CN112480265B - GNRH-I6 virus-like particle subunit vaccine - Google Patents

Abstract

The invention belongs to the field of molecular biology, and discloses a GNRH-I6-Q beta virus-like particle subunit vaccine, and provides a composition, which comprises: a virus-like particle Q β (VLP) and a GNRH-I6 repeat polypeptide; wherein the Q β (VLP) and the one GNRH-I6 repeat polypeptide are linked to each other by Sotase A enzyme catalysis. Experiments prove that the subunit vaccine prepared from the recombinant protein expressed by the invention has high antigen purity and good safety, is free from pathogenicity to animals such as mice and the like, and is easy to pass safety evaluation.

Description

GNRH-I6 virus-like particle subunit vaccine

Technical Field

The present invention relates to the fields of molecular biology, virology, immunology and medicine. The present invention provides a composition comprising: a virus-like particle Q β (VLP) and a GnRH-I6 repeat polypeptide protein, wherein the Q β (VLP) and the GnRH-I6 repeat polypeptide protein are linked to each other to form the GnRH-I6-Q β virus-like particle.

Background

In 1971, baba et al, yes Amoss, discovered GnRH-I in the hypothalamus of pigs and sheep, respectively, and determined its molecular structure, and named Luteinizing Hormone Releasing Hormone (LHRH) because of its function of promoting the pituitary release of Luteinizing Hormone (LH). Later studies have found that this hormone also promotes the release of Follicle Stimulating Hormone (FSH) from the pituitary, and is therefore also known as follicle stimulating hormone-releasing hormone (FSHRH). Collectively, they are known as gonadotropin releasing hormone (GnRH-I).

Since the discovery and molecular structure of GnRH-I in the 70's of the 20 th century, at least 24 families of GnRH-I have been discovered to date, and the amino acid sequence of GnRH-I is highly conserved among all mammals. The amino acid sequence is pyroglutamic acid (PGlu) -histidine (His) -tryptophan (Trp) -serine (Ser) -tyrosine (Tyr) -glycine (Gly) -leucine (Leu) -arginine (Arg) -proline (Pro) -glycinate (Gly-NH 2), and the molecular weight is 1.181KDW.

The physiological dose of GnRH-I can promote the increase of gonadotropin concentration (such as the slight shift of FSH and the obvious increase of LH), promote the synthesis and secretion of gonadotropin (such as estradiol, progesterone, testosterone and the like), promote the developmental maturation of follicles and ovulation or the development of testis and the maturation of sperms, and generate and maintain the second sexual characteristics. In addition, gnRH-I can also directly affect the gonads, regulate the synthetic secretion of gonadal steroid hormones, promote gametogenesis, and immunity against GnRH-derived antigens has been reported.

Autoantigen proteins are often difficult to induce an antibody response against an autoantigen. One way to increase the efficiency of vaccination is to increase the reproducibility of the antigen used. Unlike isolated proteins, viruses induce rapid and potent immune responses without any adjuvant and with and without T cell help (Bachmann and Zinkernegel, ann. Rev. Immunol:15:235-270 (1991)). They are able to trigger a much stronger immune response than their isolated components, compared to a few proteins. For B cell responses, it is well known that one of the key factors in viral immunogenicity is the repetitiveness and order of surface epitopes. Many viruses exhibit a quasicrystal surface with regularly arranged epitopes that can effectively cross-link epitope-specific immunoglobulins on B cells (Bachxnann and Zinkernagel, immunol. Today 17-553-558 (1996)). This crosslinking of B cell surface immunoglobulins is a strong activation signal that directly induces cell cycle progression and IgM antibody production. In addition, such triggered B cells are able to activate T helper cells, thereby inducing IgM antibody to IgG antibody conversion in B cells and the generation of any vaccinated long-lived B cell memory targets. (Zinkernagel, ann. Rev. Immunol.15:235-270 (1997)). Viral structures are even involved in the production of anti-antibodies in autoimmune diseases and are part of the natural response to pathogens (see Fehr, t., et al, j.exp. Med.185:1785-1792 (1997)). Thus, antigens presented by highly organized viral surfaces are capable of inducing strong antibody responses against the antigens.

VLPs are produced by gene fusion expression or by coupling exogenous antigens to VLPs as carriers by chemical coupling, and recombinant VLPs produced by the latter method are also commonly referred to as coupled VLPs.

The fusion expression method comprises the following steps of fusing exogenous antigen DNA with polypeptide gene with self-assembly capacity on the basis of preparing non-envelope VLP, and further preparing non-envelope cVLP; similar to the preparation of enveloped VLPs, different species (types) of viral or chimeric proteins are combined to form VLPs (enveloped or non-enveloped). However, the chimeric vaccine has low yield and high production cost, and is easy to cause incompatibility in product structure and components in large-scale production, so that the development of the chimeric vaccine in the vaccine is limited.

The transpeptidase SortaseA is isolated from staphylococcus aureus and is capable of selectively recognizing the specific polypeptide sequence LPXTG and cleaving the peptide bond of the amino acid at a specific site, thereby linking it to a new peptide chain and linking the antigen to the pre-assembled VLP in vitro. The method has the advantages that the size and the structure of the target antigen are not limited by conditions such as VLP monomer folding and particle assembly, and short linear peptides, peptide loops and full-length proteins, even non-protein antigens such as polysaccharides and haptens can be displayed on the surface of the VLP. The Swiss Cytos biopharmaceutical company prepares the smoking cessation vaccine by covalently coupling nicotine and the surface of bacteriophage Q beta (VLP), and the clinical evaluation result of the I/II phase shows that the nicotine-Q beta virus-like particles can induce high-titer nicotine-specific antibodies. And the single virus-like particles can be stably expressed in escherichia coli, so that the yield is improved, and the cost is reduced.

Disclosure of Invention

Aiming at the defects in the prior art, the invention provides the GNRH-I6 virus-like particle subunit vaccine which is easy to obtain by thallus culture, has high connection efficiency, no tags such as his and the like, high expression yield, convenient industrial production, strong immunogenicity and quick immunoreaction.

The invention provides a GNRH-I-AP205 protein expressed by recombinant escherichia coli, and GNRH-I6 repeat polypeptide and Q beta virus-like particles are catalyzed by sortaseA to form a recombinant protein GNRH-I6-Q beta; the recombinant protein GNRH-I6-Q beta amino acid sequence is SEQ ID NO.1.

The second aspect of the invention provides a recombinant plasmid his-MBP-TEV-GNRH-I6-PETM41, wherein the recombinant plasmid his-MBP-TEV-GNRH-I6-PETM41 is obtained by cloning the nucleotide shown in SEQ ID NO.2 into the NcoI site and the BamHI site of a prokaryotic expression vector PETM41 through a homologous recombination method.

The third aspect of the invention provides a recombinant plasmid his-TEV-Q beta-pET 28a, wherein the recombinant plasmid his-TEV-Q beta-pET 28a is obtained by cloning a nucleotide fragment shown in SEQ ID NO.3 into a prokaryotic expression vector, namely, between two enzyme cutting sites NcoI and Xho I of pET28 a.

The fourth aspect of the invention provides a recombinant plasmid his-TEV Protease-pET28a, wherein the recombinant plasmid his-TEV Protease-pET28a is obtained by cloning a nucleotide fragment shown in SEQ ID NO.4 into a prokaryotic expression vector, namely between NcoI and Xho I enzyme cutting sites of pET28 a.

The fifth aspect of the invention provides a recombinant plasmid his-Sotase A-pET28a, wherein the recombinant plasmid his-Sotase A-pET28a is obtained by cloning a nucleotide fragment shown in SEQ ID NO.5 into a prokaryotic expression vector, namely, between two enzyme cutting sites NcoI and Xho I of pET28 a.

The invention provides a preparation method of GNRH-I6-Q beta virus-like particles, which comprises the following steps:

(1) Synthesizing gene sequences in SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO.5 into a target gene, and constructing a recombinant vector, wherein the sequence is as follows:

his-MBP-TEV-GNRH-I6-PETM41；

his-TEV-Qβ-pET28a；

his-TEV Protease-pET28a；

his-Sotase A-pET28a；

(2) Transforming the recombinant plasmid with correct sequence determination in the step (1) into Escherichia coli T7shuffle to respectively obtain recombinant expression strains:

his-MBP-TEV-GNRH-I6-PETM41-T7 shuffle；

his-TEV-Qβ-pET28a-T7 shuffle；

his-TEV Protease-pET28a-T7 shuffle；

his-Sotase A-pET28a-T7 shuffle；

(3) Respectively culturing recombinant expression strains in the step II, adding IPTG (isopropyl-beta-thiogalactoside) for induction expression, collecting thalli, carrying out ultrasonic crushing, and purifying by a nickel column to respectively obtain recombinant proteins:

his-MBP-TEV-GNRH-I6 recombinant protein;

his-TEV-Q β virus-like particles;

his-TEV enzyme;

his-Sotase A enzyme;

(4) Dissolving the his-MBP-TEV-GNRH-I6 recombinant protein and the his-TEV-Q beta virus sample particles in the step three by using a PBS solution, respectively adding the his-TEV enzyme for enzyme digestion, carrying out enzyme digestion according to the proportion of 10ug of the his-TEV enzyme for enzyme digestion of 1mg of recombinant protein, carrying out oscillation enzyme digestion for 3h at 30 ℃, respectively passing enzyme digestion solutions through nickel columns, respectively concentrating penetrating solutions which are the GNRH-I6 recombinant protein and the Q beta virus sample particles by using a 3K concentration tube;

(5) And preparing the his-MBP-TEV-GNRH-I6 recombinant protein and the his-TEV-Q beta virus-like particles in the fourth step into 1mg/ml by PBS according to the weight ratio of 1:1, adding his-Sotase A enzyme in the step three, carrying out oscillation catalysis at 37 ℃ for 5 hours according to the proportion of catalyzing 2mg of mixed protein by 100ug, then passing the catalytic solution through a nickel column, and concentrating the catalytic solution into GNRH-I6-Q beta virus sample particles by using a 3K concentration tube.

Further, when the recombinant expression strain is cultured until OD600 reaches 0.6-0.8, IPTG with the final concentration of 1mM is added for induced expression for 12 hours, then the thalli are collected and mixed with 10mM PBS for ultrasonication, and then a nickel column is used for purification, 25mM imidazole is used for washing 10 column volumes, and 250mM imidazole is used for eluting target protein.

The seventh aspect of the invention provides a subunit vaccine comprising the GNRH-I6-Q β virus-like particle prepared according to the sixth aspect and a foss adjuvant, for use in the manufacture of a vaccine.

According to the invention, the Escherichia coli recombinant expression strain of the Q beta virus-like particles is constructed, so that the recombinant expression strain is successfully obtained, is easy to obtain by thallus culture, is suitable for industrial production, is added with His tags, is convenient for purifying the Q beta virus-like particles, and reduces the cost.

The invention can obtain the Q beta virus-like particles without labels by cutting the his labels with TEV enzyme. Simple operation, high efficiency and suitability for refined vaccine production.

According to the invention, the his-MBP label is cut off by TEV enzyme, and the GNRH-I6 recombinant protein without the label can be obtained. Simple operation, high efficiency and suitability for refined vaccine production

In the invention, Q beta is derived from a bacteriophage infecting escherichia coli, has the function of self-assembling phage capsid protein into nano particles, has no infectivity, has strong antigen immunity, and is a unique technology of the VLP screening platform of the applicant; the antigen European Union with the Q beta virus-like particle shows high-intensity antigenicity, can induce high-level specific antibodies in mice, rats, cats, dogs and horses, and has fast humoral immune response; the Q beta virus-like particles can be expressed by escherichia coli in large quantity, the production process is simple, and the purity of the Q beta virus-like particles is high; the GNRH-I6 recombinant protein can be expressed in a large number of soluble forms by escherichia coli, the production process is simple, and the GNRH-I6 recombinant protein has high purity; the Q beta virus-like particles are connected with the GNRH-I6 protein in vitro, the conditions are convenient to control, the connection efficiency is high, the operation is easy, and the industrial production is convenient; the escherichia coli recombinant expression strain selected by the invention has the characteristics of fast growth, easy culture, simple genetic operation, fast propagation speed, low requirement on culture conditions, cheap culture medium, capability of high-density culture, high hydrostatic pressure tolerance, convenience for industrial production and the like.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below. It should be apparent that the drawings in the following description are merely exemplary, and that other embodiments can be derived from the drawings provided by those of ordinary skill in the art without inventive effort.

FIG. 1 is a schematic diagram of a recombinant plasmid obtained by connecting the GNRH-I6 gene with the pETM41 plasmid provided in the present embodiment;

FIG. 2 is a schematic diagram of a recombinant plasmid obtained by connecting a Q beta gene with a pET28a plasmid provided in an embodiment of the present invention;

FIG. 3 is a schematic diagram of a recombinant plasmid obtained by ligating the TEV protease gene with a pET28a plasmid;

FIG. 4 is a schematic diagram of a recombinant plasmid obtained by connecting the Sortase A gene with the pET28a plasmid provided in the embodiment of the present invention;

FIG. 5 is a schematic illustration of SDS-PAGE run of GNRH-I6 protein provided in an example of the present invention;

FIG. 6 is a SDS-PAGE run of the Q.beta.virus-like particle protein provided in examples of the present invention;

FIG. 7 is a schematic SDS-PAGE gel of his-TEV enzyme protein provided in the examples of the present invention;

FIG. 8 is a schematic SDS-PAGE gel-run of his-Sortase A protein provided in the examples of the present invention;

FIG. 9 is a schematic diagram of SDS-PAGE gel running of the results of 1h of his-Sortase A catalysis of GNRH-I6 protein and Q β virus-like particles provided in the examples of the present invention;

FIG. 10 is a schematic diagram of SDS-PAGE gel running of the results of 5h catalysis of the GNRH-I6 protein and Q beta virus-like particles by his-Sortase A.

Detailed Description

In order to make the aforementioned objects, features and advantages of the present invention comprehensible, embodiments accompanied with figures are described in detail below. In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein, as those skilled in the art will recognize without departing from the spirit and scope of the present invention.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.

Molecular biology experimental methods such as enzyme digestion and homologous recombination ligation used in the examples of the present invention can be referred to in molecular cloning, second edition. The base materials for preparing the GNRH-I6-Q β virus-like particles of the present invention include: a Q beta protein nucleotide sequence, a GNRH-I6 nucleotide sequence, a TEV protease nucleotide sequence, a sortaseA nucleotide sequence, a pET28a plasmid, a PETM41 plasmid, a T7Shuffle escherichia coli strain, a small root plasmid extraction kit, an LB culture medium, IPTG, kanamycin, a nickel column, imidazole and the like. The nucleotide sequence is sent to Huada gene company to synthesize his-TEV-Q beta recombinant plasmid, his-MBP-TEV-GNRH-I6-PMET41 recombinant plasmid, his-TEV Protease-pET28a recombinant plasmid and his-sortaseA-pET28a recombinant plasmid.

Example 1

Provided is a method for preparing his-TEV-Q beta virus-like particles, which comprises the following steps:

(1) Construction of recombinant plasmid: the his tag, the TEV enzyme cutting site and the Q beta gene are directly synthesized into a pET28a vector NcoI enzyme cutting site and an XhoI enzyme cutting site by Huada gene company to obtain a recombinant plasmid his-TEV-Q beta-pET 28a;

(2) Transferring the recombinant plasmid into an expression strain: transferring the his-TEV-Q beta-pET 28a recombinant plasmid into an escherichia coli T7Shuffle expression strain to obtain a his-TEV-Q beta-pET 28a-T7Shuffle recombinant expression strain;

(3) Culturing thalli and purifying Q beta virus-like particles: culturing the his-TEV-Q beta-pET 28a-T7shuffle recombinant expression strain, and carrying out IPTG induced expression to obtain the his-TEV-Q beta virus-like particles.

Specifically, the expression and purification steps of the his-TEV-Q beta virus-like particles are as follows:

(a1) Preparation of his-TEV-Q beta-pET 28a recombinant plasmid

The plasmid-containing glycerol strain was inoculated into 5mL of 2YT medium (containing 50ug/mL kanamycin) by pipetting 5ul with a pipette, shake-cultured at 37 ℃ for 14-16 hours, and 1mL of the strain was sequenced after culture. Extracting plasmid from the residual bacterial liquid by using a small plasmid extraction kit, and detecting the concentration of nucleic acid by using a protein nucleic acid detector.

(a2) his-TEV-Q beta virus-like particle expression

Transforming T7shuffle expression strain, shaking, centrifuging and concentrating, coating kanamycin-resistant plate, culturing overnight at 37 ℃, scraping colony, inoculating 10mL 2 XYT culture medium containing ampicillin, and shaking at 37 ℃ 180r until OD value of bacterial liquid is about 0.8. The strain is transferred into 500mL 2 XYT culture medium containing ampicillin, and the strain is shaken at 37 ℃ and 180r until the OD value of the strain liquid is about 0.8. The final concentration of 0.8mM IPTG inducer was added and expression was performed overnight at 30 ℃. On the third day, the cells were collected by centrifugation at 4500rpm, 15min. And (3) ultrasonically crushing thallus 45min, opening at 3s, closing at 4s and controlling the power to be 125w. After the ultrasonic treatment is finished, collecting supernate at 9500rpm,20min and 4 ℃;

(a3) his-TEV-Q beta virus-like particle purification

The supernatant was centrifuged and purified by nickel column, which was equilibrated to 5 column volumes with PBS. Loading, washing 5 column volumes with PBS, washing 2 column volumes with 25Mm imidazole, and then washing 5 column volumes with 25Mm imidazole. Then elution was continued with 50, 100, 150, 200, 250Mm imidazole for 5 column volumes. The effluent from loading to 500mM imidazole was collected.

10uL of each pool was pipetted and added to 10uL of a2 Xprotein electrophoresis loading buffer and heated at 100 ℃ for 4min. Then protein electrophoresis is carried out, and the concentration of the collection liquid of the target strip is observed through staining. The target protein was ultrafiltered using a 3kDa ultrafiltration tube and replaced with PBS solvent.

Example 2

Referring to FIGS. 1 and 5, there is provided a method for preparing a his-MBP-TEV-GNRH-I6 recombinant protein, the method for preparing the his-MBP-TEV-GNRH-I6 recombinant protein comprising the steps of:

(1) Construction of recombinant plasmid: directly synthesizing TEV enzyme cutting sites and GNRH-I6 genes to pETM41 vector NcoI enzyme cutting sites and XhoI enzyme cutting sites by Huada gene company to obtain recombinant plasmids his-MBP-TEV-GNRH-I6-pETM41;

(2) Transferring the recombinant plasmid into an expression strain: transferring the his-MBP-TEV-GNRH-I6-pETM41 recombinant plasmid into an escherichia coli T7Shuffle expression strain to obtain a his-MBP-TEV-GNRH-I6-pETM41-T7Shuffle recombinant expression strain;

(3) And (3) culturing thalli and purifying a his-MBP-TEV-GNRH-I6 recombinant protein: culturing the his-MBP-TEV-GNRH-I6-pETM41-T7shuffle recombinant expression strain, and carrying out IPTG induced expression to obtain the his-MBP-TEV-GNRH-I6 recombinant protein.

The specific his-MBP-TEV-GNRH-I6 recombinant protein expression and purification steps are as follows:

(b1) Preparation of his-MBP-TEV-GNRH-I6-pETM41 recombinant plasmid

The plasmid-containing glycerol strain was inoculated into 5mL of 2YT medium (containing 50ug/mL kanamycin) by pipetting 5ul with a pipette, shake-cultured at 37 ℃ for 14-16 hours, and 1mL of the strain was sequenced after culture. Extracting plasmid from the residual bacterial liquid by using a small plasmid extraction kit, and detecting the concentration of nucleic acid by using a protein nucleic acid detector.

(b2) his-MBP-TEV-GNRH-I6 recombinant protein expression

Transforming T7shuffle expression strain, shaking, centrifuging and concentrating, coating kanamycin-resistant plate, culturing overnight at 37 ℃, scraping bacterial colony, inoculating 10mL kanamycin-containing 2 XYT culture medium, and shaking at 37 ℃ for 180r until the OD value of bacterial liquid is about 0.8. The strain is transferred into 500mL 2 XYT culture medium containing ampicillin, and the strain is shaken at 37 ℃ and 180r until the OD value of the strain liquid is about 0.8. The final concentration of 0.8mM IPTG inducer was added and expression was performed overnight at 30 ℃. On the third day, the cells were collected by centrifugation at 4500rpm, 15min. And (3) ultrasonically crushing thallus 45min, opening at 3s, closing at 4s and controlling the power to be 125w. After the completion of sonication, the supernatant was collected at 9500rpm,20min and 4 ℃.

(b3) Purification of his-MBP-TEV-GNRH-I6 recombinant protein

The supernatant was centrifuged and purified by nickel column, which was equilibrated to 5 column volumes with PBS. Loading, washing 5 column volumes with PBS, washing 2 column volumes with 25Mm imidazole, and then washing 5 column volumes with 25Mm imidazole. Thereafter, elution was carried out with imidazole of 50, 100, 150, 200, 250Mm for 5 column volumes in succession. The effluent from loading to 500mM imidazole was collected.

10uL of each pool was pipetted and added to 10uL of a2 Xprotein electrophoresis loading buffer and heated at 100 ℃ for 4min. Then protein electrophoresis is carried out, and the concentration of the collection liquid of the target strip is observed through staining. Performing ultrafiltration on the target protein by using a 3kDa ultrafiltration tube, and replacing the target protein by a PBS (phosphate buffer solution) solvent;

example 3

Referring to fig. 2, 3, 6 and 7, there is provided a method for preparing his-TEV recombinase, comprising the steps of:

(1) Construction of recombinant plasmid: the TEV enzyme gene is directly synthesized into pET28a vector NcoI restriction enzyme site and XhoI restriction enzyme site by Huada gene company to obtain recombinant plasmid his-TEV protease-pET28a;

(2) Transferring the recombinant plasmid into an expression strain: transferring the his-TEV protease-pET28a recombinant plasmid into an escherichia coli T7Shuffle expression strain to obtain a his-TEV protease-pET28a-T7Shuffle recombinant expression strain;

(3) And (3) culturing thalli and purifying a his-TEV recombinase: culturing the his-TEV protease-pET28a-T7shuffle recombinant expression strain, and carrying out IPTG induced expression to obtain the his-TEV recombinase.

The specific his-TEV recombinase expression and purification steps are as follows:

(c1) Preparation of his-TEV protease-pET28a recombinant plasmid

The plasmid-containing glycerol was inoculated into 5mL of 2YT medium (containing 50ug/mL kanamycin) by sucking 5ul of the plasmid-containing glycerol using a pipette, shake-cultured at 37 ℃ for 14-16 hours, and 1mL of the culture was sequenced. Extracting plasmid from the residual bacterial liquid by using a small plasmid extraction kit, and detecting the concentration of nucleic acid by using a protein nucleic acid detector.

(c2) his-TEV recombinase expression

Transforming T7shuffle expression strain, shaking, centrifuging and concentrating, coating kanamycin-resistant plate, culturing overnight at 37 ℃, scraping bacterial colony, inoculating 10mL kanamycin-containing 2 XYT culture medium, and shaking at 37 ℃ for 180r until the OD value of bacterial liquid is about 0.8. The culture medium is transferred into 500mL 2 XYT culture medium containing ampicillin, and the culture medium is shaken at 37 ℃ and 180r until the OD value of the bacterial liquid is about 0.8. The final concentration was added with 0.8mM IPTG inducer and expressed overnight at 30 ℃. On the third day, the cells were collected by centrifugation at 4500rpm, 15min. And (3) ultrasonically crushing thallus 45min, opening at 3s, closing at 4s and controlling the power to be 125w. After the completion of sonication, the supernatant was collected at 9500rpm,20min and 4 ℃.

(c3) his-TEV recombinase purification

The supernatant was centrifuged and purified by nickel column, which was equilibrated to 5 column volumes with PBS. Loading, washing 5 column volumes with PBS, washing 2 column volumes with 25Mm imidazole, and then washing 5 column volumes with 25Mm imidazole. Thereafter, elution was carried out with imidazole of 50, 100, 150, 200, 250Mm for 5 column volumes in succession. The effluent from loading to 500mM imidazole was collected.

10uL of each pool was pipetted and added to 10uL of a2 Xprotein electrophoresis loading buffer and heated at 100 ℃ for 4min. Then protein electrophoresis is carried out, and the concentration of the collection liquid of the target strip is observed through staining. Carrying out ultrafiltration on the target protein by using a 3kDa ultrafiltration tube, and replacing the target protein by a PBS solvent;

example 4

Referring to fig. 4 and 8, there is provided a method for preparing his-sortaseA recombinase, comprising the steps of:

(1) Construction of recombinant plasmid: the sortaseA enzyme gene is directly synthesized into pET28a vector NcoI enzyme cutting site and XhoI enzyme cutting site by Huada gene company to obtain recombinant plasmid his-sortaseA-pET28a;

(2) Transferring the recombinant plasmid into an expression strain: transferring the his-sortaseA-pET28a recombinant plasmid into an escherichia coli T7Shuffle expression strain to obtain a his-sortaseA-pET28a-T7Shuffle recombinant expression strain;

(3) Culturing thalli and purifying his-sortaseA recombinase: culturing the his-sortase A-pET28a-T7shuffle recombinant expression strain, and carrying out IPTG induced expression to obtain a his-sortase A recombinase.

The specific his-sortaseA recombinase expression and purification steps are as follows:

(d1) Preparation of his-sortaseA-pET28a recombinant plasmid

The plasmid-containing glycerol strain was inoculated into 5mL of 2YT medium (containing 50ug/mL kanamycin) by pipetting 5ul with a pipette, shake-cultured at 37 ℃ for 14-16 hours, and 1mL of the strain was sequenced after culture. Extracting plasmid from the residual bacterial liquid by using a small plasmid extraction kit, and detecting the concentration of nucleic acid by using a protein nucleic acid detector.

(d2) his-sortaseA recombinase expression

Transforming T7shuffle expression strain, shaking, centrifuging and concentrating, coating kanamycin-resistant plate, culturing overnight at 37 ℃, scraping bacterial colony, inoculating 10mL kanamycin-containing 2 XYT culture medium, and shaking at 37 ℃ for 180r until the OD value of bacterial liquid is about 0.8. The strain is transferred into 500mL 2 XYT culture medium containing ampicillin, and the strain is shaken at 37 ℃ and 180r until the OD value of the strain liquid is about 0.8. The final concentration of IPTG inducer was added at 0.8mM and expression was performed overnight at 30 ℃. On the third day, the cells were collected by centrifugation at 4500rpm, 15min. And (4) ultrasonically crushing thallus 45min, opening 3s, closing 4s and keeping the power at 125w. After the completion of sonication, the supernatant was collected at 9500rpm,20min and 4 ℃. (ii) a

(d3) his-sortaseA recombinase purification

The supernatant was centrifuged and purified by nickel column, which was 5 column volumes equilibrated with PBS. Loading, washing with PBS for 5 column volumes, washing with 25Mm imidazole for 2 column volumes, and then washing with 25Mm imidazole for 5 column volumes. Then elution was continued with 50, 100, 150, 200, 250Mm imidazole for 5 column volumes. The effluent from loading to 500mM imidazole was collected.

10uL of each pool was pipetted and added to 10uL of a2 Xprotein electrophoresis loading buffer and heated at 100 ℃ for 4min. Then protein electrophoresis is carried out, and the concentration of the collection liquid of the target strip is observed through staining. Carrying out ultrafiltration on the target protein by using a 3kDa ultrafiltration tube, and replacing the target protein by a PBS solvent;

example 5

The his-TEV-Q beta virus-like particles in example 1 and the his-MBP-TEV-GNRH-I6 recombinant proteins in example 2 are dissolved in PBS (10 mM), respectively added with the his-TEV recombinase in example 3 for enzyme digestion, the enzyme digestion is carried out according to the proportion of 10ug TEV enzyme digestion to 1mg recombinant proteins, the enzyme digestion is carried out by shaking at 30 ℃ for 3h, then the enzyme digestion liquid is respectively passed through a nickel column, the penetrated liquid is GNRH-I6 recombinant proteins and Q beta virus-like particles, while the his-TEV enzyme and the incompletely digested recombinant proteins are hung on the nickel column, and the penetrated liquid, namely the GNRH-I6 recombinant proteins and the Q beta virus-like particles are respectively concentrated by a 3K concentration tube.

Example 6

Referring to fig. 9 and 10, a method for preparing GNRH-I6-Q β virus-like particles is provided, wherein GNRH-I6 recombinant protein and Q β virus-like particles in example 5 are prepared into 1mg/ml with PBS (10 mM), according to the following ratio of 1:1, adding his-Sotase A5 enzyme obtained in the embodiment 4, carrying out shaking catalysis at 37 ℃ for 5 hours according to the proportion of catalyzing 2mg of mixed protein by 100ug, then passing the catalytic solution through a nickel column, and obtaining GNRH-I6-Q beta virus-like particles through the permeation solution, wherein the his-Sotase A recombinase is hung on the nickel column, and the permeation solution, namely the GNRH-I6-Q beta virus-like particles, is concentrated by a 3K concentration tube.

Example 7

A method for immunizing a GNRH-I6-Q β virus-like particle subunit vaccine is provided, wherein GNRH-I6-Q β virus-like particles are mixed with an aluminum hydroxide adjuvant for immunization, as described in example 6. On day 0, 14, 28, eight weeks old male C57BV/6 mice (five mice per group) were immunized with 50ug of GNRH-I6-Q β virus-like particle vaccine, while the GNRH-I6-Q β unadjuvanted group, the adjuvant pbs negative group, the simple Q β experimental group, and the simple GNRH-I6 experimental group were set. anti-GNRH-I6 recombinant protein antibody titers and testosterone levels were measured in these mice. On day 70 post immunization, mice were sacrificed and testis weight was determined.

Example 8

Mouse anti-GNRH-I6 antibody titer determination

Sera were collected from immunized and control mice at different time points during the experiment. anti-GNRH-I6 IgG antibody titers were determined by ELISA as follows. Coating 96-well plates with 2ug/ml GnRH-I6 deg.C overnight at 100ul per well, washing 5 times daily with 1/1000PBST, adding 300ul 2% BSA blocking plates, blocking 2h at 37 deg.C, then washing 5 times with 1/1000PBST, and mixing the mouse sera at 1:500 was diluted for the initial double gradient, 1:1000,1:2000,1:4000,1:8000,1:16000,1:32000,1:64000,1:128000,1: 256000% of the dilution was BSA. Incubate with shaking at room temperature for 45min from high to low dilution, 100ul per well. The plate was washed 5 times with 1/1000 PBST. HRP-labeled goat anti-mouse polyclonal antibody was used as a secondary antibody, and dilution 1:5000, 100ul of a 1% skimmed milk powder per well was added to the dilution, and the mixture was incubated at room temperature for 45min with shaking. TMB color development 5-10min,2M sulfuric acid termination, and direct reading at the wavelength of 450 nm. Optical Density (OD) was determined in a 450nm ELISA reader (BioRad Benchmark). The maximum OD was calculated using these data ₄₅₀ Serum dilution.

Table 1 shows that in the non-adjuvanted group of immunized male mice with GNRH-I6-Q β virus-like particles, the mean titer reached 64000 at 28 th day, and that the mean titer remained 64000 after the 3 rd needle boost and did not increase significantly. The mean titer of the GNRH-I6-Q β virus-like particle adjuvant group had reached 256000 to the maximum titer at day 28, after which the titer remained at 256000 levels throughout. While the titer of the pure GNRH-I6 immune group is only 16000 days, the results clearly show that the GNRH-I6-Q beta virus-like particles can induce the titer of antibodies against GNRH, and the immune effect is better when the adjuvant is added.

TABLE 1

Testosterone levels in mouse serum

Sera were collected from immunized and control mice at various time points during the above experiment. Testosterone levels in mouse serum were determined using testosterone-Elisa (IBL, burgh, germany).

Table 2 shows that in mice immunized with GNRH-I6-Q β virus-like particles without aluminium hydroxide adjuvant, the 47 th balance testosterone level was greatly suppressed (< 0.5 ng/ml) after immunization, and the level was still below 0.5ng/ml at day 70. In GNRH-I6-Q β alumina supplemented immunized mice, the average testosterone level dropped to <0.2ng/ml on day 28. The mean level of hormones in the central nervous system was about 30-fold lower in the group of GNRH-I6+ aluminum hydroxide-immunized mice compared to the control group. This clearly demonstrates the neutralizing activity of the induced antibody response.

TABLE 2

Weight of testis

Mice were sacrificed on day 70, the testes removed and weighed, and then fixed in 4% formaldehyde.

Table 3 shows that the testis weight was greatly reduced, on average by more than 50%, in mice immunized with GnRH-I6 on day 70. The mice receiving GNRH-I6-Q β virus-like particles and aluminum hydroxide adjuvant had an 87% reduction in testicular weight compared to the control group, which clearly demonstrated the neutralizing activity of the induced antibody response.

TABLE 3

The sequence is as follows:

SEQ ID NO.1：

MAKLETVTLGNIGKDGKQTLVLNPRGVNPTNGVASLSQAGAVPALEKRVTVSVSQPSRNRKNYKVQVKIQNPTACTANGSCDPSVTRQAYADVTFSFTQYSTDEERAFVRTELAALLASPLLIDAIDQLNPAYGGGGSLPETGGGEHWSYGLRPGGGGGSEHWSYGLRPGGGGGSEHWSYGLRPGGGGGSEHWSYGLRPGGGGGSEHWSYGLRPGGGGGSEHWSYGLRPG*

SEQ ID NO.2：

GAGAATCTTTATTTTCAGGGCGGTGGTGAACACTGGAGCTACGGTTTGAGACCCGGGgggggcgggGGATCCGAACACTGGAGCTACGGTTTGAGACCCGGGgggggcgggGGATCCGAACACTGGAGCTACGGTTTGAGACCCGGGgggggcgggGGATCCGAACACTGGAGCTACGGTTTGAGACCCGGGgggggcgggGGATCCGAACACTGGAGCTACGGTTTGAGACCCGGGgggggcgggGGATCCGAACACTGGAGCTACGGTTTGAGACCCGGGtag

SEQ ID NO.3：

CACCACCATCACCATCACGAGAATCTTTATTTTCAGGGCATGGCAAAATTAGAGACTGTTACTTTAGGTAACATCGGGAAAGATGGAAAACAAACTCTGGTCCTCAATCCGCGTGGGGTAAATCCCACTAACGGCGTTGCCTCGCTTTCACAAGCGGGTGCAGTTCCTGCGCTGGAGAAGCGTGTTACCGTTTCGGTATCTCAGCCTTCTCGCAATCGTAAGAACTACAAGGTCCAGGTTAAGATCCAGAACCCGACCGCTTGCACTGCAAACGGTTCTTGTGACCCATCCGTTACTCGCCAGGCATATGCTGACGTGACCTTTTCGTTCACGCAGTATAGTACCGATGAGGAACGAGCTTTTGTTCGTACAGAGCTTGCTGCTCTGCTCGCTAGTCCTCTGCTGATCGATGCTATTGATCAGCTGAACCCAGCGTATGGTGGTGGTGGTTCTCTTCCTGAGACCGGCTGA

SEQ ID NO.4：

CATCATCATCATCATCATATGggagagagcttgttcaagggaccaagggattacaacccaattagctctaccatttgccatttgacaaacgagtctgatggacataccacatctctgtacggaatcggattcggaccttttattattaccaacaagcatctgttcagaagaaataacggtacacttctcgtgcaatctttgcatggtgtgttcaaggtcaagaatacaactacacttcaacaacatcttatcgatggaagagacatgatcatcattagaatgccaaaggatttcccaccttttcctcagaagttgaaattcagagagccacaaagagaagagagaatctgccttgtgacaaccaacttccaaactaagtctatgtctagcatggtgtcagacacttcatgcacattcccttcatctgatggtatcttctggaagcattggattcaaacaaaggatggtcaatgtggatcaccacttgtgtctacaagagatggttttatcgttggtattcattcagcttctaatttcacaaatacaaacaattacttcacaagcgtgccaaagaacttcatggagctgctcacaaatcaagaggctcaacaatgggtttctggatggagacttaatgctgattcagtgttgtggggaggtcataaggttttcatgagcaagcctgaggaaccttttcaaccagttaaggaggctacacagcttatgaatgagttggtttactctcaa

SEQ ID NO.5：

CATCATCATCATCATCATATGCAGGCAAAACCACAGATTCCAAAAGATAAAAGCAAAGTTGCAGGTTATATCGAAATTCCAGATGCAGATATCAAAGAACCAGTCTATCCAGGCCCGGCAACCCGTGAACAGCTGAACCGTGGTGTGAGCTTTGCAGAAGAAAATGAAAGCCTGGATGATCAGAACATTAGCATTGCAGGTCATACCTTTATTGATCGTCCGAATTATCAGTTTACCAATCTGAAAGCAGCAAAAAAAGGTAGCATGGTTTATTTTAAAGTTGGTAATGAAACCCGTAAATATAAAATGACCAGCATTCGTAATGTTAAACCGACCGCAGTTGGTGTTCTGGATGAACAGAAAGGTAAAGATAAACAGCTGACCCTGATTACCTGTGATGATTATAATGAAGAAACCGGTGTTTGGGAAACCCGTAAAATTTTTGTTGCAACCGAAGTTAAA

SEQUENCE LISTING

<110> Shenzhen Hertz Life science and technology Limited

<120> GNRH-I6 virus-like particle subunit vaccine

<130> 2021

<160> 5

<170> PatentIn version 3.5

<210> 1

<211> 230

<212> PRT

<213> Artificial sequence

<400> 1

Met Ala Lys Leu Glu Thr Val Thr Leu Gly Asn Ile Gly Lys Asp Gly

1 5 10 15

Lys Gln Thr Leu Val Leu Asn Pro Arg Gly Val Asn Pro Thr Asn Gly

20 25 30

Val Ala Ser Leu Ser Gln Ala Gly Ala Val Pro Ala Leu Glu Lys Arg

35 40 45

Val Thr Val Ser Val Ser Gln Pro Ser Arg Asn Arg Lys Asn Tyr Lys

50 55 60

Val Gln Val Lys Ile Gln Asn Pro Thr Ala Cys Thr Ala Asn Gly Ser

65 70 75 80

Cys Asp Pro Ser Val Thr Arg Gln Ala Tyr Ala Asp Val Thr Phe Ser

85 90 95

Phe Thr Gln Tyr Ser Thr Asp Glu Glu Arg Ala Phe Val Arg Thr Glu

100 105 110

Leu Ala Ala Leu Leu Ala Ser Pro Leu Leu Ile Asp Ala Ile Asp Gln

115 120 125

Leu Asn Pro Ala Tyr Gly Gly Gly Gly Ser Leu Pro Glu Thr Gly Gly

130 135 140

Gly Glu His Trp Ser Tyr Gly Leu Arg Pro Gly Gly Gly Gly Gly Ser

145 150 155 160

Glu His Trp Ser Tyr Gly Leu Arg Pro Gly Gly Gly Gly Gly Ser Glu

165 170 175

His Trp Ser Tyr Gly Leu Arg Pro Gly Gly Gly Gly Gly Ser Glu His

180 185 190

Trp Ser Tyr Gly Leu Arg Pro Gly Gly Gly Gly Gly Ser Glu His Trp

195 200 205

Ser Tyr Gly Leu Arg Pro Gly Gly Gly Gly Gly Ser Glu His Trp Ser

210 215 220

Tyr Gly Leu Arg Pro Gly

225 230

<210> 2

<211> 285

<212> DNA

<213> Artificial sequence

<400> 2

gagaatcttt attttcaggg cggtggtgaa cactggagct acggtttgag acccgggggg 60

ggcgggggat ccgaacactg gagctacggt ttgagacccg gggggggcgg gggatccgaa 120

cactggagct acggtttgag acccgggggg ggcgggggat ccgaacactg gagctacggt 180

ttgagacccg gggggggcgg gggatccgaa cactggagct acggtttgag acccgggggg 240

ggcgggggat ccgaacactg gagctacggt ttgagacccg ggtag 285

<210> 3

<211> 471

<212> DNA

<213> Artificial sequence

<400> 3

caccaccatc accatcacga gaatctttat tttcagggca tggcaaaatt agagactgtt 60

actttaggta acatcgggaa agatggaaaa caaactctgg tcctcaatcc gcgtggggta 120

aatcccacta acggcgttgc ctcgctttca caagcgggtg cagttcctgc gctggagaag 180

cgtgttaccg tttcggtatc tcagccttct cgcaatcgta agaactacaa ggtccaggtt 240

aagatccaga acccgaccgc ttgcactgca aacggttctt gtgacccatc cgttactcgc 300

caggcatatg ctgacgtgac cttttcgttc acgcagtata gtaccgatga ggaacgagct 360

tttgttcgta cagagcttgc tgctctgctc gctagtcctc tgctgatcga tgctattgat 420

cagctgaacc cagcgtatgg tggtggtggt tctcttcctg agaccggctg a 471

<210> 4

<211> 747

<212> DNA

<213> Artificial sequence

<400> 4

catcatcatc atcatcatat gggagagagc ttgttcaagg gaccaaggga ttacaaccca 60

attagctcta ccatttgcca tttgacaaac gagtctgatg gacataccac atctctgtac 120

ggaatcggat tcggaccttt tattattacc aacaagcatc tgttcagaag aaataacggt 180

acacttctcg tgcaatcttt gcatggtgtg ttcaaggtca agaatacaac tacacttcaa 240

caacatctta tcgatggaag agacatgatc atcattagaa tgccaaagga tttcccacct 300

tttcctcaga agttgaaatt cagagagcca caaagagaag agagaatctg ccttgtgaca 360

accaacttcc aaactaagtc tatgtctagc atggtgtcag acacttcatg cacattccct 420

tcatctgatg gtatcttctg gaagcattgg attcaaacaa aggatggtca atgtggatca 480

ccacttgtgt ctacaagaga tggttttatc gttggtattc attcagcttc taatttcaca 540

aatacaaaca attacttcac aagcgtgcca aagaacttca tggagctgct cacaaatcaa 600

gaggctcaac aatgggtttc tggatggaga cttaatgctg attcagtgtt gtggggaggt 660

cataaggttt tcatgagcaa gcctgaggaa ccttttcaac cagttaagga ggctacacag 720

cttatgaatg agttggttta ctctcaa 747

<210> 5

<211> 462

<212> DNA

<213> Artificial sequence

<400> 5

catcatcatc atcatcatat gcaggcaaaa ccacagattc caaaagataa aagcaaagtt 60

gcaggttata tcgaaattcc agatgcagat atcaaagaac cagtctatcc aggcccggca 120

acccgtgaac agctgaaccg tggtgtgagc tttgcagaag aaaatgaaag cctggatgat 180

cagaacatta gcattgcagg tcataccttt attgatcgtc cgaattatca gtttaccaat 240

ctgaaagcag caaaaaaagg tagcatggtt tattttaaag ttggtaatga aacccgtaaa 300

tataaaatga ccagcattcg taatgttaaa ccgaccgcag ttggtgttct ggatgaacag 360

aaaggtaaag ataaacagct gaccctgatt acctgtgatg attataatga agaaaccggt 420

gtttgggaaa cccgtaaaat ttttgttgca accgaagtta aa 462

Claims (7)

1. A subunit vaccine comprising GNRH-I6-Q β virus-like particles and an aluminum hydroxide adjuvant;

wherein the GNRH-I6-Q beta amino acid sequence is SEQ ID NO.1;

the GNRH-I6-Q beta virus-like particle is prepared by the following method:

(1) Synthesizing gene sequences in SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO.5 into a target gene, and constructing a recombinant vector, wherein the sequence is as follows:

his-MBP-TEV-GNRH-I6-PETM41；

his-TEV-Qβ-pET28a；

his-TEV Protease-pET28a；

his-Sortase A-pET28a；

(2) Transforming the recombinant plasmid with correct sequence determination in the step (1) into Escherichia coli T7shuffle to respectively obtain recombinant expression strains:

his-MBP-TEV-GNRH-I6-PETM41-T7shuffle；

his-TEV-Qβ-pET28a-T7shuffle；

his-TEV Protease-pET28a-T7shuffle；

his-Sortase A-pET28a-T7shuffle；

(3) Respectively culturing recombinant expression strains in the step II, adding IPTG (isopropyl-beta-thiogalactoside) for induction expression, collecting thalli, carrying out ultrasonic crushing, and purifying by a nickel column to respectively obtain recombinant proteins:

his-MBP-TEV-GNRH-I6 recombinant protein;

his-TEV-Q β virus-like particles;

his-TEV enzyme;

his-Sortase A enzyme;

(4) Dissolving the his-MBP-TEV-GNRH-I6 recombinant protein and the his-TEV-Q beta virus sample particles in the step three by using a PBS solution, respectively adding the his-TEV enzyme for enzyme digestion, carrying out enzyme digestion according to the proportion of 1mg of recombinant protein digested by 10ug of the his-TEV enzyme, carrying out oscillation enzyme digestion for 3h at 30 ℃, respectively passing enzyme digestion solutions through nickel columns, respectively concentrating penetrating fluids which are the GNRH-I6 recombinant protein and the Q beta virus sample particles by using a 3K concentration tube;

(5) And preparing the his-MBP-TEV-GNRH-I6 recombinant protein and the his-TEV-Q beta virus-like particles in the fourth step into 1mg/ml by using PBS respectively, and mixing the two components according to a volume ratio of 1:1, mixing, adding his-Sortase A enzyme in the step three, carrying out oscillation catalysis at 37 ℃ for 5 hours according to the proportion of catalyzing 2mg of mixed protein by 100ug, then passing the catalytic solution through a nickel column, penetrating the catalytic solution to obtain GNRH-I6-Q beta virus-like particles, and concentrating by using a 3K concentration tube.

2. The subunit vaccine of claim 1, wherein the recombinant expression strain is cultured until OD600 reaches 0.6-0.8, then IPTG (1 mM IPTG) is added to the recombinant expression strain for induced expression for 12h, then the collected bacterial cells are mixed with 10mM PBS, the bacterial cells are crushed by ultrasound, and then a nickel column is used for purification, 25mM imidazole is used for washing 10 column volumes, and 250mM imidazole is used for eluting target protein.

3. The subunit vaccine of claim 1, wherein the his-MBP-TEV-GNRH-I6-PETM41 is obtained by cloning the nucleotide shown in SEQ ID NO.2 into the prokaryotic expression vector between the enzyme cutting sites NcoI and BamHI of PETM41 by homologous recombination.

4. The subunit vaccine of claim 1, wherein his-TEV-Q β -pET28a is obtained by cloning the nucleotide fragment represented by SEQ ID No.3 into the prokaryotic expression vector between the two restriction sites NcoI and XhoI of pET28 a.

5. The subunit vaccine of claim 1, wherein his-TEV Protease-pET28a is obtained by cloning the nucleotide fragment represented by SEQ ID No.4 into the prokaryotic expression vector between the two restriction sites NcoI and XhoI of pET28 a.

6. The subunit vaccine of claim 1, wherein the his-Sortase a-pET28a is obtained by cloning the nucleotide fragment shown in SEQ ID No.5 into the prokaryotic expression vector between the two restriction sites NcoI and XhoI of pET28 a.

7. The subunit vaccine of claim 1, for use in the production of a vaccine.

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