: ROWLAND, Bertram I

(B) Registration number: 20015

(C) Information/file number: A61127-1/BIR(ix) Telecom information:

(A) Tel: 415-781-1989

(B) Telex: 415-398-3249 (2) Sequence 1 data: (i) Sequence characteristics:

(A) Length: 6 amino acids

(B) type: amino acid

(C) Chain type: single

(D) Topological structure: linear (ii) Molecular type: peptide (xi) Sequence description: Sequence 1

Gly Ala Gly Ala Gly Ser(2) sequence 2 information: (i) sequence characteristics:

(A) Length: 5 amino acids

(B) type: amino acid

(C) Chain type: single

(D) Topological structure: linear (ii) Molecular type: peptide (xi) Sequence description: Sequence 2

Gly Val Gly Val Pro

1 5(2) Sequence 3 data: (i) Sequence characteristics:

(A) Length: 7 AA

(B) Type: AA

(C) Chain type: single

(D) Topological structure: linear (ii) Molecular type: peptide (xi) Sequence description: Sequence 3

Ala Lys Leu Xaa Leu Ala Glu

1 5(2) Sequence 4 data: (i) Sequence characteristics:

(A) Length: 64AA

(B) Type: AA

(C) Chain type: single

(D) Top Park Structure: Linear (II) Molecular Type: Peptide (XI) sequence description: Sequence 4gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Sera Gly Ala Gly Ala1 5 10 15gly Serra Gly Sera Gly Serg Val Val Val Val Val Val Val Gly Val Pro Gly Val Gly

20 25 30Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Lys Gly Val

35 40 45Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro

50 55 60 (2) Sequence 5 data: (i) Sequence characteristics:

(A) Length: 64 AA

(B) Type: AA

(C) Chain type: single

(D) Top Park Structure: Linear (II) Molecular Type: Peptide (XI) sequence description: Sequence 5gly Ala GLY Ala Gly Ser Gly Ala Gly Ala Gly Ala Gly Ala1 5 10 15gly Ser Gly Ala Gly Serg Val Val Val Val Val Val Val Val Val Gly Val Pro Gly Val Gly

20 25 30Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Glu Gly Val

35 40 45Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro

50 55 60 (2) Sequence 6 information: (i) Sequence characteristics:

(A) Length: 31bp

(B) type: nucleic acid

(C) Chain type: single

(D) Topological structure: linear (ii) Molecular type: cDNA (xi) Sequence description: Sequence 6 (2) Sequence 7 data:

(i) Sequential features:

(A) Length: 35bp

(B) type: nucleic acid

(C) Chain type: single

(D) Topology: linear

(ii) Molecular type: cDNA

(xi) Sequence description: Sequence 7

ACTCGGATGT ACATGCAGGC ACCCGCTCCA GAGCC 35(2) Sequence 8 information:

(i) Sequential features:

(A) Length: 192bp

(B) type: nucleic acid

(C) Chain type: double

(D) Topology: linear

(ii) Molecular type: cDNA

(xi)序列描述：序列8GGTGCCGGTT CTGGAGCTGG CGCGGGCTCT GGAGTAGGTG TGCCAGGTGT AGGAGTTCCG    60GGTGTAGGCG TTCCGGGAGT TGGTGTACCT GGAGTGGGTG TTCCAGGCGT AGGTGTGCCC    120GGGGTAGGAG TACCAGGGGT AGGCGTGCCT GGAGCGGGTG CTGGTAGCGG CGCAGGCGCG    180GGCTCTGGAG CG                                                        192(2)序列9资料：

(i) Sequential features:

(A) Length: 65AA

(B) Type: AA

(C) Chain type: single

(D) Topology: linear

(ii) Molecule type: peptide

(XI) Sequence description: Sequence 9gly Ala Gly Ser Gly Ala Gly Ala Gly Val Gly Val Pro GLY1 5 10 15VAL VAL GLE GLY VAL VAL VAL VAL VAL VAL VAL VLE GLE

20 25 25 30Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val

35 40 45Gly Val Pro Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly

50 60Ala65(2) Sequence 10 information:

(i) Sequential features:

(A) Length: 201bp

(B) type: nucleic acid

(C) Chain type: single

(D) Topology: linear

(ii) Molecular type: cDNA

(xi)序列描述：序列10ATGGCAGCGA AAGGGGACCG GGCTCTGGTG TTGGAGTGCC AGGTGTCGGT GTTCCGGGTG                 60TAGGCGTTCC GGGAGTTGGT GTACCTGGAA AGGTGTTCCG GGGGTAGGTG TGCCGGGCGT                 120TGGAGTACCA GGTGTAGGCG TCCCGGGAGC GGGTGCTGGT AGCGGCGCAG GCGCGGGCTC                 180TTTCCGCTAA AGTCCTGCCG T                                                           201(2)序列11资料：

(i) Sequential features:

(A) Length: 35bp

(B) type: nucleic acid

(C) Chain type: single

(D) Topology: linear

(ii) Molecular type: cDNA

(xi) Sequence description: Sequence 11

AAGAAGGAGA TATCATATGG CAGCGAAAGG GGACC 35(2) Sequence 12 information:

(i) Sequential features:

(A) Length: 37bp

(B) type: nucleic acid

(C) Chain type: single

(D) Topology: linear

(ii) Molecular type: cDNA

(xi) Sequence description: Sequence 12

CGCAGATCTT TAAATTACGG CAGGACTTTA GcGGAAA 37(2) Sequence 13 information:

(i) Sequential features:

(A) Length: 192bp

(B) type: nucleic acid

(C) Chain type: double

(D) Topology: linear

(ii) Molecular type: cDNA

(xi)序列描述：序列13GGTGCCGGTT CTGGAGCTGG CGCGGGCTCT GGTGTTGGAG TGCCAGGTGT CGGTGTTCCG         60GGTGTAGGCG TTCCGGGAGT TGGTGTACCT GGAAAAGGTG TTCCGGGGGT AGGTGTGCCG         120GGCGTTGGAG TACCAGGTGT AGGCGTCCCG GGAGCGGGTG CTGGTAGCGG CGCAGGCGCG         180GGCTCTGGAG CG                                                             192(2)序列14资料：

(i) Sequential features:

(A) Length: 64AA

(B) Type: AA

(C) Chain type: single

(D) Topology: Linear

(ii) Molecule type: peptide

(XI) Sequence description: Sequence 14gly Ala Gly Ser Gly Ala Gly Ala Gly Val Gly Val Pro GLY1 5 10 15VAL VAL VAL VAL VAL VAL VAL VAL VAL VLLY LYS

20 25 25 30Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly

35 40 45Val Pro Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Ala

50 55 60(2) Sequence 15 information:

(i) Sequential features:

(A) Length: 884AA

(B) Type: AA

(C) Chain type: single

(D) Topology: linear

(ii) Molecule type: peptide

(XI) Sequence description: Sequence 15MET ASP Pro Val Val Leu Gln ARG ARG ARG ARP GLU Asn Pro Gly Val1 5 10 15thr Gln Leu ARA Ala His Pro PHE ALA Ser Ala Phe Pro Pro

20 25 30Met Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Val Gly Val Pro

35 40 45Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly

50 55 60LYS GLE VAL VAL VAL VAL VAL VAL VAL VAL VAL VAL VAL VAL65 70 75 80Gly Val Pro Gly Ala Gly Serra Gly Ala Gly Sergly

85 90 95Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Val Gly Val Pro

100 105 110Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly

115 120 125Lys Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val

130 135 140Gly Val Pro Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly145 150 155 160ALA GLY Ala GLY Ala GLY Ala Gly Val Val Val Val Val Val Val Val Val Val Val Val Val Val Pro.

165 170 175Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly

180 185 190Lys Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val

195 200 205Gly Val Pro Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly

210                 215                 220Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Val Gly Val Pro225                 230                 235                 240Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly

245 250 255Lys Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val

260 265 270Gly Val Pro Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly

275 280 285Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Val Gly Val Pro

290 295 300Gly Val Gly Val Pro Gly VAL VA1 Pro Gly Val Val Val Pro Gly305 315 320lys Gly Val Val Val Val Val Val Val Val Val Val.

325 330 335Gly Val Pro Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly

340 345 350Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Val Gly Val Pro

355 360 365Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly

370 375 380Lys Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val385 390 0 4 395

Gly Val Pro Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly

405 410 415Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Val Gly Val Pro

420 425 430Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly

435 440 445Lys Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val

450                 455                 460Gly Val Pro Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly465                 470                 475                 480Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Val Gly Val Pro

485 490 495Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly

500 505 510Lys Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val

515 520 525Gly Val Pro Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly

530                 535                 540Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Val Gly Val Pro545                 550                 555                 560Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly

S65 570 575Lys Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val

580 585 590Gly Val Pro Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly

595 600 605Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Val Gly Val Pro

610 615 620Gly Val Gly Val Pro Gly Val Val Val Val Val Val Val Pro Gly625 630 640lys Gly Val Val Val Val Val Val Val Val Val Val.

645 650 655Gly Val Pro Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly

660 665 670Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Val Gly Val Pro

675 680 685Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly

690 695 700lys Gly Val Pro Gly Val Gly Val Pro Gly Val Val Val Val 705 715 720Gly Val Pro Gly Ala Gly Sera Gly Ala Gly Serge

725 730 735Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Val Gly Val Pro

740 745 750Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly

755 760 765Lys Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val

770 775 780Gly Val Pro Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly 785 795 800ALA GLE Ala Gly Ala Gly Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Pro

805 810 815Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly

820 825 830Lys Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val

835 840 845Gly Val Pro Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly

850 855 860ALA GLY ALA MET ASP Pro Gly ARG TYR GLN ASP Leu ARG Serg Serg Serg Serg Serg Serg Serg Serg Serg Serg 865 875 870his His Hwan

(i) Sequential features:

(A) Length: 1065AA

(B) Type: AA

(C) Chain type: single

(D) Topology: linear

(ii) Molecule type: protein

(XI) Sequence description: Sequence 16MET ASP PRO Val Val Val Leu Gln ARG ARG ASP TRP GLU ASN Pro Gly Val1 5 10 15thr Gln Leu ARA Ala His Pro PHE ALA Ser Ala Phe Pro Pro

20 25 25 30Met Gly Ala His Gly Pro Ala Gly Pro Lys Gly Ala His Gly Pro Ala

35 40 45Gly Pro Lys Gly Ala Gln Gly Pro Ala Gly Pro Gly Gly Ala Gln Gly

50 55 55pro Ala Gly Pro Gly GLY GLN GLN GLY Pro Ala Gly Pro Gly GLY GLY GLY ALA65 70 75 80GLN GLY Pro Ala GLY GLN GLN GLN GLY Pro Gly Pro Gly

85 90 95Gly Ala Gln Gly Pro Ala Gly Pro Gly Gly Ala Gln Gly Pro Ala Gly

100 105 110Pro Gly Gly Ala Gln Gly Pro Ala Gly Pro Gly Gly Ala Gln Gly Pro

115 120 125Ala Gly Pro Gly Gly Ala Gln Gly Pro Ala Gly Pro Gly Gly Ala Gln

130 135 140Gly Pro Ala Gly Pro Gly GLY GLN GLN GLY Pro Ala Gly Pro Gly Gly145 150 155 160ALA GLN GLY Pro GLY GLY GLN GLN GLY Pro Ala GLY PRO

165 170 175Gly Gly Ala Gln Gly Pro Ala Gly Pro Gly Gly Ala Gln Gly Pro Ala

                                                                                                                                          

195 200 205Pro Ala Gly Pro Gly Gly Ala Gln Gly Pro Ala Gly Pro Gly Gly Ala

210 215 220GLN GLY Pro Gly Pro Gly GLY Ala Gln GLY Pro Ala Gly Pro Gly 225 235 240GLY Ala Gln Gly Pro Gly GLN GLN GLN GLN GLN GLN GLN GLN

245 250 255Pro Gly Gly Ala Gln Gly Pro Ala Gly Pro Gly Gly Ala His Gly Pro

260 265 270Ala Gly Pro Lys Gly Ala His Gly Pro Ala Gly Pro Lys Gly Ala His

275 280 285Gly Pro Ala Gly Pro Lys Gly Ala His Gly Pro Ala Gly Pro Lys Gly

290                 295                 300Ala Gln Gly Pro Ala Gly Pro Gly Gly Ala Gln Gly Pro Ala Gly Pro305                 310                 315                 320Gly Gly Ala Gln Gly Pro Ala Gly Pro Gly Gly Ala Gln Gly Pro Ala

325 330 335Gly Pro Gly Gly Ala Gln Gly Pro Ala Gly Pro Gly Gly Ala Gln Gly

340 345 350Pro Ala Gly Pro Gly Gly Ala Gln Gly Pro Ala Gly Pro Gly Gly Ala

355 360 365Gln Gly Pro Ala Gly Pro Gly Gly Ala Gln Gly Pro Ala Gly Pro Gly

370                 375                 380Gly Ala Gln Gly Pro Ala Gly Pro Gly Gly Ala Gln Gly Pro Ala Gly385                 390                 395                 400Pro Gly Gly Ala Gln Gly Pro Ala Gly Pro Gly Gly Ala Gln Gly Pro

405 410 415Ala Gly Pro Gly Gly Ala Gln Gly Pro Ala Gly Pro Gly Gly Ala Gln

420 425 430Gly Pro Ala Gly Pro Gly Gly Ala Gln Gly Pro Ala Gly Pro Gly Gly

435 440 445Ala Gln Gly Pro Ala Gly Pro Gly Gly Ala Gln Gly Pro Ala Gly Pro

450 455 460Gly Gln GLN GLY Pro Ala Gly Pro GLY GLY Ala Gln GLN GLY Pro 470 475 480GLY GLY GLY Ala GLN GLN GLY Pro GLY GLY Ala Gln Gly.

485 490 495Pro Ala Gly Pro Gly Gly Ala Gln Gly Pro Ala Gly Pro Gly Gly Ala

500 505 510Gln Gly Pro Ala Gly Pro Gly Gly Ala His Gly Pro Ala Gly Pro Lys

515 520 525Gly Ala His Gly Pro Ala Gly Pro Lys Gly Ala His Gly Pro Ala Gly

530 535 540pro lys Gly Ala His GLY Pro Ala Gly Pro Lys GLY Ala Gln Gly Pro545 550 560ALA GLY Pro GLN GLN GLY Pro GLY GLY GLY GLN GLN

565 570 575Gly Pro Ala Gly Pro Gly Gly Ala Gln Gly Pro Ala Gly Pro Gly Gly

580 585 590Ala Gln Gly Pro Ala Gly Pro Gly Gly Ala Gln Gly Pro Ala Gly Pro

595 600 605Gly Gly Ala Gln Gly Pro Ala Gly Pro Gly Gly Ala Gln Gly Pro Ala

610 615 620gly Pro GLY GLY Ala Gln Gly Pro Ala Gly Pro Gly GLN GLN GLN GLN GLN GLN GLN GLN GLN GLN GLN GLN GLN GLN GLN GLN GLN GLN GLN GLN GLN GLN GLN GLN 635 640PRO Ala GLY GLY PRE GE GLN GLY GLY GLY GLY GLY GE ALN -P's 6LA that

645 650 655Gln Gly Pro Ala Gly Pro Gly Gly Ala Gln Gly Pro Ala Gly Pro Gly

660 665 670Gly Ala Gln Gly Pro Ala Gly Pro Gly Gly Ala Gln Gly Pro Ala Gly

675 680 685Pro Gly Gly Ala Gln Gly Pro Ala Gly Pro Gly Gly Ala Gln Gly Pro

690 695 700ALA GLE PRO GLY GLY Ala Gln Gly Pro Ala Gly Pro GLY GLN705 715 720GLY Pro Ala GLY GLY GLN GLY Pro Gly Pro Gly Gly Gly

725 730 735Ala Gln Gly Pro Ala Gly Pro Gly Gly Ala Gln Gly Pro Ala Gly Pro

740 745 750Gly Gly Ala Gln Gly Pro Ala Gly Pro Gly Gly Ala Gln Gly Pro Ala

755 760 765Gly Pro Gly Gly Ala His Gly Pro Ala Gly Pro Lys Gly Ala His Gly

770 775 780Pro Ala Gly Pro Lys Gly Ala His Gly Pro Ala Gly Pro Lys Gly Ala785 795 800his Gly Pro Ala Gly Pro Lys GLN GLY Pro Gly Pro Gly Gly Gly Gly Gly

805 810 815Gly Ala Gln Gly Pro Ala Gly Pro Gly Gly Ala Gln Gly Pro Ala Gly

820 825 830Pro Gly Gly Ala Gln Gly Pro Ala Gly Pro Gly Gly Ala Gln Gly Pro

835 840 845Ala Gly Pro Gly Gly Ala Gln Gly Pro Ala Gly Pro Gly Gly Ala Gln

850 855 860Gly Pro Ala Gly Pro Gly GLY GLN GLN GLY Pro Ala Gly Pro Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly 875 875 880ALA GLN GLY Pro GLY GLN GLN GLY Pro Gly Pro GLY Pro GLY Pro Gly Pro

885 890 895Gly Gly Ala Gln Gly Pro Ala Gly Pro Gly Gly Ala Gln Gly Pro Ala

900 905 910Gly Pro Gly Gly Ala Gln Gly Pro Ala Gly Pro Gly Gly Ala Gln Gly

915 920 925Pro Ala Gly Pro Gly Gly Ala Gln Gly Pro Ala Gly Pro Gly Gly Ala

930 935 940GLN GLY Pro Gly Pro Gly GLY Ala Gln GLY Pro Ala Gly Pro Gly945 955 960Gly Ala Gln Gly Pro Gly GLN GLN GLN GLN GLN GLN GLN GLN

965 970 975Pro Gly Gly Ala Gln Gly Pro Ala Gly Pro Gly Gly Ala Gln Gly Pro

980 985 990Ala Gly Pro Gly Gly Ala Gln Gly Pro Ala Gly Pro Gly Gly Ala Gln

995 1000 1005Gly Pro Ala Gly Pro Gly Gly Ala Gln Gly Pro Ala Gly Pro Gly Gly

1010 1015 1020ALA HIS GLY Pro Ala Gly Pro Lys Gly Ala His Gly Pro Ala Gly Pro 1025 1035 1040lys Gly Ala Gly ARG Tyr Gln Leu Serg Tyr

1045 1050 1055His Tyr Gln Leu Val Trp Cys Cys Lys

                                                                                                             

(i) Sequential features:

(A) Length: 4AA

(B) Type: AA

(C) Chain type: single

(D) Topology: Linear

(ii) Molecule type: peptide

(xi) Sequence description: Sequence 17

Pro Leu Gly Pro

l(2) Sequence 18 information:

(i) Sequential features:

(A) Length: 22AA

(B) Type: AA

(C) Chain type: single

(D) Topology: linear

(ii) Molecule type: peptide

(xi) Sequence description: Sequence 18

Gly Ala Gly Ala Gly Ser Gly Val Gly Val Pro Leu Gly Pro Leu Gly

l 5 10 15

Pro Gly Val Gly Val Pro

          20(2) Sequence 19 data:

(i) Sequential features:

(A) Length: 24AA

(B) Type: AA

(C) Chain type: single

(D) Topology: linear

(ii) Molecule type: peptide

(xi) Sequence description: Sequence 19

Gly Ala Gly Ala Gly ser Gly Val Gly Val Pro Gly Phe Phe Val Arg

1 5 10 15

Ala Arg Arg Gly Val Gly Val Pro

                                                         

(i) Sequential features:

(A) Length: 32bp

(B) type: nucleic acid

(C) Chain type: single

(D) Topology: linear

(ii) Molecular type: other nucleic acids

(A) Description: /desc="Synthetic"

(xi) Sequence description: Sequence 20GATCTTCGAT CTCATCACCA TCACCATCAC TA 32 (2) Sequence 21 information:

(i) Sequential features:

(A) Length: 32bp

(B) type: nucleic acid

(C) Chain type: single

(D) Topology: linear

(ii) Molecular type: other nucleic acids

(A) Description: /desc="Synthetic"

(xi) Sequence description: Sequence 21TGCTTAGTGA TGGTGATGGT GATGAGATCG AA 32 (2) Sequence 22 data:

(i) Sequential features:

(A) Length: 93bp

(B) type: nucleic acid

(C) Chain type: single

(D) Topology: linear

(ii) Molecular type: other nucleic acids

(A) Description: /desc="Synthetic"

(xi) Sequence description: Sequence 22ATGGCAGCGA AAGGGGACCG GTGCCGGCGC AGGTAGCGGA GCCGGTGCGG GCTCAAAAAG 60GGCTCTGGTG CCTTTCCGCT AAAGTCCTGC CGT 93 (2) Sequence 23 data:

(i) Sequential features:

(A) Length: 162bp

(B) type: nucleic acid

(C) Chain type: double

(D) Topology: linear

(ii) Molecular type: other nucleic acids

(A) Description: /desc="Synthetic"

(xi)序列描述：序列23GGGCTCTGGT GTTGGAGTGc CAGGTGTCGG TGTTCCGGGT GTAGGCGTTC CGGGAGTTGG    60TGTACCTGGA AAAGGTGTTC CGGGGGTAGG TGTGCCGGGC GTTGGAGTAC CAGGTGTAGG    120CGTCCCGGGA GCGGGTGCTG GTAGCGGCGC AGGCGCGGGC TC                       162(2)序列24资料：

(i) Sequential features:

(A) Length: 54AA

(B) Type: AA

(C) Chain type: single

(D) Topology: linear

(ii) Molecule type: peptide

(xi) Sequence description: Sequence 24Gly ser Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Vall 5 l0 l0 15Pro Gly Val Gly Val Gly Pro Gly pro Gly Lys

20 25 25 30Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Ala Gly Ala Gly Ser

35 40 45Gly Ala Gly Ala Gly Ser

50(2) Sequence 25 data:

(i) Sequential features:

(A) Length: 1002AA

(B) Type: AA

(C) chain type: single

(D) Top Park Structure: Linear (II) Molecular Type: Protein (XI) sequence description: Sequence 25MET ASP Pro Val Val Val Leu Gln ARG ARG ARG ARP Glu Asn Pro Gly Val1 5 10 15thr Gln Leu Ala Ala Ala Ala His Pro Pro Phe Ala Ser Asp Pro

20 25 25 30Met Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Val Gly

35 40 45Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val

50 55 60pro Gly Lys Gly Val Pro Gly Val Val Val Val Val Val Pro65 70 75 80Gly Val Val Vly Ala Gly Ala Gly Ala Gly Ala Gly Gly

85 90 95Ser Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro

100 105 110Gly Val Gly Val Pro Gly Lys Gly Val Pro Gly Val Gly Val Pro Gly

115 120 125Val Gly Val Pro Gly Val Gly Val Pro Gly Ala Gly Ala Gly Ser Gly

130 135 140

Ala Gly Ala Gly Ser Gly Val Gly Val Pro Gly Val Gly Val Pro Gly145 150 155 160Val Gly Val Pro G Val Gly L Pro G Pro G

                                                                                         

180 185 190Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Val Gly Val Pro Gly Val

195 200 205Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Lys Gly

210 215 220val Pro Gly Val Val Val Val Val Val Val Val Val 2225 235 240pro Gly Ala GLY Ala Gly Ala Gly Val Val Val Gly Gly Gly

245 250 255Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val

260 265 270Pro Gly Lys Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro

275 280 285Gly Val Gly Val Pro Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly

290                 295                 300Ser Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro305                 310                 315                 320Gly Val Gly Val Pro Gly Lys Gly Val Pro Gly Val Gly Val Pro Gly

325 330 335Val Gly Val Pro Gly Val Gly Val Pro Gly Ala Gly Ala Gly Ser Gly

340 345 350Ala Gly Ala Gly Ser Gly Val Gly Val Pro Gly Val Gly Val Pro Gly

355 360 365Val Gly Val Pro Gly Val Gly Val Pro Gly Lys Gly Val Pro Gly Val

370 375 380Gly Val Pro Gly Val Gly Val Pro Gly Val Val Val Pro Gly Ala Gly385 395 400ALA GLY Ala GLY Ala GLY Val Val Val Val Val Val

405 410 415Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Lys Gly

420 425 430Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val

435 440 445Pro Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Val Gly

450                 455                 460Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val465                 470                 475                 480Pro Gly Lys Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro

485 490 495Gly Val Gly Val Pro Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly

500 505 510Ser Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro

515 520 525Gly Val Gly Val Pro Gly Lys Gly Val Pro Gly Val Gly Val Pro Gly

530                 535                 540Val Gly Val Pro Gly Val Gly Val Pro Gly Ala Gly Ala Gly Ser Gly545                 550                 555                 560Ala Gly Ala Gly Ser Gly Val Gly Val Pro Gly Val Gly Val Pro Gly

565 570 575Val Gly Val Pro Gly Val Gly Val Pro Gly Lys Gly Val Pro Gly Val

580 585 590Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Ala Gly

595 600 605Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Val Gly Val Pro Gly Val

610 615 620Gly Val Pro Gly Val Gly Val Pro Gly Val Val Val Pro Gly Lys Gly625 635 640VAL PRO GLY VAL VAL VAL VAL VAL VAL GLY VAL VAL

645 650 655Pro Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Val Gly

660 665 670Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val

675 680 685Pro Gly Lys Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro

690                 695                 700Gly Val Gly Val Pro Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly705                 710                 715                 720Ser Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro

725 730 735Gly Val Gly Val Pro Gly Lys Gly Val Pro Gly Val Gly Val Pro Gly

740 745 750Val Gly Val Pro Gly Val Gly Val Pro Gly Ala Gly Ala Gly Ser Gly

755 760 765Ala Gly Ala Gly Ser Gly Val Gly Val Pro Gly Val Gly Val Pro Gly

770 775 780VAL GLY VAL VAL VAL VAL VAL PRO GLY LYS GLY VAL VAL785 795 800GLY VAL VAL VAL VAL VAL VAL VAL ALA GLY Ala Gly

805 810 815Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Val Gly Val Pro Gly Val

820 825 830Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Lys Gly

835 840 845Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val

850 855 860Pro Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Val Gly865 875 880val Pro Gly Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val

885 890 895Pro Gly Lys Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro

900 905 910Gly Val Gly Val Pro Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly

915 920 925Ser Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro

930 935 940Gly Val Gly Val Pro GLY LYS GLY VAL VAL VAL VAL VAL VAL PRO GLY945 955 960VAL GLY VAL VAL VAL VAL VAL VAL ALA GLY Ala Gly Ser Gly

965 970 975Ala Gly Ala Gly Ser Gly Ala Gly Ala Met Asp Pro Gly Arg Tyr Gln

980 985 990Asp Leu Arg Ser His His His His His His His

995 1000(2) sequence 26 data:

(i) Sequential features:

(A) Length: 378bp

(B) Type: AA

(C) Chain type: single

(D) Topology: linear

(ii) Molecule type: protein

(XI) Sequence description: Sequence 26MET ASP Pro Val Val Leu Gln ARG ARG ASP TRP GLU Asn Pro Gly Val1 5 10 15thr Gln Leu ARA Ala His Pro PHE Ala Phe Pro Pro

20 25 25 30Met Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Val Gly

35 40 45Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val

50 55 60pro Gly Lys Gly Val Pro Gly Val Val Val Val Val Val Pro65 70 75 80Gly Val Val Vly Ala Gly Ala Gly Ala Gly Ala Gly Gly

85 90 95Ser Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro

100 105 110Gly Val Gly Val Pro Gly Lys Gly Val Pro Gly Val Gly Val Pro Gly

115 120 125Val Gly Val Pro Gly Val Gly Val Pro Gly Ala Gly Ala Gly Ser Gly

130 135 140ALA GLY Ala Gly Ser Gly Val Gly Val Pro Gly Val Val Pro Gly145 150 155 160VAL VAL VAL VAL VAL VAL LYS GLY VAL VAL VAL Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Vall

165 170 175Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Ala Gly

180 185 190Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Val Gly Val Pro Gly Val

195 200 205Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Lys Gly

210 215 220val Pro Gly Val Val Val Val Val Val Val Val Val 2225 235 240pro Gly Ala GLY Ala Gly Ala Gly Val Val Val Gly Gly Gly

245 250 255Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val

260 265 270Pro Gly Lys Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro

275 280 285Gly Val Gly Val Pro Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly

290                 295                 300Ser Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro305                 310                 315                 320Gly Val Gly Val Pro Gly Lys Gly Val Pro Gly Val Gly Val Pro Gly

325 330 335Val Gly Val Pro Gly Val Gly Val Pro Gly Ala Gly Ala Gly Ser Gly

340 345 350Ala Gly Ala Gly Ser Gly Ala Gly Ala Met Asp Pro Gly Arg Tyr Gln

355 360 365Asp Leu Arg Ser His His His His His His His His

370 375(2) sequence 27 information:

(i) Sequential features:

(A) Length: 141bp

(B) type: nucleic acid

(C) Chain type: single

(D) Topological structure: linear (ii) Molecular type: other nucleic acids

(A)描述：/desc＝“合成的”(xi)序列描述：序列27ATGGCAGCGA AAGGGGACCG CCGGTGCGGG CTCTGGTGTT GGAGTGCCGC TGGGTCCTCT                    60TGGCCCAGGT GTCGGTGTTC CGGGTGTAGG CGTTCCGGGA GTTGGTGTAC CTGGAAAAGG                    120TTTCCGCTAA AGTCCTGCCG T                                                              141(2)序列28资料：

(i) Sequential features:

(A) Length: 181bp

(B) type: nucleic acid

(C) Chain type: single

(D) Topology: linear

(ii) Molecular type: other nucleic acids

(A) Description: /desc="Synthetic"

(xi)序列描述：序列28GGGCTCTGGT GTTGGAGTGC CGCTGGGTCC TCTTGGCCCA GGTGTCGGTG TTCCGGGTGT                 60AGGCGTTCCG GGAGTTGGTG TACCTGGAAA AGGTGTTCCG GGGGTAGGTG TGCCGGGCGT                 120TGGAGTACCA GGTGTAGGCG TCCCGGGAGC GGGTGCTGGT AGCGGCGCAG GCGCGGGCTC                 180T                                                                                 181(2)序列29资料：

(i) Sequential features:

(A) Length: 60 AA

(B) Type: AA

(C) Chain type: single

(D) Topology: linear

(ii) Molecule type: peptide

(xi) Sequence description: Sequence 29Gly Ser Gly Val Gly Val Pro Leu Gly Pro Leu Gly Pro Gly Val Gly1 5 10 10 Val 15 Val Pro Gly L Val Gly G Pro G

20 25 25 30Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro

35 40 45Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser

50 55 60(2) Sequence 30 information:

(i) Sequential features:

(A) Length: 936 AA

(B) Type: AA

(C) Chain type: single

(D) Topology: linear

(ii) Molecule type: protein

(XI) Sequence description: Sequence 30MET ASP Pro Val Val Leu Gln ARG ARG ARP TRP GLU Asn Pro Gly Vall 5 10 15thr Gln Leu ARA Ala His Pro PHE Ala Phe Pro Pro

20 25 25 30Met Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Val Gly

35 40 45Val Pro Leu Gly Pro Leu Gly Pro Gly Val Gly Val Pro Gly Val Gly

50 55 60VAL Pro GLY VAL VAL PRO GLY LYS GLY VAL VAL VAL VAL VAL65 70 75 80pro Gly Val Val Val Val Val Val Ala Gly Ala Gly Gly

85 90 95Ser Gly Ala Gly Ala Gly Ser Gly Val Gly Val Pro Leu Gly Pro Leu

100 105 110Gly Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val

115 120 125Pro Gly Lys Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro

130 135 140Gly Val Gly Val Pro Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly145 150 155 160SER GLY VAL VAL PRO Leu Gly Pro GLY Val Val Val Val Val

165 170 175Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Lys Gly Val Pro

180 185 190Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly

195 200 205Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Val Gly Val Pro

210                 215                 220Leu Gly Pro Leu Gly Pro Gly Val Gly Val Pro Gly Val Gly Val Pro225                 230                 235                 240Gly Val Gly Val Pro Gly Lys Gly Val Pro Gly Val Gly Val Pro Gly

245 250 255Val Gly Val Pro Gly Val Gly Val Pro Gly Ala Gly Ala Gly Ser Gly

260 265 270Ala Gly Ala Gly Ser Gly Val Gly Val Pro Leu Gly Pro Leu Gly Pro

275 280 285Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly

290 295 300lys Gly Val Pro Gly Val Gly Val Pro Gly Val Val Val Val 305 315 320Gly Val Pro Gly Ala Gly Serra Gly Ala GLY Ser Gly

325 330 335Val Gly Val Pro Leu Gly Pro Leu Gly Pro Gly Val Gly Val Pro Gly

340 345 350Val Gly Val Pro Gly Val Gly Val Pro Gly Lys Gly Val Pro Gly Val

355 360 365Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Ala Gly

370 375 380ALA GLY Ala Gly Ala Gly Val Val Gly Val Prou Gly385 395 400PRO Leu Gly Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val

405 410 415Gly Val Pro Gly Lys Gly Val Pro Gly Val Gly Val Pro Gly Val Gly

420 425 430Val Pro Gly Val Gly Val Pro Gly Ala Gly Ala Gly Ser Gly Ala Gly

435 440 445Ala Gly Ser Gly Val Gly Val Pro Leu Gly Pro Leu Gly Pro Gly Val

450 455 460Gly Val Pro Gly Val Gly Val Pro Gly Val Val Val Pro Gly Lys Gly465 475 480VAL PRO GLY VAL VAL VAL VAL VAL VAL GLY VAL VAL

485 490 495Pro Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Val Gly

500 505 510Val Pro Leu Gly Pro Leu Gly Pro Gly Val Gly Val Pro Gly Val Gly

515 520 525Val Pro Gly Val Gly Val Pro Gly Lys Gly Val Pro Gly Val Gly Val

530 535 540pro GLY VAL VAL VAL Pro Gly Val Gly Val Pro Gly Ala Gly Ala Gly545 550 560SER GLE GLY ALA GLY VAL VAL VAL PRLY Pro Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu Leu

565 570 575Gly Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val

580 585 590Pro Gly Lys Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro

595 600 605Gly Val Gly Val Pro Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly

610 615 620SER GLE VAL VAL VAL PRO Leu Gly Pro Leu Gly Pro Gly Val Val 625 635 640pro Gly Val Val Val Val Val Lys Gly Val Val Val Val Val Val Val Val Val Pro

645 650 655Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly

660 665 670Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Val Gly Val Pro

675 680 Leu Gly Pro Leu Gly Pro Gly Val Gly Val Pro Gly Val Gly Val Pro

690 695 700Gly Val Gly Val Pro GLY LYS GLY VAL VAL VAL VAL VAL VAL PRO GLY705 715 720VAL VAL VAL VAL VAL VAL VAL VAL GLY ALA GLY Ser Gly

725 730 735Ala Gly Ala Gly Ser Gly Val Gly Val Pro Leu Gly Pro Leu Gly Pro

740 745 750Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly

755 760 765Lys Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val

770 775 780Gly Val Pro Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly 785 795 800Val Gly Val Pro Uu Gly Pro GLY Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Pro Gly

805 810 815Val Gly Val Pro Gly Val Gly Val Pro Gly Lys Gly Val Pro Gly Val

820 825 830Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Ala Gly

835 840 845Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Val Gly Val Pro Leu Gly

850 860pro Leu Gly Pro Gly Val Val Val Pro Gly Val Val Val Val865 875 880gly Val Pro GLY Val Val Val Val Val Val Val Gly

885 890 895Val pro Gly Val Gly Val Pro Gly Ala Gly Ala Gly Ser Gly Ala Gly

900 905 910Ala Gly Ser Gly Ala Gly Ala Met Asp Pro Gly Arg Tyr Gln Asp Leu

915 920 925Arg Ser His His His His His His His

930 935(2) sequence 31 information:

(i) Sequential features:

(A) Length: 147bp

(B) type: nucleic acid

(C) Chain type: single

(D) Topology: linear

(ii) Molecular type: other nucleic acids

(A) Description: /desc="Synthetic"

(xi)序列描述：序列31ATGGCAGCGA AAGGGGACCG CCGGTGCGGG CTCTGGTGTT GGAGTGCCAG GCTTCTTTGT                 60ACGTGCACGC CGTGGTGTCG GTGTTCCGGG TGTAGGCGTT CCGGGAGTTG GTGTACCTGG                 120AAAAGGTTTC CGCTAAAGTC CTGCCGT                                                     147(2)序列32资料：

(i) Sequential features:

(A) Length: 186bp

(B) type: nucleic acid

(C) Chain type: single

(D) Topology: linear

(ii) Molecular type: other nucleic acids

(A) Description: /desc="Synthetic"

(xi)序列描述：序列32GGGCTCTGGT GTTGGAGTGC CAGGCTTCTT TGTACGTGCA CGCCGTGGTG TCGGTGTTCC                    60GGGTGTAGGC GTTCCGGGAG TTGGTGTACC TGGAAAAGGT GTTCCGGGGG TAGGTGTGCC                    120GGGCGTTGGA GTACCAGGTG TAGGCGTCCC GGGAGCGGGT GCTGGTAGCG GCGCAGGCGC                    180GGGCTC                                                                               186(2)序列33资料：

(i) Sequential features:

(A) Length: 62 AA

(B) Type: AA

(C) Chain type: single

(D) Topology: linear

(ii) Molecule type: peptide

(XI) Sequence description: Sequence 33Gly Ser Gly Val Gly Val Pro Gly PHE VAL ARG ALA ARG GLY1 5 10 15VAL GLY VAL VAL VAL VAL VAL VAL VAL VLE GLY LYS

20 25 25 30Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly

35 40 45Val Pro Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser

50 55 60(2) sequence 34 information:

(i) Sequential features:

(A) Length: 966AA

(B) Type: AA

(C) Chain type: single

(D) Topology: linear

(ii) Molecule type: protein

(XI) Sequence description: Sequence 34MET ASP Pro Val Val Leu Gln ARG ARG ASP TRP GLU Asn Pro Gly Val1 5 10 15thr Gln Leu ARA Ala His Pro PHE Ala Phe Pro Pro

20 25 25 30Met Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Val Gly

35 40 45Val Pro Gly Phe Phe Val Arg Ala Arg Arg Gly Val Gly Val Pro Gly

50 55 60VAL VAL VAL VAL VAL VAL VAL PRO GLY LYS GLY VAL VAL VAL65 70 75 80GLY VAL VAL VAL VAL VAL VAL VAL VAL VLA GLY ALA GLY.

85 90 95Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Val Gly Val Pro Gly Phe

100 105 110Phe Val Arg Ala Arg Arg Gly Val Gly Val Pro Gly Val Gly Val Pro

115 120 125Gly Val Gly Val Pro Gly Lys Gly Val Pro Gly Val Gly Val Pro Gly

130 135 140VAL GLY VAL Pro Gly Val Gly Val Pro Gly Ala Gly Aka GLY Serge145 150 155 160ALA GLY Ala Gly Val Val Phe Phe Phe Val Ala Ala Ala Ala Ala Ala Ala Ala Ala

165 170 175Arg Arg Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val

180 185 190Pro Gly Lys Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro

195 200 205Gly Val Gly Val Pro Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly

210 215 220SER GLE VAL VAL VAL Pro GLY PHE PHE VAL ARG ARG ARG GLY VAL 2225 235 240GLY VAL VAL VAL VAL VAL VAL VLS GLS GLS GLE

245 250 255Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val

260 265 270Pro Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Val Gly

275 280 285Val Pro Gly Phe Phe Val Arg Ala Arg Arg Gly Val Gly Val Pro Gly

290 295 300Val Gly Val Pro Gly Val Gly Val Pro Gly Lys Gly Val Pro Gly Val 305 315 320Gly Val Val Val Val Val Val Val Ala Gly Val Ala Gly Gly

325 330 335Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Val Gly Val Pro Gly Phe

340 345 350Phe Val Arg Ala Arg Arg Gly Val Gly Val Pro Gly Val Gly Val Pro

355 360 365Gly Val Gly Val Pro Gly Lys Gly Val Pro Gly Val Gly Val Pro Gly

370 375 380Val Gly Val Pro Gly Val Gly Val Pro Gly Ala Gly Ala Gly Ser Gly 385 395 400ALA GLE Ala Gly Val Val Phe Phe Phe Val Alg Ala

405 410 415Arg Arg Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val

420 425 430Pro Gly Lys Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro

435 440 445Gly Val Gly Val Pro Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly

450 455 460r Gly Val Gly Val Pro Gly PHE PHE VAL ARG ARG ARG GLY VAL465 475 480GLY VAL VAL VAL VAL VAL VAL VAL VAL LYS GLS GLE

485 490 495Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val

500 505 510Pro Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Val Gly

515 520 525Val Pro Gly Phe Phe Val Arg Ala Arg Arg Gly Val Gly Val Pro Gly

530 535 540Val Gly Val Val Val Val Val Pro Gly Lys Gly Val Val545 550 560Gly Val Val Val Val Val Val Val Ala Gly

565 570 575Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Val Gly Val Pro Gly Phe

580 585 590Phe Val Arg Ala Arg Arg Gly Val Gly Val Pro Gly Val Gly Val Pro

595 600 605Gly Val Gly Val Pro Gly Lys Gly Val Pro Gly Val Gly Val Pro Gly

610 615 620VAL GLY VAL VAL VAL VAL VAL PRO GLY Ala Gly Ala Gly Sergly625 635 640ALA GLY ALA GLY VAL VAL VAL PHE VAL ARG ALA

645 650 655Arg Arg Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val

660 665 670Pro Gly Lys Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro

675 680 685Gly Val Gly Val Pro Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly

690 695 700ser Gly Val Gly Val Pro Gly PHE PHE VAL ARG ARG ARG GLE Val705 715 720gly Val Val Val Val Val Val Val Val Val Lys Gry

725 730 735Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val

740 745 750Pro Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Val Gly

755 760 765Val Pro Gly Phe Phe Val Arg Ala Arg Arg Gly Val Gly Val Pro Gly

770 775 780VAL GLY VAL VAL VAL VAL VAL PRO GLY LYS GLY VAL VAL785 795 800GLY VAL VAL VAL VAL VAL VAL VAL ALA GLY Ala Gly

805 810 815Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Val Gly Val Pro Gly Phe

820 825 830Phe Val Arg Ala Arg Arg Gly Val Gly Val Pro Gly Val Gly Val Pro

835 840 845Gly Val Gly Val Pro Gly Lye Gly Val Pro Gly Val Gly Val Pro Gly

850 855 860Val Gly Val Pro Gly Val Gly Val Pro Gly Ala Gly Ala Gly Ser Gly865 870 0 5 8 8 7

Ala Gly Ala Gly Ser Gly Val Gly Val Pro Gly Phe Phe Val Arg Ala

885 890 895Arg Arg Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val

900 905 910Pro Gly Lys Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro

915 920 925Gly Val Gly Val Pro Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly

930 935 940ser Gly Ala Gly Ala Met ASP Pro Gly ARG TYR GLN ASP Leu ARG Ser945 955 960HIS HIS HIS HIS HIS

     965(2) Sequence 35 data:

(i) Sequential features:

(A) Length: 11AA

(B) Type: AA

(C) Chain type: single

(D) Topology: linear

(ii) Molecule type: peptide

(xi) Sequence description: Sequence 35 Arg Gly Arg Gly Arg Gly Lys Gly Lys Gly Lys1 5 10

Claims (28)

1. method of keeping isolating living tissue proximity relations, this method comprises:

When this isolating tissue is contact relation, using a kind of precursor composition sentences to this separation living tissue it is combined, this precursor composition comprises polyfunctional crosslinking agent and protein polymer, and this polymer contains 3 repetitives to fifteen amino acid of the structural protein that derives from least a natural generation of at least 40% percentage by weight, in this protein polymer, have two heteroatom functional groups at least, and this heteroatom functional group and the reaction of this cross-linking agent, thereby this precursor composition can generate the binding compositions with intensive bond properties;

Kept proximity relations and should separate living tissue.

2. the method for claim 1, wherein said heteroatom functional group is amino.

3. the method for claim 1, wherein said heteroatom functional group is that aminoacid and polyfunctional compound's reaction of the natural generation in this protein polymer but do not form said composition results.

4. the method for claim 1, wherein said unit is selected from GAGAGS (sequence 01), GVGVP (sequence 02) and GXX, the X ' s in the formula be identical or different and can be any aminoacid, but have at least 10% and the aminoacid that is no more than 60% quantity be proline.

5. method as claimed in claim 4, wherein said unit are GAGAGS (sequence 01) and GVGVP (sequence 02) and form a kind of block copolymerization albumen.

6. the method for claim 1, wherein said application comprise a kind of energy and the difunctional compound that should polyfunctional cross-linking compounds reacts.

7. method as claimed in claim 6, wherein said difunctional compound are lysine or arginine.

8. method of keeping isolating living tissue proximity relations, it comprises:

When this isolating tissue is proximity relations, using a kind of precursor composition sentences to this separation living tissue it is combined, this precursor composition comprises multifunctional cross-linking agent and a kind of repetitive GAGAGS (sequence 01) of 70% weight and protein block copolymer of GVGVP (sequence 02) of containing at least of a kind of energy and amino reaction, wherein in two unit, there is a seed amino acid to be contained at least one lysine at least or arginic insertion group replaces by at least one lysine or arginine or at least two, thereby this precursor composition can be used to form the binding compositions with intensive bond properties;

And this isolating living tissue remains on proximity relations.

9. method as claimed in claim 8, wherein, said proteinic each lysine and arginic equivalent arrive in the scope of 40KD between 1.

10. method as claimed in claim 8, wherein, each block of said block copolymer have at least two unit and GAGAS (sequence 01) to the ratio of GVGVP (sequence 02) between 1: in the scope of 1-16.

11. method as claimed in claim 8, wherein, said cross-linking agent has multiple amino reactive group, and this group is selected from aldehyde radical, isocyano, isothiocyanic acid root and activatory carboxyl.

12. method as claimed in claim 11, wherein, said reactive group is an isocyano.

13. method as claimed in claim 8 wherein, comprises lysine or arginine at least in the said application.

14. a method of keeping isolating living tissue proximity relations, this method comprises:

When this isolating tissue is proximity relations, using a kind of precursor composition sentences to this isolating living tissue it is combined, this precursor composition comprises the multifunctional cross-linking agent and the protein block copolymer that is at least 30KD that are selected from glutaraldehyde and polymethylene vulcabond, this copolymer contains the GAGAGS (sequence 01) of 70% percentage by weight and the repetitive of GVGVP (sequence 02) at least, wherein in two unit, there is a seed amino acid to be replaced at least by at least one lysine or arginine, this copolymer has lysine and the arginine of equivalent between the 3-15KD scope, and this precursor composition is the binding compositions that has intensive bond properties for formation;

And this isolating living tissue keeps proximity relations.

15. one kind seals method damaged in the living tissue, this method comprises:

Use a kind of precursor composition at this fault location, it comprises multifunctional cross-linking agent and protein polymer, and this polymer contains 3-15 amino acid whose repetitive of the structural protein that derive from least a natural generation of at least 40% percentage by weight, wherein having an aminoacid to be contained the insertion groups that amino acid whose heteroatom functional is rolled into a ball or at least two contain heteroatom functional group at least in two unit replaces, thereby this precursor composition can be used for forming the binding compositions with intensive bond properties;

Thereby seal this defective.

16. method as claimed in claim 15, wherein, said unit is selected from GAGAGS (sequence 01), GVGVP (sequence 02) and GXX, X ' s in the formula is identical or different and X can be any aminoacid, but have at least 10% and the X ＇ s that is no more than 60% quantity be proline.

17. method as claimed in claim 15, wherein, said unit is GAGAGS (sequence 01) and GVGVP (sequence 02) and exists to form the proteic form of block polymer.

18. method as claimed in claim 15, wherein, said tissue is to be selected from tremulous pulse, vein, hair and blood blood vessel, lung, the blood vessel wall of cerebral dura mater or rectum.

19. test kit that contains polyfunctional cross-linking agent and protein polymer, this polymer contains the amino acid whose repetitive of 3-15 of the structural protein that derives from least a natural generation of 40% percentage by weight at least, wherein in two unit, there is a seed amino acid to be comprised that amino acid whose heteroatom functional group replaces at least, and this heteroatom functional group and the reaction of this cross-linking agent.

20. test kit as claimed in claim 19, wherein, said heteroatom functional group is amino.

21. test kit as claimed in claim 19, wherein, said heteroatom functional group is the aminoacid of the natural generation in this protein polymer and a kind of polyfunctional chemical compound reaction and do not form said composition results.

22. test kit as claimed in claim 19, wherein, said unit is selected from GAGAGS (sequence 01), GVGVP (sequence 02) and GXX, X ' s in the formula is same or different and X is any aminoacid, but have at least 10% and X ' s of being no more than 60% quantity be proline.

23. test kit as claimed in claim 22, wherein, said unit is GAGAGS (sequence 01) and GVGVP (sequence 02) and exists to form the proteinic form of block polymer.

24. test kit as claimed in claim 19, wherein, said cross-linking agent has the multiple aldehyde radical that is selected from, isocyano, the reactive group of isothiocyanic acid root and activatory carboxyl.

25. test kit that comprises the protein block copolymer that is at least 30KD, this copolymer contains the GAGAGS (sequence 01) of 70% percentage by weight and the repetitive of GVGVP (sequence 02) at least, wherein have a seed amino acid to be replaced by at least a lysine or arginine in two unit at least, this copolymer has equivalent lysine or arginine and be selected from glutaraldehyde and the cross-linking agent of aliphatic vulcabond in the scope of 1-40KD.

26. crosslinked protein compositions, the feature of this protein before crosslinked is 3 to 15 amino acid whose repetitives that contain the structural protein that derives from least a natural generation of 40% percentage by weight at least, wherein in two unit, there is a seed amino acid to be comprised that amino acid whose heteroatom functional group replaces at least, and this heteroatom functional group energy and at least one aldehyde radical, isocyano, isothiocyanic acid root and activatory carboxyl reaction.

27. cross-linked proteins compositions as claimed in claim 26, wherein, said proteinic feature also comprises the GAGAGS (sequence 01) of at least 70% percentage by weight and the repetitive of GVGVP (sequence 02), wherein have a seed amino acid to be replaced by at least one lysine or arginine in two unit at least, this copolymer has lysine and the arginine of equivalent between 1-20KD.

28. a method of keeping isolating living tissue proximity relations, this method comprises:

At this isolating tissue is when being in contact condition, using a kind of precursor composition sentences to this isolating living tissue it is combined, what this precursor composition comprised the reaction of (1) a kind of energy and dual functional chemical compound comprises glycine 2-amino ethyl ester, lysine cholinester or glycine 1, the multifunctional cross-linking agent of 3-propylene diester, and this difunctional compound is to react with amino, and the protein polymer of 3 to 15 amino acid whose repetitives of (2) a kind of structural protein that derives from least a natural generation that contains 40% percentage by weight at least, in this protein polymer, contain two amido functional groups at least, and amido functional group can react with this cross-linking agent, thereby this precursor composition is used to form the compositions with intensive bond properties;

And this isolating living tissue keeps in touch relation.