KR100711143B1 - Recombinant Polypeptide for Immunocastration and Vaccine Comprising the Same - Google Patents

Abstract

The present invention relates to a recombinant polypeptide, and more particularly, to a recombinant polypeptide capable of inducing an immune response against gonadotropin-releasing hormone (GnRH) in animals and an immuno castration method using the same.

Immune castration, gonadotropin releasing hormone, Grimsby virus, VP6, CDV

Description

Recombinant Polypeptide for Immunocastration and Vaccine Comprising the Same}

The present invention relates to a recombinant polypeptide, and more particularly, to a recombinant polypeptide capable of inducing an immune response against gonadotropin-releasing hormone (GnRH) in animals and an immuno castration method using the same.

Immature castration has been successfully performed in poultry, allowing pets to solve problems related to surgical castration. The inventors have invented a vaccine that regulates higher hormones for the castration of pets and inhibits the development of the testes.

About 20 years ago, immunocastration on various types of poultry has been proposed as an alternative to economic castration and surgical castration of hydrogen and boars in terms of prevention of animal abuse (Oonk et al., 1998; Ladd. 1993). ), Little attention was paid to pets. However, even in pets, surgical castration has been performed continuously for the following purposes. Pet castration, for example, can help reduce the roaming, digging, sexual and violent behavior caused by sexual impulses and prevents testicular cancer, prostatic cancer and rectal cancers. The purpose is to prevent excessive population growth of pets. Surgical castration is a simple and easy method, but the risk of systemic anesthesia and animal abuse are controversial.

Immunocastration has been developed as a method to solve the problems of the traditional surgical castration technique described above. Australia, meanwhile, became the first country to perform immunocastration instead of surgically castration ( Pig progress , 16 (3): 8 (2000)).

Most immunocastration methods are carried out using a vaccine capable of inducing an immune response against GonRadotropin Releasing Hormone (GnRH).

WO 92/19746 discloses a method for immunocastration of animals using recombinant polypeptides comprising GnRH or analogs thereof and T-cell epitopes, WO 02 / 22659 discloses A peptide that comprises a modified GnRH decapeptide sequence which allows for a an immunogenic response that allows for discrimination between different types of GnRH, preferably between GnRH-I and GnRH-II, and a method of immunocasting in pigs using the same . On the other hand, USP 5837268 discloses a method for improving immunogenicity against GnRH using chimeric proteins including leukotoxin and GnRH polypeptide.

However, there is still a need for a vaccine that, when administered to animals as a vaccine against GnRH, can induce a stronger immune response and achieve a castration effect.

Throughout this specification many patent documents and articles are referenced and their citations are indicated. The disclosures of cited patent documents and articles are incorporated herein by reference in their entirety, so that the level of the technical field to which the present invention belongs and the contents of the present invention are more clearly explained.

The present inventors have made intensive research efforts to improve immune castration vaccines. As a result, VP6 (Redmond et al., 1990) of Grimsby virus (GRV) (Lee et al., 2003) and CDV (canine distemper) Improved immunity when using P35) (Ghosh et al., 2001) in the P35 region, which has been reported as a potential T-cell epitope of the virus, as a carrier for GnRH, and with appropriate modification of the peptide derived from GnRH The present invention has been completed by confirming that immunocastration vaccines can be produced at relatively low cost while having immunogenicity.

Accordingly, it is an object of the present invention to provide a recombinant polypeptide capable of inducing an immune response to GnRH in an animal.                         

Another object of the present invention to provide a vaccine composition for immuno castration comprising the recombinant polypeptide described above.

Another object of the present invention to provide an immunocastration method using the vaccine composition for immunocastration described above.

According to one aspect of the present invention, the present invention provides a pharmaceutical composition comprising: (a) a peptide derived from gonadotropin-releasing hormone (GnRH) or a multimer of the peptide; And (b) VP6 protein of Grimsby virus (hereafter GRV) or P35 of canine measles virus (hereafter CDV), and provides a recombinant polypeptide capable of inducing an immune response to GnRH in an animal.

The present inventors have studied to develop a peptide that can effectively induce an immune response to GnRH in various mammals, and confirmed that the peptide or a variant thereof performs this function in a certain sequence obtained from the sequences of various GnRH.

In the recombinant polypeptides of the invention, such fusion forms of GnRH-derived peptides or multimers of these peptides with VP6 of GRV or P35 of CDV can be constructed by various methods known in the art.

According to a preferred embodiment of the present invention, the peptide used in the present invention has the amino acid sequence shown in SEQ ID NO: 1. According to another variant of the invention, the peptide of the present invention has a sequence of point mutations of 1-3 amino acid sequences of the amino acid sequence shown in SEQ ID NO: 1, wherein the point mutation is a peptide of SEQ ID NO: 1 It is intended to induce higher immunogenicity for GnRH.

Preferred examples of the mutated amino acid sequence represented by SEQ ID NO: 1 of the peptides of the present invention are peptides having the amino acid sequence represented by SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4.

The recombinant polypeptide of the present invention preferably has two or more copies rather than one copy of the above-described amino acid sequence, in which case it has one or more of the peptides described above. It is more preferable. With such multiple copies, the immune reaction to GnRH that is triggered is greater. Such multimer-typed polypeptide preferably comprises one or more peptides selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4. More preferably two or more peptides. According to a most preferred embodiment of the present invention, the multimer of the peptide comprises an amino acid sequence represented by SEQ ID NO: 6.

The VP6 protein of GRV or P35 of CDV included in the recombinant polypeptide of the present invention serves as a carrier.

In the recombinant polypeptide of the present invention, the GnRH-derived peptide and the VP6 protein may be linked in the order of VP6-GnRH and P35-GnRH or in the order of GnRH-VP6 and GnRH-P35, preferably in the order of VP6-GnRH and P35-GnRH It is connected to.

As the carrier protein of the present invention, both VP6 and P35 may be used, but in the dog vaccinated against CDV, it is preferable to use P35 as a carrier rather than VP6.

The recombinant polypeptide of the present invention has an advantage of effectively inducing an immune response to GnRH in a challenged host, and has an advantage of being able to be manufactured at low cost.

According to another aspect of the present invention, the present invention is (a) the recombinant polypeptide described above; And (b) a pharmaceutically acceptable carrier.

Since the vaccine composition of the present invention includes the above-described recombinant polypeptide of the present invention as an active ingredient, the content common to the above-described recombinant polypeptide is omitted in order to avoid the complexity due to excessive duplication of the present specification.

Pharmaceutically acceptable carriers included in the pharmaceutical compositions of the present invention are those commonly used in the preparation, such as lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, Calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, and the like It doesn't happen. The pharmaceutical composition of the present invention may further include lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives and the like in addition to the above components.

The pharmaceutical composition of the present invention can be administered orally or parenterally, and in the case of parenteral administration, it can be administered by intravenous injection, subcutaneous injection, intramuscular injection, etc., preferably by intramuscular injection.

Suitable dosages of the pharmaceutical compositions of the invention vary depending on factors such as the formulation method, mode of administration, age, weight, sex, morbidity, condition of food, time of administration, route of administration, rate of excretion and response to reaction, Usually a skilled practitioner can easily determine and prescribe a dosage effective for the desired treatment or prophylaxis.

The pharmaceutical compositions of the present invention may be prepared in unit dose form by formulating with a pharmaceutically acceptable carrier and / or excipient according to methods which can be easily carried out by those skilled in the art. Or may be prepared by incorporating into a multi-dose container. In this case, the formulation may be in the form of a solution, suspension or emulsion in an oil or an aqueous medium, or may be in the form of extracts, powders, granules, tablets or capsules, and may further include a dispersant or stabilizer.

The vaccine composition of the present invention preferably further comprises an adjuvant. Examples of adjuvants include aluminum hydroxide, Freud's complete or incomplete adjuvant, DEAE dextran, levamisol, PCG and poly I: C or poly A: U.

The invention of the present invention may induce a humoral or optionally cell-mediated immune response and / or a combination of the two immune responses.

Various embodiments of the vaccine compositions of the present invention may be referred to Remington's Pharmaceutical Science , 18th ed., Mach Publishing, Easton, Pennsylvania, USA.

According to another aspect of the present invention, the present invention provides a method for immune castration comprising administering to the mammal the vaccine composition of the present invention described above.

The method of the present invention can be applied to any mammal, for example, but not limited to cattle, pigs, dogs, cats, goats, sheep, horses, rabbits or mice.

In the method of the present invention, administration is preferably performed two or more times. For example, booster injections are performed 1-4 times at 1-10 week intervals after initial vaccination.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it is to those of ordinary skill in the art that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention. Will be self-evident.

Example 1

In order to examine the conserved regions of GnRH, we compared the conserved regions of GnRH of previously known animals to select the peptide with the highest similarity ( QHWSYGLRPG ) and based on them. The base sequence of the GnRH peptide for use in the invention was determined. The designed peptides were designed such that the conserved sequence consisted of GnRH-like peptides repeated six times. In order to introduce the synthetic DNA into the plasmid vector, the Sph I site was inserted into the restriction site. In order to increase the antigenicity of the GnRH-like peptide, the base sequence of the GnRH was changed by an oint mutation.

Example 2

After selecting a protein having better adjuvanticity than leukotoxin of Hemophilus somnus , the GnRH synthesized in Example 1 can be combined to provide the most preferable vaccine protein of the present invention. A vector system was prepared. In general, ovalbumin is known to have a good immune adjuvant effect, but according to the researches of the present inventors, since rotavirus VP6 protein also has an immune adjuvant effect corresponding to ovalbumin, VP6 protein may be substituted for GnRH peptide. Used as a carrier. Also, in dogs vaccinated with the CDV vaccine, the P35 protein of CDV was more than doubled as a carrier compared to VP6, and in dogs vaccinated with measles vaccine, the effect was increased. Even if a castration vaccine is applied, the castration vaccine has a low effect and is safe.

Example 2-1 :

First, GR virus and CD virus, which were separated and stored in Konkuk University's veterinary microbiology department, were obtained and subjected to RT-PCR using the virus as a template to amplify CD35 P35 under the following conditions. For PCR of P35, the primers include a forward primer having a base sequence of 5'-GAATTCACTGCTGCTCAG-3 '(18-mer) and a base sequence of 5'-GTCGACTCAGAGCTCATT-3' (18-mer). Reverse primer having a was used. PCR conditions were denatured step (denaturation step): 95 ℃, 40 seconds; Annealing step: 48 ° C., 40 seconds; And extension step: the reaction was completed by performing 35 cycles of these three steps at 72 ° C. and 70 sec. The amplified PCR product was digested with EcoRI and SalI, and the pGEX-4T1 vector (Pharmacia), which was digested with the same restriction enzymes, was mixed at an appropriate ratio, followed by ligation buffer (Roche Diagnostics GmbH) and T4 DNA ligue. Ligase (Roche Diagnostics GmbH) was added to react for 1 hour at room temperature. Subsequently, the reaction was transformed into E. coli DH5a (Promega) treated with CaCl2, and then cultured in LB medium containing ampicillin (100 mg / ml), and then strains having ampicillin resistance were selected and each plasmid DNA was isolated. The plasmid in which P35 was inserted was isolated by using the method of Birnboim & Doly, (1979) and analyzed by restriction enzyme analysis. The plasmid thus separated and identified was named "pGSTP35".

Example 2-2 :

The pGSTP35 prepared in Example 2-1 was digested with SacI and SalI, and the GnRH-like gene of Example 1 was digested with restriction enzymes SacI and SalI and mixed at an appropriate ratio. The mixture was ligated in the same manner as in Example 2-1, and then transformed into E. coli DH5a. Subsequently, the cells were cultured in LB medium containing ampicillin (100 mg / ml), and then strains having ampicillin resistance were selected and plasmid DNA was isolated. Plasmids incorporating GnRH-like peptides and P35 were isolated using the method of Birnboim & Doly, (1979) and confirmed using restriction enzyme analysis. In pGSTP35GnRH, GnRH-like genes are linked in-frame behind the P35 gene.

Example 3

Plasmids were extracted from E. coli DH5a transformants of pGSTVP6GnRH and pGSTP35GnRH cloned in Example 2-2. Each of the extracted plasmids was transformed into E. coli BL21 (DE3) pLysS, and then cultured in LB medium containing ampicillin (100 mg / ml), and then, strains having ampicillin resistance were selected and plasmid DNA was isolated. And the presence of the corresponding plasmid was confirmed.

E. coli BL21 (DE3) pLysS transformants for each plasmid were seeded in LB liquid medium containing 100 mg / ml final concentration of ampicillin and seeded at 37 ° C. for 16 hours. Seed cultures were inoculated to 1% in 100 ml LB liquid medium containing ampicillin (final concentration 100 mg / ml) and incubated at 37 ° C. for 1.5 hours, followed by addition of IPTG (final concentration 0.5 mM) and 3- Expression was induced for 4 hours. After the incubation was terminated, the cells were recovered by centrifugation at 4500 x g for 40 minutes. In order to identify the protein expressed primarily, the cells recovered were ultrasonically crushed and 0.8% polyacrylamide gel electrophoresis was used to confirm the expression of proteins corresponding to each plasmid construct as shown in FIG. 7.

Furthermore, the protein was separated from the recovered E. coli in the following manner: 3.5 ml of suspension solution (100 mM NaCl; 50 mM / Tris / HCl, pH 8.0; 1 mM EDTA, pH 8.0) was added to the cell precipitate. After preparing the cell suspension, the suspended cells were disrupted for 20 minutes by repeated pulses of 9 seconds and pauses of 9 seconds using an ultrasonic crusher. The lysate was centrifuged (17,000 × g, 4 ° C., 40 minutes) to discard the supernatant, and 3 ml Lysis solution (1% lysozyme, 3% Triton X-100) was added to the obtained pellet. The solution was well suspended and crushed again in the same manner as the previous example, followed by incubation for 30 minutes with gentle mixing at room temperature. Inclusion bodies were separated by centrifugation (17,000 × g, 4 ° C., 40 minutes), and homogenized by addition of 1 ml osmotic shock solution (suspension solution containing 8M urea). The homogenized protein solution was carefully stirred at 4 ° C. for 1 hour and then centrifuged (17,000 × g, 4 ° C., 40 minutes) to recover the supernatant. Dialysis (4 ° C., 24 hours) was performed using phosphate buffer to remove urea and the like present in the supernatant.

Example 4

The efficacy of the castration vaccine was examined using the proteins of pGSTVP6GnRH and pGSTP35GnRH obtained in Example 3.                     

Freund's adjuvant was used as an adjuvant. CFA (complete Freund's adjuvant) was used for initial immunization and Incomplete Freund's adjuvant (IFA) was used for 2, 3 and 4th immunization. The same amount of the protein and the immunoadjuvant (adjuvant) was emulsified using a tuberculin syringe and inoculated by taking the required amount. Inoculation was carried out by intramuscular injection (IM) method and the doses were 50 μg, 100 μg and 200 μg. Therefore, the experimental group was composed of 9 groups), and each group was composed of 2 rats. The experimental animals were WD rats and were acclimated for 1 week after being imported at 3 weeks of age.

Immunization was initiated at 4 weeks of age and inoculated 4 times at 2 week intervals, and 1 week after the last inoculation, the reproductive organs were examined.

Significant castration vaccine effects have been shown in immunoassays using recombinant proteins isolated from Escherichia coli, including the pGSTVP6GnRH construct.

Among the test groups, the testicular and epididymal contraction effects were reduced in testes and epididymis in proportion to the increase in the use of pGSTVP6GnRH recombinant protein 100 μg, 200 μg and 400 μg. It was observed that the same recombinant protein inhibits the production of prostate and seminal vesicles.

Having described the specific part of the present invention in detail, it is apparent to those skilled in the art that the specific technology is merely a preferred embodiment, and the scope of the present invention is not limited thereto. Thus, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

As described in detail above, the present invention provides a recombinant polypeptide capable of inducing an immune response to GnRH in an animal, and an immuno castration method using the same.

<110> Nexgen Biotechnologies, Inc. <120> Recombinant Polypeptide for Immunocastration and Vaccine          Comprising the Same <130> P <160> 5 <170> KopatentIn 1.71 <210> 1 <211> 10 <212> PRT <213> peptide derived from gonadotropin releasing hormone in a variety of animals <400> 1 Gln His Trp Ser Tyr Gly Leu Arg Pro Gly   1 5 10 <210> 2 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> peptide with point-mutated amino acids in peptide of SEQ ID NO: 1 <400> 2 Gln His Trp Ser Gly Gly Leu Arg Pro Gly   1 5 10 <210> 3 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> peptide with point-mutated amino acids in peptide of SEQ ID NO: 1 <400> 3 Glu His Trp Ser Tyr Gly Leu Arg Pro Gly   1 5 10 <210> 4 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> peptide with point-mutated amino acids in peptide of SEQ ID NO: 1 <400> 4 Glu Asp Trp Ser Tyr Gly Leu Arg Pro Gly   1 5 10 <210> 5 <211> 68 <212> PRT <213> Artificial Sequence <220> <223> mutimer of GnRH-like peptide <400> 5 Gln His Trp Ser Gly Gly Leu Arg Pro Gly Gly Ser Ser Glu His Trp   1 5 10 15 Ser Tyr Gly Leu Arg Pro Gly Ser Gly Gln His Trp Ser Tyr Gly Leu              20 25 30 Arg Pro Gly Gly Gly Ser Glu Asp Trp Ser Tyr Gly Leu Arg Pro Gly          35 40 45 Ser Gln His Trp Ser Tyr Gly Leu Gln His Trp Ser Tyr Gly Leu Arg      50 55 60 Pro Gly Met Leu  65

Claims (12)

(a) SEQ ID NO: 2, SEQ ID mutated to induce high immunogenicity against GnRH from the amino acid sequence of SEQ ID NO: 1 peptide derived from gonadotropin-releasing hormone (GnRH), SEQ ID NO: 1 peptide A peptide of NO: 3 or SEQ ID NO: 4 or a multimer of said peptide; And (b) VP6 protein of Grimsby virus (GRV) or P35 of dog measles virus (CDV), wherein the recombinant polypeptide is capable of inducing an immune response to gonadotropin-releasing hormone in animals. delete delete delete The method of claim 1, wherein the multimer of the peptide comprises at least one peptide selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4. Recombinant polypeptides. The method of claim 1, wherein the multimer of the peptide comprises at least two peptides selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4. Recombinant polypeptides. delete The recombinant polypeptide of claim 6, wherein the multimer of the peptide comprises an amino acid sequence represented by SEQ ID NO: 5. Vaccine compositions for immunocastration comprising: (a) the recombinant polypeptide of any one of claims 1, 5, 6 and 8; And (b) a pharmaceutically acceptable carrier. 10. The vaccine composition of claim 9, wherein the vaccine composition further comprises an adjuvant. The method of claim 9, comprising administering the vaccine composition of claim 9 to a mammal other than a human. 12. The method of claim 11, wherein the mammal is a cow, pig, dog, cat, goat, sheep, horse, rabbit or rat.