CN116162173A - GnRH6-CRM197 recombinant protein castration vaccine and preparation method thereof - Google Patents
GnRH6-CRM197 recombinant protein castration vaccine and preparation method thereof Download PDFInfo
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- A61P15/08—Drugs for genital or sexual disorders; Contraceptives for gonadal disorders or for enhancing fertility, e.g. inducers of ovulation or of spermatogenesis
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Abstract
The invention discloses a GnRH6-CRM197 recombinant protein castration vaccine and a preparation method thereof, wherein the mammal castration recombinant protein vaccine contains a GnRH recombinant protein antigen, and the amino acid sequence of the GnRH recombinant protein antigen is shown as a sequence table seq_1. The GnRH6-CRM197 recombinant protein castration vaccine contains a mammalian GnRH sequence, wherein the mammal comprises dogs, cats, pet pigs, holland pigs and mice. The mammalian castration recombinant protein vaccine contains a canine GnRH sequence of sequence table seq_2. The mammalian castration recombinant protein vaccine contains a goat CRM197 sequence of a sequence table seq_3. The invention discloses construction, transformation expression and purification of a recombinant GnRH6-CRM197 protein vaccine and acquisition of engineering bacteria BL21 (DE 3) -PET21 a-GnRH6-CRM197. After the vaccine is used for immunizing male animals, testes are atrophic, so that the purpose of controlling animal fertility is achieved.
Description
Technical Field
The invention relates to the field of molecular vaccinology, in particular to a GnRH6-CRM197 recombinant protein castration vaccine and a preparation method thereof.
Background
The traditional castration is a surgical castration method which has higher requirements on sites, environment, operation and the like, meanwhile, side effects are generated in the operation, such as a certain death caused by high infection rate, certain difficulty is brought to China with huge annual stock output of livestock, in addition, the operation cost is higher, animal welfare is not facilitated, the continuous thousand years of surgical castration is challenged by the requirements of large-scale and standardized livestock production, and researches show that the immune castration has the advantages of convenience, high efficiency, reversibility, safety and the like, so that the immune castration technology is most hopeful to become a substitute of the traditional castration.
Hypothalamic Gonadotropin-releasing hormone (GnRH) plays a decisive role in reproductive endocrine regulation, and its main biological function is to control synthesis and secretion of luteinizing hormone (1uteinizing hormone,LH) and follicle stimulating hormone (Fo 11ic1e-stim mu 1ating hormone,FSH) by anterior pituitary cells, i.e. the genital axis. Studies have shown that FSH and LH are closely related to reproductive activities such as oestrus, mating, etc., and testicular development, spermatogenesis, etc., in male animals, so that by immunizing GnRH, production of antibodies to GnRH is induced, in vivo neutralization of GnRH thereby reducing GnRH levels, resulting in reduced synthesis of LH and FSH, with concomitant disruption of oestrus, testicular development and spermatogenesis, eventually achieving the objective of sterilization. In the research aspect of GnRH immune castration vaccine, early chemical synthetic peptide vaccine is formed by connecting GnRH monomer and carrier protein, but the vaccine constructed by GnRH monomer is almost in all immunized animals, the immune effect is unstable, the immunogenicity is not strong, the reaction difference of the same animal species in the same batch of tests on the immunization of the vaccine is large, and some animals do not even react to the vaccine. Fang Fugui et al developed studies on the immunogenicity of GnRH hexamers and found that it was immunogenic and that it was necessary to select a carrier protein that enhanced the immunogenicity of GnRH in order to further enhance its active immune effects. The research proves that the diphtheria toxin mutant CRM197 can be used as carrier protein to enhance the immune effect, is a good carrier of weak antigen, and is connected with the diphtheria toxin mutant CRM197 to enhance the immunogenicity on the basis of GnRH hexamer. At present, a large number of reports about GnRH vaccines are made at home and abroad, but no commercial application is achieved, and the reasons are poor immunogenicity and high cost. Moreover, the research on the combined application of GnRH hexamer and CRM197 protein is not reported at present, so that a fusion protein vaccine of GnRH hexamer and CRM197 with strong immunogenicity and low cost is necessary to be developed.
Disclosure of Invention
The invention aims to provide a GnRH6-CRM197 recombinant protein castration vaccine and a preparation method thereof, which improve the immunogenicity of low molecular weight antigens, improve the immune effect of the antigens and avoid the unstable effect of polypeptides.
In one aspect of the invention, the invention provides a GnRH recombinant protein antigen. According to the embodiment of the invention, the amino acid sequence of the GnRH recombinant protein antigen is shown as a sequence table seq_1.
In another aspect of the invention, the invention provides a GnRH6-CRM197 recombinant protein castration vaccine. According to an embodiment of the present invention, the mammalian castration recombinant protein vaccine comprises the GnRH recombinant protein antigen.
In addition, the GnRH6-CRM197 recombinant protein castration vaccine according to the embodiment of the invention can also have the following additional technical characteristics:
in some embodiments of the invention, the GnRH6-CRM197 recombinant protein castration vaccine contains mammalian GnRH sequences, including canine, feline, pet porcine, netherlands porcine, mice.
In some embodiments of the invention, the mammalian castration recombinant protein vaccine comprises the canine GnRH sequence of seq_2 of the sequence listing.
In some embodiments of the invention, the mammalian castration recombinant protein vaccine contains the goat CRM197 sequence of sequence listing seq_3.
In some embodiments of the invention, the GnRH6-CRM197 recombinant protein castration vaccine comprises a GnRH6-CRM197 transformed e.coli expressed recombinant protein of sequence listing seq_1.
In another aspect of the invention, the invention provides a GnRH6-CRM197 for the transformation of E.coli. According to the embodiment of the invention, the GnRH6-CRM197 transformed E.coli is preserved in China general microbiological culture Collection center (CGMCC) at the date of 6 months and 19 of 2022, the preservation number is CGMCC No.25027, the classification is named as Escherichia coli, the specific name is BL21 (DE 3) -PET21a-GnRH6-CRM197, the preservation address is North Chen and West road No. 1 of the Korean area of Beijing city No. 3, the post code is 100101, and the GnRH6-CRM197 transformed E.coli expresses the recombinant protein containing the amino acid of the sequence table seq_1.
In another aspect of the invention, the invention provides a method for constructing a GnRH6-CRM197 transformed E.coli. According to an embodiment of the invention, the method comprises the following steps:
(1) Designing a recombinant CRM197 sequence comprising the selected GnRH hexamer based on the mammalian GnRH sequence and the goat CRM197 sequence;
(2) The DNA sequence with BamH I and Xho I cleavage sites is designed and synthesized by adopting a codon optimization system, and the DNA product after BamH I and Xho I double cleavage is connected with a carrier PET21a which is also digested, so as to obtain a connecting solution;
(3) And then transferring the connection solution into escherichia coli DH5 alpha, screening to obtain a plasmid PET21a-GnRH6-CRM197, transferring the plasmid GnRH6-CRM197 into escherichia coli BL21 (DE 3) strain to obtain engineering bacteria BL21 (DE 3) -PET21a-GnRH6-CRM197, and finally expressing recombinant protein containing amino acid of a sequence table seq_1, namely converting the GnRH6-CRM197 into escherichia coli.
In another aspect of the invention, the invention provides a method for preparing a GnRH6-CRM197 recombinant protein castration vaccine. According to the embodiment of the invention, the GnRH6-CRM197 converted escherichia coli is subjected to bacterial breaking purification to obtain the recombinant protein containing the amino acid of the sequence table seq_1, namely the GnRH6-CRM197 recombinant protein castration vaccine.
In another aspect of the invention, the invention provides the use of a GnRH6-CRM197 recombinant protein castration vaccine. According to an embodiment of the invention, the GnRH6-CRM197 recombinant protein castration vaccine is for use in mammalian fertility control.
Compared with the prior art, the invention has the beneficial effects that:
1) The invention adopts the recombination mode of GnRH hexamer and CRM197 protein, improves the immunogenicity of low molecular weight antigen and the immune effect of antigen, and avoids the unstable effect of polypeptide; meanwhile, the recombinant GnRH6-CRM197 protein is used, so that the cost of a polypeptide synthesis preparation is reduced, and the use is more convenient.
2) The BL21 (DE 3) -PET21a-GnRH6-CRM197 engineering bacteria prepared by the invention are different from other engineering bacteria in the preparation process, and the conditions of the engineering bacteria such as culture time, culture temperature, revolution, induced expression time and temperature, bacterial breaking, purification process and the like are special for the engineering bacteria, so that the engineering bacteria are only suitable for the preparation conditions of the engineering bacteria; the new engineering bacteria are developed, the preparation time is shorter, the preparation conditions are simpler, the preparation cost is lower, and the engineering bacteria are suitable for large-scale production, and have further development value and good industrialization prospect; the castration vaccine prepared by the new engineering bacteria has stronger immunogenicity, reduces the stress of animals, is more convenient to use, has the advantages of safety, convenience, mild action effect and the like, can be directly used for intramuscular injection of animals, is simple and easy to operate, achieves more ideal effects in controlling the development and reproductive function of reproductive organs of animals, better promotes the growth of meat animals, and further promotes the development of animal castration technology.
Drawings
FIG. 1 is a map of plasmid PET21a-GnRH6-CRM197 in example 1 of the present invention;
FIG. 2 is a gel electrophoresis analysis chart of recombinant protein GnRH6-CRM197 (71.3 kDa) in example 1 of the present invention, wherein 1 refers to Merker band, 2 refers to GnRH6-CRM197 pre-purification band, and 3 refers to GnRH6-CRM197 post-purification band;
FIG. 3 is a diagram showing Western blot (Westernblot) analysis of purified recombinant protein GnRH6-CRM197 (71.3 kDa) in example 1 of the present invention, wherein 1 is Mark band and 2 is GnRH6-CRM197 (71.3 kDa) and GnRH antibody reaction band;
FIG. 4 is a testis representation of male rat testis from control (left) and immunized (right) groups of example 2 according to the present invention;
fig. 5 is a drawing of histological observation of male rat testes of the immune group and the control group provided by the embodiment of the invention, wherein the microscope magnification of the drawing a and the drawing C is 100×, the microscope magnification of the drawing B and the drawing D is 400×, the drawing a and the drawing B are the control group, the drawing C and the drawing D are the test group, a is spermatogenic cell, B is spermatocyte, C is sperm cell, D is sperm, e is vacuolation, and f is seminiferous tubule.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and fully with reference to the accompanying drawings, in which it is evident that the embodiments described are only some, but not all embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1
A construction method of GnRH6-CRM197 recombinant protein castration vaccine comprises the following steps:
(1) Construction of plasmid PET21a-GnRH6-CRM197
Based on the domestic canine GnRH sequence (sequence listing seq_2) and the goat CRM197 sequence (sequence listing seq_3), a recombinant CRM197 sequence was designed that contained the selected GnRH hexamer. The lyophilized powder of the BamH I and Xho I cleavage site-carrying DNA sequence (shown as seq_4) synthesized from Beijing, the Biotech Co., ltd was dissolved in sterilized ultra pure water according to the specification, and the dissolved DNA and PET21a plasmids were simultaneously double digested with BamH I and Xho I, respectively, in a double digestion system of GnRH6-CRM197 gene fragment (100 ng/. Mu.L) or PET21a (100 ng/. Mu.L) 40. Mu.L, respectively, and in a system of 10 XCutSmart Buffer 10. Mu.L, bamH I and Xho I each 1. Mu.L and sterilized ultra pure water 48. Mu.L, respectively. The target fragment was separated by digestion at 37℃for 10 hours and by electrophoresis, the target band was excised by a knife, and purified by a gel recovery kit.
The GnRH6-CRM197 gene fragment and the PET21a carrier fragment are connected overnight at 4 ℃ by using T4 ligase according to the mol ratio of 5:1, the connection mixture is used for transforming escherichia coli DH5 alpha and then cultured overnight on LB solid medium containing 100 mug/mL ammonia Xilin (amp+), several monoclonal sequencing is selected, and the bacteria with correct sequence are selected to extract plasmids, namely the plasmid PET21a-GnRH6-CRM197.
(2) Inducible expression and purification of GnRH6-CRM197 vaccine proteins
(1) Preparation of bacteria: e.coli BL21 (DE 3) was transformed with the above-mentioned plasmid PET21a-GnRH6-CRM197 by a conventional method, cultured on LB solid medium containing amp+ (100. Mu.g/mL) for 12-15 hours, and one transformant was picked up and cultured in LB liquid medium (10 mL) containing amp+ (100. Mu.g/mL) with shaking at 37℃and 200rpm for 16 hours; 1mL of the bacterial liquid after 16h culture was taken, 100mL of fresh LB liquid medium containing amp+ (100. Mu.g/mL) was added, the culture was continued by shaking at 200rpm at 37℃for 12h with the final concentration of 1.0mmol/L of IPTG added when the OD600 was 1.0, and the bacterial pellet was collected by centrifugation at 4000g and 4℃for 45min at 200 rpm.
(2) And (3) breaking bacteria: adding 8mL (accounting for 8% of the total volume of the thalli) of non-denatured lysate into 100mL of bacterial sediment for resuspension, freezing the resuspended thalli at-20 ℃ or directly using the thalli for ultrasound, and adding a protease inhibitor PMSF (diluted according to the volume ratio of 1:100-1:1000, and the working concentration is 17-174 mug/mL) during ultrasound; the ultrasonic power is 200W, the ultrasonic power is 4s, the intermittent ultrasonic power is 2s, and the ultrasonic power is 20min; after the ultrasonic treatment is finished, 10000g of bacterial liquid is centrifuged for 30min at 4 ℃, the supernatant is discarded, urea solution (8 mol/L) is added into the sediment, 5-10 mL of urea (8 mol/L) solution is added into the sediment according to 100mL of bacterial sediment for dissolution, the solution is dissolved for 12h at 4 ℃, after the bacterial liquid is completely dissolved, the bacterial liquid is centrifuged for 10min at 5000g of rotation speed at 4 ℃, the supernatant is taken, and the supernatant is subjected to nickel column purification, so that the GnRH6-CRM197 recombinant protein castration vaccine is obtained.
Wherein, the plasmid extraction and the double enzyme digestion electrophoresis recovery use the corresponding kit of Beijing Aidelai life technology Co., ltd; a competent cell method for transforming plasmid and DNA ligation solution into Escherichia coli; all restriction enzymes and ligases were purchased from "beijing new intel biotechnology limited".
FIG. 1 is a schematic diagram of plasmid PET21a-GnRH6-CRM197 of example 1 of the present invention, wherein the DNA sequence with BamHI and XhoI cleavage sites was synthesized by a codon optimization system, and the DNA product was ligated with the same digested vector PET21a after BamHI and XhoI double cleavage. The purified protein was detected by SDS-PAGE electrophoresis (electrophoresis at 80V for 30min followed by electrophoresis at 120V for 60 min) and its molecular weight was determined, as shown in FIG. 2, and the purified protein was analyzed by WB, confirming that it was the antigen protein GnRH6-CRM197 having the amino acid sequence in sequence Listing seq_1. The non-denaturing lysate was at a final concentration of 50mmol/LTris >500mmol/LNaC1, pH7.5. As shown in FIG. 3, the developed GnRH6-CRM197 has good reactivity with GnRH antibodies.
Sequence table seq_1
QHWSGG1RPGGGSEHWSYG1RPGGGSEHWSYG1RPGGGSEDWSYG1RPGGGSEHWSYG1RPGGGSEHWSYG1RPGGGGSGADDVVDSSKSFVMENFSSYHGTKPGYVDSIQKGIQKPKSGTQGNYDDDWKEFYSTDNKYDAAGYSVDNENP1SGKAGGVVKVTYPG1TKV1A1KVDNAETIKKE1G1S1TEP1MEQVGTEEFIKRFGDGASRVV1S1PFAEGSSSVEYINNWEQAKA1SVE1EINFETRGKRGQDAMYEYMAQACAGNRVRRSVGSS1SCIN1DWDVIRDKTKTKIES1KEHGPIKNKMSESPNKTVSEEKAKQY1EEFHQTA1EHPE1SE1KTVTGTNPVFAGANYAAWAVNVAQVIDSETADN1EKTTAA1SI1PGIGSVMGIADGAVHHNTEEIVAQSIA1SS1MVAQAIP1VGE1VDIGFAAYNFVESIIN1FQVVHNSYNRPAYSPGHKTQPF1HDGYAVSWNTVEDSIIRTGFQGESGHDIKITAENTP1PIAGV11PTIPGK1DVNKSKTHISVNGRKIRMRCRAIDGDVTFCRPKSPVYVGNGVHAN1HVAFHRSSSEKIHSNEISSDSIGV1GYQKTVDHTKVNSK1S1FFEIKS
Sequence listing seq_2
MEPIPK1VAG1111TFCVVSCSGQHWSYG1RPGGKRNAEH1IDSFQEMAKE1DQPAEPQH1ECTIHKPRPP1RD1RGA1ES1IEEETGQKRI
Sequence listing seq_3
GADDVVDSSKSFVMENFSSYHGTKPGYVDSIQKGIQKPKSGTQGNYDDDWKEFYSTDNKYDAAGYSVDNENP1SGKAGGVVKVTYPG1TKV1A1KVDNAETIKKE1G1S1TEP1MEQVGTEEFIKRFGDGASRVV1S1PFAEGSSSVEYINNWEQAKA1SVE1EINFETRGKRGQDAMYEYMAQACAGNRVRRSVGSS1SCIN1DWDVIRDKTKTKIES1KEHGPIKNKMSESPNKTVSEEKAKQY1EEFHQTA1EHPE1SE1KTVTGTNPVFAGANYAAWAVNVAQVIDSETADN1EKTTAA1SI1PGIGSVMGIADGAVHHNTEEIVAQSIA1SS1MVAQAIP1VGE1VDIGFAAYNFVESIIN1FQVVHNSYNRPAYSPGHKTQPF1HDGYAVSWNTVEDSIIRTGFQGESGHDIKITAENTP1PIAGV11PTIPGK1DVNKSKTHISVNGRKIRMRCRAIDGDVTFCRPKSPVYVGNGVHAN1HVAFHRSSSEKIHSNEISSDSIGV1GYQKTVDHTKVNSK1S1FFEIKS
Sequence listing seq_4
GGATCCCAGCATTGGAGCGGTGGTCTGCGTCCTGGTGGTGGTAGTGAACATTGGAGCTATGGTCTGCGTCCGGGTGGTGGTTCTGAACATTGGAGTTATGGTCTGCGCCCTGGTGGTGGCTCTGAAGATTGGAGTTATGGCCTGCGTCCGGGCGGTGGTAGTGAACATTGGTCATACGGTCTGCGTCCAGGTGGTGGTAGCGAACATTGGTCTTATGGTCTGCGTCCGGGTGGCGGTGGTAGCGGTGCAGATGATGTTGTTGATAGCAGTAAAAGTTTTGTGATGGAAAATTTTAGTAGTTATCATGGTACGAAACCGGGTTATGTTGATAGCATTCAGAAAGGTATTCAGAAACCGAAAAGCGGTACCCAGGGTAATTATGATGATGATTGGAAAGAATTTTATAGTACCGATAACAAATATGATGCCGCAGGTTATTCCGTGGATAATGAAAATCCGCTGAGCGGCAAAGCAGGTGGTGTTGTTAAAGTGACATATCCGGGTCTGACGAAAGTGCTGGCCCTGAAAGTTGATAATGCGGAAACCATTAAAAAGGAACTGGGTCTGAGCCTGACCGAACCGCTGATGGAACAGGTGGGTACAGAAGAATTTATTAAACGTTTTGGCGATGGTGCTAGCCGTGTTGTTCTGAGCCTGCCTTTTGCCGAAGGTAGCTCTTCAGTTGAATATATTAATAACTGGGAACAGGCAAAAGCCCTGAGCGTGGAACTGGAAATTAATTTTGAAACACGTGGTAAACGTGGTCAGGATGCAATGTATGAATATATGGCACAGGCATGTGCAGGTAACCGTGTTCGTCGTAGCGTAGGTAGTAGTCTGAGTTGTATTAATCTGGATTGGGATGTTATTCGTGATAAAACAAAAACAAAAATCGAAAGCCTGAAAGAACATGGTCCGATTAAAAATAAAATGAGCGAAAGCCCGAATAAAACCGTTAGCGAAGAAAAAGCAAAACAGTATCTGGAAGAATTTCATCAGACCGCACTGGAACATCCGGAACTGAGCGAACTGAAAACAGTGACAGGTACCAACCCGGTGTTTGCCGGTGCAAATTATGCAGCATGGGCAGTGAATGTTGCACAGGTGATTGATAGCGAAACCGCAGATAACCTGGAAAAAACCACCGCAGCACTGAGCATTCTGCCGGGTATTGGTAGCGTTATGGGTATTGCAGATGGTGCAGTTCATCATAATACCGAAGAAATTGTTGCACAGAGCATTGCACTGAGCAGCCTGATGGTTGCACAGGCAATTCCGCTGGTTGGTGAACTGGTTGATATTGGTTTTGCAGCATATAATTTTGTTGAAAGCATTATTAATCTGTTTCAGGTTGTTCATAATAGCTATAATCGTCCGGCATATAGCCCGGGTCATAAAACCCAGCCGTTTCTGCATGATGGTTATGCAGTTAGCTGGAATACCGTTGAAGATAGCATTATTCGTACCGGTTTTCAGGGTGAAAGCGGTCATGATATTAAAATTACCGCAGAAAATACCCCGCTGCCGATTGCAGGTGTTCTGCTGCCGACCATTCCGGGTAAACTGGATGTTAATAAAAGCAAAACCCATATTAGCGTTAATGGTCGTAAAATTCGTATGCGTTGTCGTGCAATTGATGGTGATGTTACCTTTTGTCGTCCGAAAAGCCCGGTTTATGTTGGTAATGGTGTTCATGCAAATCTGCATGTTGCATTTCATCGTAGCAGCAGCGAAAAAATTCATAGCAATGAAATTAGCAGCGATAGCATTGGTGTTCTGGGTTATCAGAAAACCGTTGATCATACCAAAGTTAATAGCAAACTGAGCCTGTTTTTTGAAATTAAAAGCCTCGAG
Example 2
Use of GnRH6-CRM197 recombinant protein castration vaccine:
20 adult male rats were selected, 10 control and test groups were immunized initially at 5 months of age and boosted 2 weeks apart, each at 300 micrograms dose, 3 total immunizations, and sacrificed 2 months after the last immunization. Testis samples were taken for preparation of sections and hematoxylin-eosin staining for histological structure.
Evaluation of immunocastration results for GnRH6-CRM197 vaccine:
obvious vacuolation of the testis tissue occurred in rats of the test group. The vacuolated rat testis seminiferous tubule has obvious change, the seminoma cell and the spermatid cell are not distinct in level, and no spermatid is generated. The testis seminiferous tubules of the rats in the control group are normal in structure, normal spermatogenic cells, spermatocytes, spermatids and sperms are arranged in the seminiferous tubules, the level is clear, and a large amount of sperms are produced. It can be seen that GnRH6-CRM197 inhibited the development of rat testis and eventually atrophy, as shown in FIG. 4, the immunized group of rat testis was smaller, indicating that the developed GnRH6-CRM197 was very immunogenic. As shown in FIG. 5, the testis tissue of the immunized group rat is obviously cavitation denatured, the number of sperm-producing cells is reduced, no sperm is produced, and the GnRH6-CRM197 can inhibit the development of rat testis and finally shrink, so that the developed GnRH6-CRM197 has strong immunogenicity.
The foregoing is merely illustrative and explanatory of the invention, as it is well within the scope of the invention, as it is intended to provide those skilled in the art with various modifications, additions and substitutions to the specific embodiments disclosed and those skilled in the art without departing from the scope of the invention as disclosed in the accompanying claims.
Claims (10)
1. A GnRH recombinant protein antigen, characterized by: the amino acid sequence of the GnRH recombinant protein antigen is shown as a sequence table seq_1.
2. A GnRH6-CRM197 recombinant protein castration vaccine characterized by: the mammalian castration recombinant protein vaccine comprises the GnRH recombinant protein antigen of claim 1.
3. The GnRH6-CRM197 recombinant protein castration vaccine of claim 2, wherein: the GnRH6-CRM197 recombinant protein castration vaccine contains a mammalian GnRH sequence, wherein the mammal comprises dogs, cats, pet pigs, holland pigs and mice.
4. The GnRH6-CRM197 recombinant protein castration vaccine of claim 3 wherein: the mammalian castration recombinant protein vaccine contains a canine GnRH sequence of sequence table seq_2.
5. The GnRH6-CRM197 recombinant protein castration vaccine of claim 2, wherein: the mammalian castration recombinant protein vaccine contains a goat CRM197 sequence of a sequence table seq_3.
6. The GnRH6-CRM197 recombinant protein castration vaccine of claim 2, wherein: the GnRH6-CRM197 recombinant protein castration vaccine comprises GnRH6-CRM197 converted escherichia coli expressed recombinant protein of a sequence table seq_1.
7. A GnRH6-CRM197 transformed escherichia coli, characterized by: the GnRH6-CRM197 transformed Escherichia coli is preserved in China general microbiological culture collection center (CGMCC) at the date of 6-19 of 2022, the preservation number is CGMCC No.25027, the classification is Escherichia coli, the classification is specifically BL21 (DE 3) -PET21a-GnRH6-CRM197, and the GnRH6-CRM197 transformed Escherichia coli expresses recombinant protein containing amino acid of the sequence table seq_1.
8. A method of constructing a GnRH6-CRM197 encoding escherichia coli according to claim 7, comprising the steps of:
(1) Designing a recombinant CRM197 sequence comprising the selected GnRH hexamer based on the mammalian GnRH sequence and the goat CRM197 sequence;
(2) The DNA sequence with BamH I and Xho I cleavage sites is designed and synthesized by adopting a codon optimization system, and the DNA product after BamH I and Xho I double cleavage is connected with a carrier PET21a which is also digested, so as to obtain a connecting solution;
(3) And then transferring the connection solution into escherichia coli DH5 alpha, screening to obtain a plasmid PET21a-GnRH6-CRM197, transferring the plasmid GnRH6-CRM197 into escherichia coli BL21 (DE 3) strain to obtain engineering bacteria BL21 (DE 3) -PET21a-GnRH6-CRM197, and finally expressing recombinant protein containing amino acid of a sequence table seq_1, namely converting the GnRH6-CRM197 into escherichia coli.
9. A preparation method of GnRH6-CRM197 recombinant protein castration vaccine is characterized by comprising the following steps: the GnRH6-CRM197 converted escherichia coli of claim 7 or 8 is subjected to bacterial breaking purification to obtain recombinant protein containing amino acid of a sequence table seq_1, namely the GnRH6-CRM197 recombinant protein castration vaccine.
10. Use of a GnRH6-CRM197 recombinant protein castration vaccine according to claim 2, characterized in that: the GnRH6-CRM197 recombinant protein castration vaccine is used for controlling the fertility of mammals.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116987201A (en) * | 2023-09-27 | 2023-11-03 | 贝格探索(成都)科技有限公司 | Multimeric recombinant protein for regulating and controlling reproductive capacity of mammal, preparation method and application |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2001049317A2 (en) * | 2000-01-05 | 2001-07-12 | Aventis Pasteur Limited | Enhancing the immune response to an antigen by presensitizing with an inducing agent prior to immunizing with the inducing agent and the antigen |
| CN103405764A (en) * | 2005-12-13 | 2013-11-27 | 葛兰素史密丝克莱恩生物有限公司 | Vaccine compositions comprising a saponin adjuvant |
| CN108904788A (en) * | 2018-06-14 | 2018-11-30 | 广州源博医药科技有限公司 | GnRH- alexin recombinates castration vaccine and its preparation |
| CN112500456A (en) * | 2020-12-02 | 2021-03-16 | 深圳赫兹生命科学技术有限公司 | Castration AP205 virus-like particle subunit vaccine |
| CN113388041A (en) * | 2020-03-12 | 2021-09-14 | 厦门大学 | SARS-CoV-2S tripolymer protein with early-stage conformation before fusion and its application |
-
2022
- 2022-10-28 CN CN202211336837.4A patent/CN116162173A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2001049317A2 (en) * | 2000-01-05 | 2001-07-12 | Aventis Pasteur Limited | Enhancing the immune response to an antigen by presensitizing with an inducing agent prior to immunizing with the inducing agent and the antigen |
| CN103405764A (en) * | 2005-12-13 | 2013-11-27 | 葛兰素史密丝克莱恩生物有限公司 | Vaccine compositions comprising a saponin adjuvant |
| CN108904788A (en) * | 2018-06-14 | 2018-11-30 | 广州源博医药科技有限公司 | GnRH- alexin recombinates castration vaccine and its preparation |
| CN113388041A (en) * | 2020-03-12 | 2021-09-14 | 厦门大学 | SARS-CoV-2S tripolymer protein with early-stage conformation before fusion and its application |
| CN112500456A (en) * | 2020-12-02 | 2021-03-16 | 深圳赫兹生命科学技术有限公司 | Castration AP205 virus-like particle subunit vaccine |
Non-Patent Citations (2)
| Title |
|---|
| "《中国生物制品学杂志》2015年第28卷主题词索引", 中国生物制品学杂志, no. 12, 20 December 2015 (2015-12-20) * |
| 杨亚萍: "GnRH六聚体-麦芽糖结合蛋白（MBP-GnRH-6）的融合表达及鉴定", 安徽农业大学硕士学位论文, 21 September 2007 (2007-09-21), pages 1 - 47 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116987201A (en) * | 2023-09-27 | 2023-11-03 | 贝格探索(成都)科技有限公司 | Multimeric recombinant protein for regulating and controlling reproductive capacity of mammal, preparation method and application |
| CN116987201B (en) * | 2023-09-27 | 2023-12-08 | 贝格探索(成都)科技有限公司 | Multimeric recombinant protein for regulating and controlling reproductive capacity of mammal, preparation method and application |
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