CN116590300A - Fusion gene for animal castration, expression vector, gene vaccine and application thereof - Google Patents
Fusion gene for animal castration, expression vector, gene vaccine and application thereof Download PDFInfo
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- CN116590300A CN116590300A CN202310550032.8A CN202310550032A CN116590300A CN 116590300 A CN116590300 A CN 116590300A CN 202310550032 A CN202310550032 A CN 202310550032A CN 116590300 A CN116590300 A CN 116590300A
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- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
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Abstract
The invention belongs to the technical field of genetic engineering, and particularly relates to a fusion gene for animal castration, an expression vector, a genetic vaccine and application thereof. The fusion gene comprises an S-GnRH gene, an S-NKB gene and a KISS1 gene from upstream to downstream, wherein the S-GnRH gene and the S-NKB gene are respectively formed by connecting an HBsAg-S antigen gene on the upstream of the GnRH gene and the NKB gene, and a 2A peptide gene is connected among the S-GnRH gene, the S-NKB gene and the KISS1 gene. The fusion gene provided by the invention can simultaneously express GnRH, NKB and KISS1 hormone after being transferred into an animal body, has strong immunogenicity and quick induction immune response, can quickly excite the animal body to generate a neutralizing antibody of the corresponding hormone, can reduce the concentration reduction level of endogenous hormone to less than half of that of a contemporaneous control group in about 2 weeks, and ensures that the aim of quick castration is achieved under the conditions of reducing the number of times of enhancing immunity and reducing injection dosage.
Description
Technical Field
The invention relates to the technical field of genetic engineering, in particular to a fusion gene for animal castration, an expression vector, a genetic vaccine and application thereof.
Background
Castration, which is a technique for removing the reproductive system of animals or losing sexual function, aims to eliminate sexual desire and reproductive capacity of animals, and enables the sexuality of the animals to become mild and durable, so that the animals are convenient to raise and manage; the meat quality of the meat animal is improved, and the yield is increased; and the mating behavior in the herd can be controlled, thereby being beneficial to the propagation and breeding of fine breeds. The most common castration technology for animals achieves the aim of castrating animals by castration and removal of testes of male animals, and the castration process comprises the steps of preparation before operation, sterilization, operation implementation, hemostasis sterilization and the like, is complex in operation, time-consuming and labor-consuming, and can easily cause high stress and surgical wound infection of the animals, further increase the morbidity of the animals and even lead to death of the animals. In addition, a long recovery period is required after the operation, and the normal growth of animals is greatly affected. Therefore, the simple and low-cost castration method is of great significance.
In recent years, hormone immunocastration has great potential for use. Hormone immune castration is mainly targeted by genital hormone members in the hypothalamic-pituitary-gonadal axis (target gene or target protein), and antibody "neutralization" is responsible for immune castration. The technical key of immune castration lies in the selection of immune targets, wherein gonadotropin releasing hormone (GnRH), follicle Stimulating Hormone (FSH) and Luteinizing Hormone (LH) are commonly used for immune castration, for example, by actively immunizing animals with exogenous GnRH, inducing the bodies of the animals to generate a large amount of anti-GnRH specific antibodies, neutralizing the GnRH in vivo and further reducing the GnRH level, leading to reduced synthesis of LH and FSH, causing imbalance of the reproductive endocrine system, reducing synthesis and secretion of the reproductive hormone and destruction of testicular development and spermatogenesis, and finally achieving the aim of immune castration. Active immunization by GnRH can avoid a plurality of shortages of surgical castration technology, and is a safe and friendly method which is most hopeful to replace surgical castration. However, monomeric GnRH vaccines have not shown significant efficacy in reducing testicular quality, serum testosterone, and other reproductive parameters, both in single and multiple injections. Therefore, in order to achieve the aim of immune castration of the developed vaccine, it is necessary to increase the expression efficiency of the vaccine in the body or to find a new immune castration target.
The KISS1 gene is also called as a KISS gene, is a neuropeptide positioned upstream of GnRH, and plays a decisive role in reproduction control switch by controlling secretion of GnRH. Proved by researches, the gene vaccine fused with the KISS1 and the GnRH can obviously reduce the reproductive performance of animals, but has slower hormone level effect, the testosterone level can be reduced to nearly half of the initial concentration after 6 weeks of immunization, the estradiol level is reduced by about 40 percent, and a higher immunization dosage is needed, and multiple times of boosting is needed, so that the immunogenicity is to be enhanced, and the operation complexity and the use convenience of a castration technology are enhanced.
In conclusion, the development of the composite gene vaccine which has good immunogenicity, quick immune effect and can effectively improve castration effect has important application value.
Disclosure of Invention
Aiming at the problems of low immunogenicity and slow immune effect of the gene vaccine for animal castration in the prior art, the invention provides a fusion gene, an expression vector and a gene vaccine for animal castration and application thereof, so as to solve or at least alleviate part of the problems in the prior art.
The invention is realized by the following technical scheme:
a first aspect of the present invention provides a fusion gene for castration in an animal, comprising, from upstream to downstream, an S-GnRH gene formed by linking an HBsAg-S antigen gene upstream of a GnRH gene, an S-NKB gene formed by linking an HBsAg-S antigen gene upstream of an NKB gene, and a KISS1 gene linked with a 2A peptide gene between the S-GnRH gene and the S-NKB gene and between the S-NKB gene and the KISS1 gene.
Further, the DNA sequence of the GnRH gene is shown as SEQ ID NO:1 is shown in the specification; the DNA sequence of the NKB gene is shown in SEQ ID NO:2 is shown in the figure; the DNA sequence of the KISS1 gene is shown as SEQ ID NO: 3.
Further, the DNA sequence of the HBsAg-S antigen gene is shown in SEQ ID NO:4 is shown in the figure; the DNA sequence of the 2A peptide gene is shown in SEQ ID NO: shown at 5.
Further, a first restriction site is arranged on the upstream of the S-GnRH gene, a second restriction site is arranged on the upstream of the 2A peptide gene, a third restriction site is arranged on the downstream of the KISS1 gene, and the first restriction site, the second restriction site and the third restriction site are different.
Still further, the first cleavage site is GCTAGC, the second cleavage site is AAGCTT, and the third cleavage site is CTCGAG.
Still further, the full-length DNA sequence of the fusion gene is shown in SEQ ID NO: shown at 6.
In a second aspect the present invention provides an expression vector for castration in an animal, the expression vector carrying a fusion gene as described above.
Further, the starting vector of the expression vector is a eukaryotic expression vector pvax-asd.
In a third aspect, the invention provides a genetic vaccine for animal castration, which comprises a vaccine vector carrying the expression vector for animal castration, wherein the vaccine vector is a live attenuated viral vector or a live attenuated bacterial vector.
Further, the vaccine carrier is attenuated salmonella choleraesuis C500.
The fourth aspect of the invention provides the application of the fusion gene for animal castration, the expression vector for animal castration and the gene vaccine for animal castration in preparing medicines for controlling animal oestrus expression.
Further, the animal estrus manifestations include reducing animal estrus aggressiveness and/or controlling fertility.
The invention has the advantages and positive effects that:
the fusion gene provided by the invention can simultaneously and respectively express exogenous hormones GnRH, NKB and KISS1 after being transferred into an animal body, has strong immunogenicity and quick induction immune response, can quickly excite the animal body to generate a neutralizing antibody of corresponding hormone, is combined with endogenous hormones (GnRH, NKB and KISS 1) to reduce the concentration of the neutralizing antibody, can quickly and effectively reduce testosterone level and estradiol level through cascade reaction by reducing the upstream hormone level, can realize that the corresponding hormone level is reduced to less than half of a synchronous control group in about 2 weeks, further effectively reduces the production of spermatogenic cells and sperms, blocks the sperms, realizes the aim of inhibiting the reproductive capacity of male mice in a short time, ensures that the aim of quick castration is achieved under the conditions of reducing the number of enhancing immunity and reducing the injection dosage, and ensures that the castration technology is more optimized and more convenient.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for the description of the embodiments will be briefly described below, and it is apparent that the drawings in the following description are only some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a flow chart showing construction of expression vectors for coexpression of GnRH gene, NKB gene and KISS1 gene according to an embodiment of the present invention;
FIG. 2 is a graph showing the change of GnRH antibody level with immunization time after a male mouse is immunized by different genetic vaccines according to the embodiment of the present invention;
FIG. 3 is a graph showing the variation of KISS1 antibody levels with time after immunization of male mice with different genetic vaccines according to an embodiment of the present invention;
FIG. 4 is a graph showing the variation of NKB antibody levels with time after a male mouse is immunized with different genetic vaccines according to an embodiment of the present invention;
FIG. 5 is a graph showing the variation of testosterone (T) levels with time after immunization of male mice with different genetic vaccines according to an embodiment of the present invention;
FIG. 6 is a graph showing the change of estradiol (E2) level with time after a male mouse is immunized by different genetic vaccines according to the embodiment of the present invention;
FIG. 7 is a diagram showing semen smear of a control group (a), a double-expressed gene vaccine group (b) and a triple-expressed gene vaccine group (c) according to an embodiment of the present invention;
FIG. 8 shows testis HE stained sections of control (a), double expressed gene vaccine (b) and triple expressed gene vaccine (c) according to the examples of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be described in further detail with reference to the following examples. The examples described herein are intended to illustrate the invention only and are not intended to limit the invention.
Various modifications to the precise description of the invention will be readily apparent to those skilled in the art from the information contained herein without departing from the spirit or scope of the appended claims. It is to be understood that the scope of the invention is not limited to the defined processes, properties or components, as these embodiments, as well as other descriptions, are merely illustrative of specific aspects of the invention. Indeed, various modifications of the embodiments of the invention which are obvious to those skilled in the art or related fields are intended to be within the scope of the following claims.
For a better understanding of the present invention, and not to limit its scope, all numbers expressing quantities, percentages and other values used in the present invention are to be understood as being modified in all instances by the term "about". Accordingly, unless indicated otherwise, the numerical parameters set forth in the specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained. Each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. In addition, the terms "comprising," "including," "containing," "having," and the like are intended to be non-limiting, as other steps and other ingredients may be added that do not affect the result.
In order that the above-recited objects, features and advantages of the present invention will become more readily apparent, a more particular description of the invention briefly described above will be rendered by reference to specific embodiments thereof which are illustrated in the appended drawings.
In the early study of the laboratory (patent publication No. CN112048459A, the invention name: a salmonella choleraesuis, vaccine and application thereof, the publication date: 12 th month 8 th week of 2020), the fusion form of pVAX-S/GnRH-2a/KISS1-asd is constructed, and the fusion form of pVAX-S/GnRH-2a/KISS1-asd is transferred into salmonella choleraesuis C500 strain to form a genetic vaccine, after the genetic vaccine is injected, the gonadotropin releasing hormone (GnRH) gene and the KISS1 gene can be simultaneously expressed in animals, exogenous hormones GnRH and KISS1 are produced, the genetic vaccine can effectively regulate the sex hormone level and oestrogen behavior of the animals after the first immunization and boosting for 2 times (boosting every two weeks), but the effective immunization dose of the genetic vaccine is larger, the immunogenicity is to be boosted, and the onset rate is slower, the study shows that after the first immunization, the sex hormone level is measured every 2 weeks, 4 weeks, 6 weeks, the high-dose group ketone level is respectively reduced by 27%, 39.5%, 35.5%, 25% and 35.36% of the sex hormone level is reduced by the same period as that of the control group, namely, the high dose is reduced by 35.20%, and the immune level is respectively, and the immune level is reduced by the dose is higher than 35.35% and the dose 11 CFU) can reduce testosterone and estradiol levels to a lower level after 6 weeks of immunization, thereby requiring high doses and multiple booster immunizations to achieve a better immune castration, which increases the complexity of vaccine use to some extent.
Neurokinin B (NKB) (GenBank accession number: XM_ 004006562.5) is also known as neuromedin K (neuromedin K), which is a member of the tachykinin family, and the tachykinin receptors are mainly three, namely NK1R, NK2R and NK3R, and the coding process of the neurokinin B is respectively participated by Tacrl, tacr2 and Tacr3 genes, so that the NKB plays a biological effect mainly through NK3R receptor binding. According to the research invention, three neuropeptides, namely kisspeptin, NKB and DYN, are co-expressed in hypothalamic arciform nucleus, after exogenous injection of NKB or NK3R receptor agonists, the transient activation of the neuropeptides can increase the pulse release times of GnRH, and further, the in vivo LH secretion amount of the neuropeptides is induced to increase, so that cascade reaction is generated, and the genital axis is regulated and controlled. According to the invention, NKB, KISS1 and GnRH are taken as castration targets together, and after the gene vaccine formed by the co-expression of the NKB, gnRH and KISS1 genes is injected into an animal body, the immune response of the animal body can be effectively induced, and the generated specific neutralizing antibody can play a role in neutralizing corresponding hormone rapidly, so that the testosterone and estradiol levels can be reduced efficiently in a short time, and the castration purpose is realized. Thus, the present invention has been completed.
First, an embodiment of the present invention provides a fusion gene for castration of animals, comprising, from upstream to downstream, an S-GnRH gene formed by linking an HBsAg-S antigen gene upstream of a GnRH gene, an S-NKB gene formed by linking an HBsAg-S antigen gene upstream of an NKB gene, and a KISS1 gene linked with a 2A peptide gene between the S-GnRH gene and the S-NKB gene and between the S-NKB gene and the KISS1 gene. That is, the sequence of connection of the genes is as follows: the HBsAg-S antigen (S) gene, gnRH gene, 2A peptide gene, HBsAg-S antigen (S) gene, NKB gene, 2A peptide gene and KISS1 gene form S-GnRH-2A/S-NKB-2A/KISS1 fusion gene.
Genetic vaccines are novel immunization strategies that present a protein of interest to the immune system of the body by selecting as a target a foreign gene encoding the protein of interest, and then by means of viral vectors or non-viral naked DNA plasmids. The present invention is capable of allowing the CDS sequences encoding the GnRH gene, the NKB gene and the KISS1 gene to be co-expressed in a fusion gene by means of a 2A peptide, which is also referred to as a "self-cleaving" peptideThe fusion gene of the invention can simultaneously express hormones GnRH, NKB and KISS1 respectively after being transferred into an animal body, and simultaneously GnRH and NKB are connected with HBsAg-S antigen to have enhanced immunogenicity. The research data of the invention show that the research data is 3 multiplied by 10 8 The method has the advantages that compared with the prior study, the two gene vaccines expressing GnRH genes and KISS1 genes can be reduced to about 50% in 6 weeks, the same effect can be realized in about 2 weeks, the hormone level of subsequent booster immunization can be always maintained at a low level, so that the sperm cell yield is obviously lower than that of the two gene vaccines in 6 weeks, the effect of immune castration similar to that of the two gene vaccines can be realized in the first immunization, and the reproductive capacity of the male mice can be inhibited in a short time, so that the booster immunization times can be reduced, the injection dosage is reduced, and the castration technology is more optimized and more convenient.
Optionally, the DNA sequence of the GnRH gene is set forth in SEQ ID NO:1 is shown in the specification; the DNA sequence of the NKB gene is shown in SEQ ID NO:2 is shown in the figure; the DNA sequence of the KISS1 gene is shown as SEQ ID NO:3 is shown in the figure; the DNA sequence of the HBsAg-S antigen gene is shown in SEQ ID NO:4 is shown in the figure; the DA sequence of the 2A peptide gene is shown as SEQ ID NO: shown at 5.
In order to facilitate the construction of the fusion gene and the subsequent insertion of the fusion gene into an expression vector, a first cleavage site is arranged upstream of the S-GnRH gene, a second cleavage site is arranged upstream of the 2A peptide gene, a third cleavage site is arranged downstream of the KISS1 gene, and the first cleavage site, the second cleavage site and the third cleavage site are different. On the basis of pVAX-S/GnRH-2A/KISS1-asd plasmid in the early stage of the laboratory, carrying out single enzyme digestion on the pVAX-S/GnRH-2A/KISS1-asd plasmid by using an endonuclease of a second enzyme digestion site, carrying out enzyme digestion on a 2A/S-NKB gene (formed by connecting a 2A peptide gene on the upstream of the S-NKB gene) by using the same enzyme, and then inserting the 2A/S-NKB gene between the S-GnRH gene and the 2A/KISS1 gene (formed by connecting a 2A peptide gene on the upstream of the KISS1 gene) by using homologous recombination, thereby constructing fusion genes which are formed by sequentially connecting the S gene, the GnRH gene, the 2A peptide gene, the S gene, the NKB gene, the 2A peptide gene and the KISS1 gene, and the three-gene expression vectors are obtained. And (3) carrying out double enzyme digestion on the three-gene expression vector through the endonucleases of the first enzyme digestion site and the third enzyme digestion site, so as to obtain the fusion gene of the invention.
Illustratively, the first cleavage site is GCTAGC (NheI), the second cleavage site is AAGCTT (HindIII), and the third cleavage site is CTCGAG (XhoI). Thus, the full-length DNA sequence of the fusion gene is shown as SEQ ID NO: shown at 6.
Next, another embodiment of the present invention provides an expression vector for castration of animals, which carries the fusion gene as described above.
The fusion gene of the invention is operably connected with at least one expression regulatory element, the fusion gene is positioned at the downstream of the expression regulatory element such as a promoter and an enhancer to form an expression vector, then the expression vector is introduced into a vaccine vector to obtain a vaccine vector for transforming the expression vector, thus obtaining a genetic vaccine, and the genetic vaccine is injected into an animal organism to co-express and produce exogenous hormones GnRH, NKB and KISS1. The selection of expression vectors/expression regulatory elements is well known in the art and will not be described in detail herein. Plasmids are common vectors, and therefore, in the present invention, plasmids have the same meaning as vectors.
The advantages of the expression vector for animal castration over the prior art are the same as those of the fusion gene for animal castration described above over the prior art, and are not described in detail herein.
Alternatively, the expression vector comprises a eukaryotic expression vector or a viral expression vector.
In some specific embodiments, the starting vector of the expression vector is a eukaryotic expression vector pvax-asd. The construction flow of the expression vector is shown in FIG. 1. Firstly, inserting an S-GnRH gene and a 2a/KISS1 gene into a pVAX-asd vector to obtain a pVAX-S/GnRH-2a/KISS1-asd vector, then respectively enzyme-cutting the 2a/S-NKB gene and the pVAX-S/GnRH-2a/KISS1-asd vector by HindIII, and connecting the 2a/S-NKB gene into the pVAX-S/GnRH-2a/KISS1-asd vector by using a homologous recombination method to form the pVAX-S/GnRH-2a/S-NKB-2a/KISS1-asd vector.
Still another embodiment of the present invention provides a genetic vaccine for animal castration, comprising a vaccine vector carrying an expression vector for animal castration as described above, wherein the vaccine vector is one of a live attenuated viral vector (e.g., an adenovirus vector) and a live attenuated bacterial vector (e.g., a live attenuated salmonella choleraesuis).
The advantages of the animal castration gene vaccine over the prior art are the same as those of the animal castration fusion gene described above over the prior art, and are not described in detail herein.
In some specific embodiments, the vaccine vector is attenuated salmonella choleraesuis C500.
Optionally, the genetic vaccine further comprises pharmaceutically acceptable excipients, wherein the pharmaceutically acceptable excipients comprise one or more of solvents, dispersants, diluents, fillers, wetting agents, binders, disintegrants, lubricants, preservatives, suspending agents, emulsifiers, excipients, flavoring agents and carriers. Pharmaceutically acceptable excipients refer to components that do not interfere with the efficacy of the biological activity of the vaccine carrier and that are not significantly toxic to the body at the concentrations at which they are administered, including any one or a combination of at least two of solvents, dispersants, diluents, fillers, wetting agents, binders, disintegrants, lubricants, preservatives, suspending agents, emulsifiers, excipients, flavoring agents, immunoadjuvants, and the like. The use of the aforementioned components in the field of vaccines is well known in the art. For example, the solvents include, but are not limited to: water, phosphate Buffered Saline (PBS), each component may be used alone or in combination with a plurality of kinds.
The invention also provides the application of the fusion gene for animal castration, the expression vector for animal castration and the gene vaccine for animal castration in preparing medicines for controlling animal oestrus performance including but not limited to reducing animal oestrus aggression and controlling fertility.
The advantages of the fusion gene for animal castration, the expression vector for animal castration and the gene vaccine for animal castration in the preparation of the expression medicine for controlling the oestrus of animals are the same as those of the fusion gene for animal castration compared with the prior art, and are not described in detail herein.
The invention will be further illustrated with reference to specific examples. The experimental methods in which specific conditions are not specified in the following examples are generally conducted under conventional conditions, for example, those described in the molecular cloning Experimental guidelines (fourth edition) published in Cold spring harbor laboratory, or are generally conducted under conditions recommended by the manufacturer.
Examples
1. Acquisition of 2a/S-NKB Gene of interest
Referring to CDS sequence of NKB gene (accession number: XM_ 004006562.5) in GenBank, and connecting HBsAg-S antigen gene (see SEQ ID NO: 4) and 2A peptide gene (see SEQ ID NO: 5) in series upstream of CDS sequence of NKB gene (see SEQ ID NO: 2), adding NheI and HindIII cleavage sites at two ends to form NheI-2A/S-NKB-HindIII target gene fragment, sending the fragment to Wohan Jin Kairui bioengineering Co., ltd, synthesizing the synthesized fragment, inserting the synthesized fragment into pUC57 plasmid, named pUC-2A/S-NKB-sp for short, and transferring the synthesized fragment into escherichia coli to obtain positive clone.
2. E.coli positive clone culture
Activating attenuated Salmonella choleraesuis C500 (carrying pVAX-S/GnRH-2a/KISS 1-asd) stored at-80deg.C on LB solid medium; coli strain x6097 and Salmonella choleraesuis C500 lacking asd gene and crp gene were activated on LB solid medium containing DAP (final concentration 50. Mu.g/mL); coli transformed with the synthetic plasmid pUC-2a/KISS1-sp was activated on LB solid medium containing Amp (final concentration: 50. Mu.g/mL), and cultured in a constant temperature incubator at 37 ℃. After overnight growth, single colonies were picked up in sequence in the corresponding liquid LB medium, and placed in a constant temperature shaking table at 37 ℃ for culture at 220r/min overnight.
2.1pUC-2a/S-NKB-sp plasmid Small extraction procedure
(1) 1-5mL of the overnight cultured bacterial liquid was taken and added to a centrifuge tube (self-contained), and the bacteria were collected by centrifugation at 13000rpm for 30 seconds, and the whole supernatant was removed as much as possible.
(2) 250. Mu.L Buffer P1 was added to the centrifuge tube with the bacterial pellet left, and the mixture was thoroughly mixed using a pipette or vortex shaker to suspend the bacterial pellet. Note that: if the fungus blocks are not thoroughly mixed, the cracking effect is affected, so that the extraction quantity and purity are lower.
(3) 250 mu L Buffer P2 is added into the centrifuge tube, and the mixture is gently mixed up and down for 8 to 10 times, so that the thalli are fully cracked and placed for 3 to 5 minutes at room temperature. At this point the solution became clear and viscous.
(4) 250 μl Buffer E3 was added to the centrifuge tube, immediately mixed upside down for 8-10 times, and white flocculent precipitate appeared at this time, and left at room temperature for 5 minutes. Centrifugation at 13000rpm for 5min, aspiration of the supernatant, loading of the supernatant into a filtration column (Endo-reverse FM), centrifugation at 13000rpm for 1min, filtration, and collection of the filtrate in a centrifuge tube (self-contained).
(5) To the filtrate, 225. Mu.L of isopropyl alcohol was added and mixed upside down.
(6) Column balance: 200. Mu.L of Buffer PS was added to the adsorption column (Spin Columns DM) loaded in the collection tube, centrifuged at 13000rpm for 1 minute, the waste liquid in the collection tube was discarded, and the adsorption column was replaced in the collection tube.
(7) The mixed solution of the filtrate and isopropyl alcohol in step 5 was transferred to an equilibrated adsorption column (loaded into a collection tube). Centrifugation at 13000rpm for 1 minute, pouring off the waste liquid in the collection tube, and putting the adsorption column back into the collection tube again.
(8) 750. Mu.L Buffer PW (please check whether absolute ethanol has been added) was added to the column, centrifuged at 13000rpm for 1min and the waste liquid in the collection tube was decanted.
(9) The column was replaced in the collection tube and centrifuged at 13000rpm for 1 minute.
(10) The adsorption column was placed in a new collection tube, 50-100. Mu.L of Endo-Free Buffer EB was added to the middle of the adsorption membrane, and the mixture was left at room temperature for 2-5 minutes and centrifuged at 13000rpm for 2 minutes, and pUC-2A-S/NKB-sp plasmid solution was collected in a centrifuge tube. The plasmid was stored at-20 ℃.
(11) The column was replaced in the collection tube and centrifuged at 13000rpm for 1 minute.
(12) The adsorption column was placed in a new collection tube, 50-100. Mu.L of Endo-Free Buffer EB was added to the middle of the adsorption membrane, and the mixture was left at room temperature for 2-5 minutes and centrifuged at 13000rpm for 2 minutes, and pUC-2A-S/NKB-sp plasmid solution was collected in a centrifuge tube. The plasmid was stored at-20 ℃.
2.2 amplification of fragments of interest
Designing homologous recombination primer (TY-F/R) according to the target fragment 2a/S-NKB gene, and enabling two ends to contain HindIII enzyme cutting sites to obtain an amplified product HindIII-2 a/S-NKB-HindIII, wherein the primer sequences are as follows:
TY-F: GGGCAGAGGAAAATAAAGCTTATGGGCAGTGGAGAGGGC (see SEQ ID NO: 7);
TY-R: GCCCTCTCCACTGCCAAGCTTTTCCACACTGGGTGGGTACTTAA (see SEQ ID NO: 8);
3. construction of the three expression vectors pVAX-S/GnRH-2a/S-NKB-2a/KISS1-asd
3.1 ligation of plasmid pVAX-S/GnRH-2a/S-NKB-2a/KISS1-asd
Referring to FIG. 1, the HindIII-2 a/S-NKB-HindIII target gene fragment was ligated into the pVAX-S/GnRH-2a/KISS1-asd vector using homologous recombination, and the 2a/S-NKB gene fragment and the HindIII single-digested plasmid pVAX-S/GnRH-2a/KISS1-asd linearized fragment were ligated using homologous recombination, the ligation system being shown in Table 1.
TABLE 1 ligation System for plasmid pVAX-S/GnRH-2a/S-NKB-2a/KISS1-asd
The components in the above table were mixed well, centrifuged briefly and then placed on ice for 30min at 37℃and immediately transformed into E.coli x6097 in a brief ice bath.
3.2 transformation of bacteria
(1) 100. Mu.L of competent cells x6097 were thawed by removing ice from-80 ℃; adding 10. Mu.L of the ligation product; gently mixing, and ice-bathing for 30min;
(2) Transferring to 42 ℃ for heat shock for 90s, then rapidly taking out and placing on ice for 2min, wherein the centrifuge tube cannot be shaken in the process;
(3) Adding 500mL LB liquid culture medium without DAP, mixing uniformly, and placing at 37 ℃ for shake culture at 220r/min for 1h to rejuvenate bacteria;
(4) Centrifuging the rejuvenated bacterial liquid for 5min at 3000r/min, discarding part of supernatant, and reserving about 50 mu L of supernatant to resuspend the bacterial body;
(5) Gently blowing up the precipitate, mixing, transferring to LB solid medium (without DAP), spreading the liquid evenly, and placing in a 37 ℃ incubator. After 30min of incubation, the plate was inverted and incubated overnight at 37℃to see if transformed colonies were growing.
3.3 screening and identification of Positive clones
Single colonies cultured overnight after transformation were picked separately and cultured overnight with shaking at 37℃in LB liquid medium without DAP, plasmid extraction was performed using the plasmid miniextraction kit, and the plasmids after extraction were identified by restriction enzyme digestion using the corresponding restriction enzymes (NheI and XhoI, hindIII and XhoI and HindIII). Taking 10 mu L of the product after enzyme digestion, separating out a target band through 2% agarose gel electrophoresis, screening suspected recombinant plasmids, sending the plasmids to a new technology limited company of Wuhan engine science for sequencing, comparing the detection sequences with target sequences, wherein the plasmids with correct sequences are named as pVAX-S/GnRH-2a/S-NKB-2a/KISS1-asd, corresponding bacterial liquid is named as x6097 (pVAX-S/GnRH-2 a/S-NKB-2a/KIS S1-asd), and carrying out expansion culture on the bacterial liquid.
3.3.1 identification of Positive clones by digestion
The NheI and XhoI, nheI and HindIII double enzyme digestion are respectively used for single enzyme digestion, and after agarose gel irradiation, clear and bright specific bands are found in a gel diagram of the NheI and XhoI double enzyme digestion at 2000-3000 bp and are close to the size of a target fragment (2622 bp); the NheI and HindIII double-restriction gel shows two bands, approximately at 500bp and above 1000bp, approaching two target fragment sizes, 498bp and 1156bp; the HindIII single cut gel shows a specific band, approaching the 2a/S-NKB size (1156 bp) at 1000bp above.
3.3.2 sequencing identification of Positive clones
Through sequencing pVAX-S/GnRH-2a/S-NKB-2a/KISS1-asd plasmid by Wohan engine science, analysis results show that the sequence after splicing is consistent with the insertion direction and the base sequence of the fusion gene of the target sequence S-GnRH-2a/S-NKB-2a/KISS 1.
This example separates three genes by means of self-cleaving 2A peptide, enabling independent expression. The sequence of the gene linkage was S-GnRH-2a-S-NKB-2a-KISS1, and specifically, the DNA sequence of the fusion gene inserted into the three expression vectors was as follows:
note that, from upstream to downstream, the underlined wavy line indicates NheI, hindIII, hindIII and XhoI cleavage sites in order, the italic portion indicates the hepatitis b surface antigen HBsAg-S (abbreviated as S) gene, the underlined straight line indicates the 2A peptide gene, and the shaded portion indicates the GnRH gene, NKB gene, KISS1 gene in order.
4. Construction of genetic vaccine
And transferring the tri-expression vector pVAX-S/GnRH-2a/S-NKB-2a/KISS1-asd with correct sequencing into the C500 competence of salmonella choleraesuis in an electrotransformation mode to obtain the C500 competence engineering bacteria of tri-expression anti-gonadotropin releasing hormone, neurokinin B and KISS, and taking the engineering bacteria as a subsequent genetic vaccine.
5. Gene vaccine immunized mice
The gene vaccine (T2) of the invention is injected into the quadriceps femoris two-point of a 3-week-old Kunming male mouse serving as a study object, and the immune dose is 3 multiplied by 10 8 CFU, boost every two weeks after the first immunization, boost twice altogether. Salmonella choleraesuis C500 (T1) injected with an equivalent amount of the pVAX-S/GnRH-2a/KISS1-asd vector and PBS 100 microliters (C) injected were used as controls.
6. Immune results and analysis
6.1, gnRH, KISS1 and NKB antibody levels
Blood is collected every 2W, and the generation condition of GnRH, KISS1 and NKB antibodies in the immunized mice is detected by adopting an indirect ELISA method, and the specific steps are as follows:
(1) Each well of the 96-well plate was coated with standard GnRH, KISS1, NKB antigen 100 ng/100. Mu.L, and incubated overnight at 4 ℃.
(2) The reaction solution was discarded, and washed with PBST 4 times, 200. Mu.L/well for 3min each.
(3) Blocking solution (i.e., 1% BSA solution) was added at 150. Mu.L/well and incubated at 37℃for 2h.
(4) The reaction solution was discarded, and washed with PBST 4 times, 200. Mu.L/well for 3min each.
(5) 100 μl/well of plasma to be tested (1:25, 1:50, 1:100, 1:200, 1:400, 1:800, 1:1600 dilution) was added, while negative control wells, non-specific adsorption wells (and PBST instead of plasma) and zero control were set, and incubated for 90min at 37 ℃.
(6) The reaction solution was discarded, and washed with PBST 4 times, 200. Mu.L/well for 3min each.
(7) Goat anti-mouse IgG-HRP (Boolon, 1:3000 dilution) was added at 100. Mu.L/well and reacted at 37℃for 1h.
(8) The reaction solution was discarded, and washed with PBST 4 times, 200. Mu.L/well for 3min each.
(9) Adding 100 mu L/hole of single-component TMB color development liquid, and reacting for 15min at 37 ℃ in a dark place.
(10) Adding 2mol/L H 2 SO 4 The reaction was stopped at 50. Mu.L/well of stop solution, and the OD of each well was measured at a wavelength of 450nm within 15 minutes. At the same dilution, if the OD of the sample serum 450 Value of>Negative serum OD 450 Mean +2 Standard Deviation (SD), the dilution factor is the final antibody titer of the sample serum, and is determined to be positive, otherwise negative.
The results are shown in FIGS. 2-4, wherein the abscissa represents time (weeks) and the ordinate represents GnRH, KISS1 and NKB antibody titers, respectively. As can be seen from the figure, gnRH and KISS1 antibody titers increased and decreased in the post-immunization double-expression injection group (T1 group) and the triple-expression injection group (T2 group) with increasing number of immunizations, while NKB antibodies continued to increase; and the antibody titer of the immune group in each period is obviously increased (P is less than 0.01) compared with that of the blank control group.
6.2 analysis of related hormone levels
Blood was collected every 2W to detect testosterone (T) and estradiol (E2) levels, and the levels of GnRH, KISS1 and NKB in serum from mice treated differently were detected by competition ELISA according to the kit instructions, as follows:
(1) The required strips were removed from the aluminium foil bags after 20min equilibration at room temperature and the remaining strips were sealed with a self-sealing bag and returned to 4 ℃.
(2) Setting a standard substance hole and a sample hole, wherein 50 mu L of standard substances with different concentrations are added into the standard substance hole; sample Kong Xianjia to-be-measured sample 10 mu L, and then 40 mu L of sample diluent is added; blank holes are not added.
(3) In addition to the blank wells, 100. Mu.L of horseradish peroxidase (HRP) -labeled detection antibody was added to each of the standard wells and sample wells, the reaction wells were sealed with a sealing plate membrane, and incubated in an incubator at 37℃for 60min.
(4) Removing liquid, beating to dry on the water-absorbing paper, filling the washing liquid in each hole, standing for 1min, throwing away the washing liquid, beating to dry on the water-absorbing paper, and repeating the plate washing for 5 times.
(5) Adding 100 mu L/hole of single-component TMB color development liquid, and reacting for 15min at 37 ℃ in a dark place.
(6) Adding 2mol/L H 2 SO 4 The OD of each well was measured at a wavelength of 450nm in 15min at 50. Mu.L/well of the stop solution.
(7) A standard curve was drawn and the concentrations of estradiol and progesterone in the sample (calculated value multiplied by 5-position final concentration value) were calculated from the standard curve.
The results are shown in FIGS. 5-6, where the abscissa indicates time (weeks) and the ordinate indicates testosterone (T) and estradiol (E2) concentrations, respectively, and the data superscripts in the time periods (e.g., a, b, and c) are completely different and indicate significant differences (p < 0.05). As can be seen from the figure, both the three-expression injection group (T2 group) and the two-expression injection group (T1 group) were significantly reduced and the difference was extremely significant (P < 0.001) compared to the control group (C). At weeks 2-6, testosterone levels were reduced by 60.1%, 59.5% and 65.12% respectively for the three expression injection group (T2 group) compared to the contemporaneous control group, and estrogen levels were reduced by 48.26%, 42.74% and 58.41% respectively compared to the contemporaneous control group; testosterone levels were reduced by 15.27%, 24.47% and 64.06% in the double expression injection group (T1 group) compared to the contemporaneous control group, respectively, and estrogen levels were reduced by 27.46%, 20.75% and 19.58% compared to the contemporaneous control group, respectively. Although serum testosterone levels of the double-expression injection group (T1 group) and the triple-expression injection group (T2 group) were reduced by more than 50% after 6 weeks of immunization, the difference was very remarkable (P < 0.001), the triple-expression injection group (T2 group) could reduce testosterone levels to less than 50% at week 2 and could reduce estradiol levels to nearly half, whereas the double-expression injection group (T1 group) took 6 weeks to achieve reduction of serum testosterone levels to less than 50% and at this time estradiol levels were reduced by only about 20%, and it was seen that the triple-expression injection group of the present invention had a rapid onset of hormone levels.
6.3 testis Mass analysis
After 6 weeks of immunization, the cervical dislocation of the mice was killed, epididymis was taken, and morphological changes were observed. Immediately placing epididymis on one side into 1 milliliter of capacitation liquid, shearing the epididymis by using scissors and a syringe needle, placing the epididymis on a 37 ℃ incubator to enable the epididymis to be free for 30 minutes, sucking 1 drop of semen to drop on a glass slide, uniformly wiping the glass slide, naturally air-drying the epididymis, dyeing the epididymis by using coomassie brilliant blue, washing the epididymis with running water for 3 times, each time for 1 minute, sucking the surrounding water by using clean absorbent paper, and placing the epididymis under a 100-time microscope to observe sperm density.
As a result, see fig. 7, from which it was found that the number of sperm in mice injected with the double-and triple-expressed gene vaccines was significantly reduced, and the sperm density of the triple-expressed gene vaccine was the lowest.
6.4 testis tissue analysis
After 6 weeks of immunization, the cervical dislocation of the mice is killed, epididymis of the mice is adopted, and testis HE staining and slicing are carried out to observe the testis tissue morphology.
As a result, as shown in FIG. 8, it was found from the graph that the production of germ cells, particularly sperm cells, in testes of mice injected with the double-expression and triple-expression gene vaccine was significantly reduced as compared with the control group (C), that the triple-expression group was significantly less than the double-expression group, and that germ cells at each stage in the seminiferous tubules were irregularly arranged. The result shows that the three-expression gene vaccine can lead to the reduction of the seminiferous capacity of the male mice after immunization, and the effect is superior to that of the double expression group.
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, and alternatives falling within the spirit and principles of the invention.
Claims (10)
1. A fusion gene for castration of animals, comprising, from upstream to downstream, an S-GnRH gene, an S-NKB gene, and a KISS1 gene, the S-GnRH gene being formed by an upstream junction HBsAg-S antigen gene of a GnRH gene, the S-NKB gene being formed by an upstream junction HBsAg-S antigen gene of a NKB gene, and a 2A peptide gene being joined between the S-GnRH gene and the S-NKB gene and between the S-NKB gene and the KISS1 gene.
2. The fusion gene for animal castration according to claim 1, wherein the GnRH gene has a DNA sequence as set forth in SEQ ID NO:1 is shown in the specification;
the DNA sequence of the NKB gene is shown in SEQ ID NO:2 is shown in the figure;
the DNA sequence of the KISS1 gene is shown as SEQ ID NO:3 is shown in the figure;
the DNA sequence of the HBsAg-S antigen gene is shown in SEQ ID NO:4 is shown in the figure;
the DNA sequence of the 2A peptide gene is shown in SEQ ID NO: shown at 5.
3. The fusion gene for animal castration according to claim 2, wherein a first cleavage site is provided upstream of the S-GnRH gene, a second cleavage site is provided upstream of the 2A peptide gene, a third cleavage site is provided downstream of the KISS1 gene, and the first cleavage site, the second cleavage site and the third cleavage site are different.
4. The fusion gene for castration of an animal of claim 3, wherein the first cleavage site is gctag c, the second cleavage site is AAGCTT, and the third cleavage site is CTCGAG.
5. The fusion gene for animal castration of claim 4, wherein the full-length DNA sequence of the fusion gene is set forth in SEQ ID NO: shown at 6.
6. An expression vector for animal castration, wherein the expression vector for animal castration carries the fusion gene for animal castration of any one of claims 1-5.
7. The animal castration expression vector of claim 6, wherein the starting vector for the animal castration expression vector is a eukaryotic expression vector pvax-asd.
8. A genetic vaccine for animal castration, comprising a vaccine vector carrying the expression vector for animal castration according to any one of claims 6 to 7, wherein the vaccine vector is a live attenuated viral vector or a live attenuated bacterial vector.
9. The genetic vaccine for animal castration of claim 8, wherein the vaccine vector is attenuated salmonella choleraesuis C500.
10. Use of a fusion gene for animal castration according to any one of claims 1-5, an expression vector for animal castration according to any one of claims 6-7, a genetic vaccine for animal castration according to any one of claims 8-9 for the preparation of a medicament for controlling oestrus in an animal.
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