CN112480265A - GNRH-I6 virus-like particle subunit vaccine - Google Patents
GNRH-I6 virus-like particle subunit vaccine Download PDFInfo
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Abstract
The invention belongs to the field of molecular biology, and discloses a GNRH-I6-Q beta virus-like particle subunit vaccine, which provides a composition comprising: 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 not pathogenic to animals such as mice and the like, is easy to evaluate through safety, and the composition can be used for producing the vaccine and effectively inducing immune response, particularly humoral immune response.
Description
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 a 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 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-NH2), and the molecular weight is 1.181 KDW.
The physiological dose of GnRH-I can promote the increase of the concentration of gonadotropin (such as the light rise of FSH and the obvious rise of LH), promote the synthesis and secretion of the gonadotropin (such as estradiol, progesterone, testosterone and the like), promote the mature of ovarian follicles or the mature of ovules or testis and sperm, 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 can 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)). Compared to a few proteins, they are able to trigger a much stronger immune response than their isolated components. For B cell responses, it is well known that one key factor in the immunogenicity of viruses is the repetitiveness and order of surface epitopes. Many viruses exhibit a quasicrystal surface with regularly arranged epitopes that can effectively crosslink epitope-specific immunoglobulins on B cells (Bachxnann and Zinkernegel, 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)). The viral structure is even involved in the production of anti-antibodies in autoimmune diseases and is 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 able to induce 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, viral proteins or chimeric proteins of different species (types) 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 of VLP monomer folding, particle assembly and the like, and short linear peptides, peptide loops and full-length proteins, even non-protein antigens such as polysaccharide and hapten 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 repeated polypeptide and Q beta virus-like particles are catalyzed by sortaseA enzyme 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 NcoI and Xho I enzyme cutting sites 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 NcoI and Xho I restriction enzyme 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 NcoI and Xho I enzyme cutting sites 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-like 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 digesting 1mg of recombinant protein, carrying out shaking enzyme digestion for 3h at 30 ℃, then respectively passing enzyme digestion solutions through nickel columns, respectively concentrating penetrating solutions which are the GNRH-I6 recombinant protein and the Q beta virus-like particles by using a 3K concentration tube;
(5) the his-MBP-TEV-GNRH-I6 recombinant protein and the his-TEV-Q beta virus-like particles in the fourth step are prepared into 1mg/ml by PBS, and the weight ratio of the recombinant protein to the his-TEV-Q beta virus-like particles is 1: 1, mixing, 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, penetrating the catalytic solution to obtain GNRH-I6-Q beta virus-like particles, and concentrating by using a 3K concentration tube.
Further, when the recombinant expression strain is cultured until OD600 reaches 0.6-0.8, 1mM IPTG is added to carry out induction expression for 12h, then the collected strain is mixed with 10mM PBS and subjected to 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 the target protein.
In a seventh aspect, the invention provides a subunit vaccine comprising the GNRH-I6-Q β virus-like particle prepared in 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 through thallus culture, is suitable for industrial production, is added with a His tag, 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 non-label GNRH-I6 recombinant protein 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 by escherichia coli in a large amount in a soluble manner, 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, so that the condition is 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 a pETM41 plasmid provided in the examples of the present invention;
FIG. 2 is a schematic diagram of a recombinant plasmid obtained by ligating the Q.beta.gene with a pET28a plasmid;
FIG. 3 is a schematic diagram of a recombinant plasmid obtained by ligating the TEV protease gene with pET28a plasmid;
FIG. 4 is a schematic diagram of a recombinant plasmid obtained by connecting the SortaseA gene with the pET28a plasmid;
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-run of his-TEV enzyme protein provided in an example 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 illustration of SDS-PAGE run of the results of 1h of the GNRH-I6 protein and Q β virus-like particles catalyzed by his-Sortase A provided in the examples of the present invention;
FIG. 10 is a schematic illustration of SDS-PAGE run of the results of 5h catalysis of GNRH-I6 protein and Q β 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.
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 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-medium-volume plasmid minikit, 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 label, the TEV enzyme cutting site and the Q beta gene are directly synthesized into pET28a carrier NcoI enzyme cutting site and XhoI enzyme cutting site by Huada gene company to obtain recombinant plasmid his-TEV-Q beta-pET 28 a;
(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
Converting T7shuffle expression strain, shaking, centrifuging, concentrating, coating kanamycin-resistant plate, culturing overnight at 37 ℃, scraping bacterial 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 bacterial cells were collected by centrifugation at 4500rpm for 15 min. The thalli is broken by ultrasonic for 45min, the opening time is 3s, the closing time is 4s, and the power is 125 w. After the ultrasonic treatment is finished, 9500rpm is carried out for 20min, and supernatant is collected at 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 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 2X protein electrophoresis loading buffer and heated at 100 ℃ for 4 min. 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 restriction enzyme sites and GNRH-I6 gene into pETM41 vector NcoI restriction enzyme sites and XhoI restriction enzyme sites by Huada gene company to obtain recombinant plasmid his-MBP-TEV-GNRH-I6-pETM 41;
(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
T7shuffle expression strain is transformed, centrifuged and concentrated after shaking, coated with kanamycin-resistant plate, cultured overnight at 37 ℃, scraped to obtain colony, inoculated with 10mL kanamycin-containing 2 XYT culture medium, shaken at 37 ℃ and 180r until the OD value of the 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 bacterial cells were collected by centrifugation at 4500rpm for 15 min. The thalli is broken by ultrasonic for 45min, the opening time is 3s, the closing time is 4s, and the power is 125 w. After the completion of sonication, the supernatant was collected at 9500rpm for 20min at 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 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 2X protein electrophoresis loading buffer and heated at 100 ℃ for 4 min. 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 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 carrier NcoI restriction enzyme site and XhoI restriction enzyme site by Huada gene company to obtain recombinant plasmid his-TEV protease-pET28 a;
(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 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.
(c2) his-TEV recombinase expression
T7shuffle expression strain is transformed, centrifuged and concentrated after shaking, coated with kanamycin-resistant plate, cultured overnight at 37 ℃, scraped to obtain colony, inoculated with 10mL kanamycin-containing 2 XYT culture medium, shaken at 37 ℃ and 180r until the OD value of the 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 was added with 0.8mM IPTG inducer and expressed overnight at 30 ℃. On the third day, the bacterial cells were collected by centrifugation at 4500rpm for 15 min. The thalli is broken by ultrasonic for 45min, the opening time is 3s, the closing time is 4s, and the power is 125 w. After the completion of sonication, the supernatant was collected at 9500rpm for 20min at 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 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 2X protein electrophoresis loading buffer and heated at 100 ℃ for 4 min. 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 carrier NcoI enzyme cutting site and XhoI enzyme cutting site by Huada gene company to obtain recombinant plasmid his-sortaseA-pET28 a;
(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
T7shuffle expression strain is transformed, centrifuged and concentrated after shaking, coated with kanamycin-resistant plate, cultured overnight at 37 ℃, scraped to obtain colony, inoculated with 10mL kanamycin-containing 2 XYT culture medium, shaken at 37 ℃ and 180r until the OD value of the 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 was added with 0.8mM IPTG inducer and expressed overnight at 30 ℃. On the third day, the bacterial cells were collected by centrifugation at 4500rpm for 15 min. The thalli is broken by ultrasonic for 45min, the opening time is 3s, the closing time is 4s, and the power is 125 w. After the completion of sonication, the supernatant was collected at 9500rpm for 20min at 4 ℃. (ii) a
(d3) his-sortaseA recombinase purification
The supernatant was centrifuged and purified by nickel column, which was equilibrated to 5 column volumes 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 2X protein electrophoresis loading buffer and heated at 100 ℃ for 4 min. 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 protein in example 2 are dissolved in PBS solution (10mM), respectively added with the his-TEV recombinase in example 3 for enzyme digestion, the enzyme digestion is carried out according to the proportion of 1mg recombinant protein digested by 10ug TEV enzyme, the enzyme digestion is carried out at 30 ℃ for 3h by shaking, then the enzyme digestion liquid respectively passes through a nickel column, the liquid passes through GNRH-I6 recombinant protein and Q beta virus-like particles, while the his-TEV enzyme and the incompletely digested recombinant protein are hung on the nickel column, and the liquid passing through GNRH-I6 recombinant protein and 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 of example 5 are prepared into 1mg/ml with PBS (10mM), according to the ratio of 1: 1, adding the his-Sotase A5 enzyme of example 4, carrying out vibration 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 carrying out GNRH-I6-Q beta virus-like particles, hanging the his-Sotase A recombinase on the nickel column, and concentrating the penetrating solution, namely the GNRH-I6-Q beta virus-like particles by using a 3K concentration tube.
Example 7
A GNRH-I6-Q β virus-like particle subunit vaccine immunization method is provided, in which 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-week old male C57BV/6 mice (five mice per group) were immunized with 50ug GNRH-I6-Q β virus-like particle vaccine, while the GNRH-I6-Q β unadjuvanted group, the adjuvant pbs negative group, the Q β only experimental group, and the GNRH-I6 only 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
At various times during the experimentSera were collected from immunized and control mice. anti-GNRH-I6 IgG antibody titers were determined by ELISA as follows. The 96-well plates were coated with 2ug/ml GnRH-I64 degrees celsius overnight at 100ul per well, washed 5 times the following day with 1/1000PBST, added to 300ul 2% BSA blocking plate, blocked at 37 ℃ for 2h, then washed 5 times with 1/1000PBST, and the mouse sera were washed as 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 diluted with 2% 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, 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, 450nm wavelength direct reading. Optical Density (OD) was determined in a 450nm ELISA reader (BioRad Benchmark). The maximum OD was calculated using these data450Serum 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 consistently at 256000. 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 (<0.5ng/ml) after immunization, still below 0.5ng/ml on day 70. In GNRH-I6-Q β alumina-supplemented mice, mean testosterone levels dropped to <0.2ng/ml on day 28. The mean level of central nervous system hormones 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 of mice immunized with GnRH-I6 was greatly reduced, on average by more than 50% 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：
MAKLETVTLGNIGKDGKQTLVLNPRGVNPTNGVASLSQAGAVPALEKRVTVSVSQPSRNRKNYKV QVKIQNPTACTANGSCDPSVTRQAYADVTFSFTQYSTDEERAFVRTELAALLASPLLIDAIDQLN PAYGGGGSLPETGGGEHWSYGLRPGGGGGSEHWSYGLRPGGGGGSEHWSYGLRPGGGGGSEHWSY GLRPGGGGGSEHWSYGLRPGGGGGSEHWSYGLRPG*
SEQ ID NO.2：
GAGAATCTTTATTTTCAGGGCGGTGGTGAACACTGGAGCTACGGTTTGAGACCCGGGgggggcgg gGGATCCGAACACTGGAGCTACGGTTTGAGACCCGGGgggggcgggGGATCCGAACACTGGAGCT ACGGTTTGAGACCCGGGgggggcgggGGATCCGAACACTGGAGCTACGGTTTGAGACCCGGGggg ggcgggGGATCCGAACACTGGAGCTACGGTTTGAGACCCGGGgggggcgggGGATCCGAACACTG GAGCTACGGTTTGAGACCCGGGtag
SEQ ID NO.3：
CACCACCATCACCATCACGAGAATCTTTATTTTCAGGGCATGGCAAAATTAGAGACTGTTACTTT AGGTAACATCGGGAAAGATGGAAAACAAACTCTGGTCCTCAATCCGCGTGGGGTAAATCCCACTA ACGGCGTTGCCTCGCTTTCACAAGCGGGTGCAGTTCCTGCGCTGGAGAAGCGTGTTACCGTTTCG GTATCTCAGCCTTCTCGCAATCGTAAGAACTACAAGGTCCAGGTTAAGATCCAGAACCCGACCGC TTGCACTGCAAACGGTTCTTGTGACCCATCCGTTACTCGCCAGGCATATGCTGACGTGACCTTTT CGTTCACGCAGTATAGTACCGATGAGGAACGAGCTTTTGTTCGTACAGAGCTTGCTGCTCTGCTC GCTAGTCCTCTGCTGATCGATGCTATTGATCAGCTGAACCCAGCGTATGGTGGTGGTGGTTCTCT TCCTGAGACCGGCTGA
SEQ ID NO.4：
CATCATCATCATCATCATATGggagagagcttgttcaagggaccaagggattacaacccaattag ctctaccatttgccatttgacaaacgagtctgatggacataccacatctctgtacggaatcggat tcggaccttttattattaccaacaagcatctgttcagaagaaataacggtacacttctcgtgcaa tctttgcatggtgtgttcaaggtcaagaatacaactacacttcaacaacatcttatcgatggaag agacatgatcatcattagaatgccaaaggatttcccaccttttcctcagaagttgaaattcagag agccacaaagagaagagagaatctgccttgtgacaaccaacttccaaactaagtctatgtctagc atggtgtcagacacttcatgcacattcccttcatctgatggtatcttctggaagcattggattca aacaaaggatggtcaatgtggatcaccacttgtgtctacaagagatggttttatcgttggtattc attcagcttctaatttcacaaatacaaacaattacttcacaagcgtgccaaagaacttcatggag ctgctcacaaatcaagaggctcaacaatgggtttctggatggagacttaatgctgattcagtgtt gtggggaggtcataaggttttcatgagcaagcctgaggaaccttttcaaccagttaaggaggcta cacagcttatgaatgagttggtttactctcaa
SEQ ID NO.5：
CATCATCATCATCATCATATGCAGGCAAAACCACAGATTCCAAAAGATAAAAGCAAAGTTGCAGG TTATATCGAAATTCCAGATGCAGATATCAAAGAACCAGTCTATCCAGGCCCGGCAACCCGTGAAC AGCTGAACCGTGGTGTGAGCTTTGCAGAAGAAAATGAAAGCCTGGATGATCAGAACATTAGCATT GCAGGTCATACCTTTATTGATCGTCCGAATTATCAGTTTACCAATCTGAAAGCAGCAAAAAAAGG TAGCATGGTTTATTTTAAAGTTGGTAATGAAACCCGTAAATATAAAATGACCAGCATTCGTAATG TTAAACCGACCGCAGTTGGTGTTCTGGATGAACAGAAAGGTAAAGATAAACAGCTGACCCTGATT ACCTGTGATGATTATAATGAAGAAACCGGTGTTTGGGAAACCCGTAAAATTTTTGTTGCAACCGA AGTTAAA。
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 (9)
1. The GNRH-I6-Q beta protein expressed by recombinant escherichia coli is characterized in that a GNRH-I6 repeated polypeptide and a Q beta virus-like particle are catalyzed by sortaseA enzyme to form a recombinant protein GNRH-I6-Q beta; the recombinant protein GNRH-I6-Q beta amino acid sequence is SEQ ID NO. 1.
2. A recombinant plasmid his-MBP-TEV-GNRH-I6-PETM41 is characterized in that 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.
3. A recombinant plasmid his-TEV-Q beta-pET 28a is characterized in that 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 NcoI and Xho I enzyme cutting sites of pET28 a.
4. A recombinant plasmid his-TEV Protease-pET28a is characterized in that 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 NcoI and Xho I enzyme cutting sites of pET28 a.
5. A recombinant plasmid his-Sotase A-pET28a is characterized in that 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.
6. A preparation method of GNRH-I6-Q beta virus-like particles is characterized by comprising 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-like 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 digesting 1mg of recombinant protein, carrying out shaking enzyme digestion for 3h at 30 ℃, then respectively passing enzyme digestion solutions through nickel columns, respectively concentrating penetrating solutions which are the GNRH-I6 recombinant protein and the Q beta virus-like particles by using a 3K concentration tube;
(5) the his-MBP-TEV-GNRH-I6 recombinant protein and the his-TEV-Q beta virus-like particles in the fourth step are prepared into 1mg/ml by PBS, and the weight ratio of the recombinant protein to the his-TEV-Q beta virus-like particles is 1: 1, mixing, 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, penetrating the catalytic solution to obtain GNRH-I6-Q beta virus-like particles, and concentrating by using a 3K concentration tube.
7. The method of claim 6, wherein the recombinant expression strain is cultured until OD600 reaches 0.6-0.8, IPTG with a final concentration of 1mM is added for induction expression for 12h, the collected bacterial cells are mixed with 10mM PBS and then subjected to ultrasonication, and then the bacterial cells are purified by using a nickel column, washed with 25mM imidazole for 10 column volumes, and eluted with 250mM imidazole for target protein.
8. A subunit vaccine comprising the GNRH-I6-Q β virus-like particle of claim 6 and a fors adjuvant.
9. A subunit vaccine as claimed in claim 8, for use in the production of a vaccine.
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