Application of molecular chaperone TF in promoting soluble expression of VP2 structural protein of Seneca virus type A

Abstract

The present invention relates to the field of molecular biology technology, and specifically to an application of a molecular chaperone TF in promoting the soluble expression of type A Seneca virus structural protein VP2. The present invention first constructs a SVA VP2 protein recombinant expression plasmid pET28a-SVA-VP2 based on the gene sequence of the SVA CH / FJ / 2017 strain; then the pET28a-SVA-VP2 recombinant plasmid and the molecular chaperone pTf16 plasmid are co-transfected into BL21 (DE3) competent cells to successfully prepare a soluble recombinant SVA VP2 protein; the recombinant VP2 protein has good immunogenicity in both in vivo and in vitro tests, and can induce the body to produce a strong humoral immune response and cellular immune response, providing experimental materials and theoretical basis for the development of SVA subunit vaccines and diagnostic preparations.

Description

Technical Field

[0001] The invention relates to the technical field of genetic engineering, and in particular to an application of a molecular chaperone TF in promoting the soluble expression of type A Seneca virus structural protein VP2. Background Art

[0002] Senecavirus A (SVA) belongs to the genus Senecavirus of the family Picornaviridae and is a newly emerging pathogen of swine infectious diseases. SVA mainly infects pigs and is susceptible to pigs of different ages. It mainly causes severe vesicular disease in sows and death of newborn piglets, with a mortality rate of up to 30% to 70% in newborn piglets. The main clinical symptoms of SVA infection are vesicular lesions on the skin and mucous membranes of the pig's nose, oral epithelium, tongue, and hoof crown, which are very similar to the lesions caused by foot-and-mouth disease (FMD), swine vesicular exanthema (VES), swine vesicular disease (SVD), and vesicular stomatitis (VS), and are clinically difficult to distinguish. The outbreak and prevalence of swine Senecavirus disease has caused great economic losses to the global pig industry.

[0003] Compared with other pathogens, SVA has been less studied, and the immunogenicity of the protein is also in the initial stage. Although there have been reports on the research progress of various candidate vaccines such as inactivated vaccines, attenuated vaccines, nucleic acid vaccines and subunit vaccines for SVA, there is still no commercial vaccine for the prevention of SVA infection. After SVA infects the animal body, it can simultaneously activate the body's B cell and T cell responses, thereby producing a large number of neutralizing antibodies to resist and eliminate SVA infection. Studies have confirmed that mice are candidate animal models for preliminary evaluation of the immunogenicity and protective effects of SVA vaccines; Meng Yuan et al. used recombinant SVA VP1 protein combined with adjuvant to immunize mice to evaluate its immune response effect; Cui Chuan et al. fused and expressed SVA VP2 protein and detected high levels of specific antibodies and IFN-γ and IL-2 cytokine content in the serum of mice immunized with VP2 protein. As a dominant antigen that induces the body to produce neutralizing antibodies, SVA VP2 structural protein is one of the candidate antigens for SVA subunit vaccine and diagnostic preparation research. The preparation of soluble and highly active recombinant VP2 protein is the key to subunit vaccine research.

[0004] When preparing recombinant proteins through in vitro expression systems, the Escherichia coli expression system is often the preferred expression system due to its advantages such as simple operation, low production cost and high expression level. However, most recombinant proteins are expressed in the form of inclusion bodies. The main reason is that the protein aggregates rapidly during the formation process and the peptide chain folding time is short, resulting in insufficient and incomplete peptide chain folding, which leads to abnormal conformation and the formation of inclusion bodies. Previous studies have shown that the exogenous expression of SVA VP2 protein is mostly in the form of inclusion bodies, which may cause the protein to fail to fold correctly, thus affecting its normal biological function. Among them, Zhang Yongning et al. and Cui Chuan et al. prepared soluble SVA VP2 protein by adding fusion tags to the coding gene of VP2 protein using prokaryotic expression vectors pET-30a and pET-32a, respectively. However, the molecular weight of the recombinant protein prepared in this way is significantly larger than that of the normal VP2 protein, which may have a certain impact on the accurate study of the function of VP2 protein.

[0005] Molecular chaperone proteins are highly conserved proteins that promote the proper folding of other proteins in vivo. Different chaperone systems contribute to the de novo folding, transport and assembly of oligomeric complexes of proteins, as well as recovery from stress-induced unfolding. Studies have also shown that molecular chaperones can improve the soluble expression of target proteins, but the results of molecular chaperones promoting the soluble expression of target proteins also have a certain degree of randomness, and on the contrary may inhibit the soluble expression of other enzymes or proteins; and the molecular chaperone contamination phenomenon is serious during the protein purification process. Moreover, the present invention found in the research process that most molecular chaperones (such as pG-KJE8, pGro7, pKJE7, pG-Tf2, etc.) did not make the A-type Seneca virus structural protein VP2 soluble.

[0006] Trigger Factor (TF) is a ribosome-related chaperone protein that can bind to most of the nascent peptide chains at the exit of the ribosome to form a protected folding space, so that the nascent peptide chains are not affected by the leading and aggregation and fold into mature proteins with normal conformations. Although there are literatures that have published studies on the soluble expression of VP2 proteins in other viruses by the molecular chaperone TF, the differences in VP2 proteins are large due to different virus types, resulting in opposite results in their soluble expression. For example, the literature (Study on the promotion of soluble expression of feline parvovirus-like particles by four molecular chaperones) found that the molecular chaperone pTf16 (TF) can promote the soluble expression of FPV VP2 protein; the literature (Effects of five molecular chaperones on the soluble expression of partial genes of mink Aleutian virus nucleocapsid protein VP2) found that the pTf16 (TF) chaperone protein was co-expressed with mink Aleutian virus nucleocapsid protein VP2, and no increase in soluble protein was observed, indicating that pTf16 (TF) cannot promote the soluble expression of mink Aleutian virus nucleocapsid protein VP2. Summary of the invention

[0007] In view of the above problems, the present invention uses SVA VP2 protein recombinant plasmid and molecular chaperone protein TriggerFactor (TF) expression plasmid pTF16 to co-express and successfully achieve soluble expression of SVAVP2 protein. Soluble recombinant VP2 protein is purified, and the humoral immunity and cellular immune response levels induced by the recombinant protein are systematically analyzed by immunizing mice. The research results show that the recombinant VP2 protein prepared in this study has good immunogenicity and immunoreactivity, which lays a foundation for the development of new subunit vaccines and diagnostic preparations for SVA.

[0008] Specifically include the following:

[0009] In a first aspect, the present invention provides an application of a molecular chaperone protein Trigger factor in promoting the expression of the structural protein VP2 of type A Seneca virus.

[0010] In a second aspect, the present invention provides a genetically engineered bacterium that solublely expresses the structural protein VP2 of type A Senecavirus, wherein the genetically engineered bacterium contains a plasmid that expresses the structural protein VP2 of type A Senecavirus and a plasmid that expresses the molecular chaperone Triggerfactor.

[0011] Preferably, the method comprises: transforming a host cell with a plasmid expressing a molecular chaperone Trigger factor and a plasmid expressing a type A Seneca virus structural protein VP2 to obtain a genetically engineered bacterium that can simultaneously express the type A Seneca virus structural protein VP2 and the molecular chaperone protein Trigger factor.

[0012] In a third aspect, the present invention provides a method for soluble expression of recombinant type A Senecavirus structural protein VP2, the method comprising: fermenting and culturing genetically engineered bacteria that simultaneously express type A Senecavirus structural protein VP2 and molecular chaperone protein Triggerfactor, and inducing expression to obtain recombinant type A Senecavirus structural protein VP2.

[0013] Preferably, the method comprises the following steps:

[0014] Preparation of genetically engineered bacteria: Preparation of genetically engineered bacteria that simultaneously express the structural protein VP2 of type A Seneca virus and the molecular chaperone protein Trigger factor;

[0015] Induced expression: The obtained genetically engineered bacteria were fermented and cultured, and L-arabinose and IPTG were added to induce expression to obtain the recombinant A-type Seneca virus structural protein VP2.

[0016] Preferably, the preparation of the genetically engineered bacteria comprises the following steps:

[0017] (1) The codons of the VP2 gene of the structural protein of type A Seneca virus were optimized by Escherichia coli, the SVAVP2 gene was synthesized and connected to the Escherichia coli expression vector to construct a recombinant expression plasmid;

[0018] (2) Transforming the molecular chaperone pTF16 plasmid into Escherichia coli competent cells, and screening and preparing competent cells;

[0019] (3) The recombinant plasmid described in step (1) is transferred into the competent cells of step (2), and a genetically engineered bacterium that simultaneously expresses the structural protein VP2 of type A Seneca virus and the molecular chaperone protein Trigger factor is obtained by screening.

[0020] Preferably, the Escherichia coli includes BL21(DE3), BL21(DE3)pLysS, B834(DE3), BLR(DE3), JM109, XL1Blue, ER2566, Rosetta, and GI698.

[0021] Preferably, the preparation of the genetically engineered bacteria is:

[0022] (1) synthesizing the SVA VP2 gene sequence shown in SEQ ID NO.1 and ligating it into the Escherichia coli expression vector pET28a(+) to construct the recombinant expression plasmid pET28a-SVA-VP2;

[0023] (2) Synthesis of the molecular chaperone pTF16 (Cm + ) plasmid, transformed into BL21(DE3) competent cells, cultured in a medium containing chloramphenicol, and picked positive clones to obtain pTF16-BL21(DE3) competent cells;

[0024] (3) The recombinant plasmid pET-28a-SVA-VP2 described in step (1) is transferred into the pTF16-BL21 (DE3) competent cells described in step (2), cultured in a medium containing kanamycin and chloramphenicol, and positive clones are picked to obtain genetically engineered bacteria that simultaneously express the type A Seneca virus structural protein VP2 and the molecular chaperone protein Trigger factor.

[0025] Preferably, the induced expression is:

[0026] The genetically engineered bacteria that simultaneously express the type A Seneca virus structural protein VP2 and the molecular chaperone protein Trigger factor are fermented and cultured, and L-arabinose with a final concentration of 2 g / L and IPTG with a final concentration of 1 mmol / L are added to the culture to induce expression;

[0027] The bacteria were collected by centrifugation, resuspended in PBS, broken, and centrifuged to obtain the supernatant, and the recombinant Seneca virus type A structural protein VP2 was purified.

[0028] In a fourth aspect, the present invention provides a recombinant type A Seneca virus structural protein VP2 prepared by the method described in the third aspect.

[0029] In a fifth aspect, the present invention provides the use of the recombinant type A Seneca virus structural protein VP2 described in the fourth aspect in the preparation of a Seneca virus disease vaccine.

[0030] In a sixth aspect, the present invention provides a vaccine for preventing Seneca virus infection, wherein the vaccine comprises the recombinant type A Seneca virus structural protein VP2 described in the fifth aspect.

[0031] Preferably, the vaccine further comprises an adjuvant.

[0032] Preferably, the adjuvant is one or more of a chemical immune adjuvant, a microbial immune adjuvant, a plant immune adjuvant, and a biochemical immune adjuvant.

[0033] The beneficial effects of the present invention are:

[0034] (1) The present invention optimizes the prokaryotic expression codons based on the gene sequence encoding the structural protein VP2 of type A Seneca virus, and constructs the SVA VP2 protein recombinant expression plasmid pET28a-SVA-VP2; the pET28a-SVA-VP2 recombinant plasmid and the molecular chaperone pTf16 plasmid are co-transfected into BL21 (DE3) competent cells, and the expression of soluble recombinant SVAVP2 protein is successfully induced;

[0035] (2) The recombinant VP2 protein can produce a specific immune reaction with SVA positive serum. The serum antibody titer of immunized mice can reach 1:32000, the neutralizing antibody level can reach 1:128, and the spleen CD4 + and CD8 + The percentage of T lymphocytes was significantly higher than that of the control group, and the expression levels of cytokines such as IFN-γ, IL-2, IL-4 and IL-10 were significantly higher than those of the PBS control group, which can induce the body to produce stronger humoral and cellular immune responses. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 The result of double enzyme digestion of the recombinant expression plasmid; M is the DL 10000 DNA molecular marker, and 1 is the enzyme digestion product of the recombinant expression plasmid;

[0037] Figure 2SDS-PAGE analysis results of SVA VP2 recombinant protein, where M is the protein molecular weight marker, 1 is pET-28a(+) without IPTG induction, 2 is pET-28a(+) induced by IPTG, 3 is pET28a-SVA VP2 without IPTG induction, 4 is pET28a-SVA VP2 induced by IPTG, 5 is pET28a-SVA VP2 is the supernatant after bacterial lysis induced by IPTG, 6 is the precipitate after bacterial lysis induced by IPTG, 7 is pET28a-TF16-SVAVP2 without L-arabinose and IPTG induction, 8 is pET28a-TF16-SVAVP2 induced by L-arabinose and IPTG, 9 is the supernatant after bacterial lysis induced by L-arabinose and IPTG of pET28a-TF16-SVAVP2, 10 is the precipitate after bacterial lysis induced by L-arabinose and IPTG of pET28a-TF16-SVAVP2;

[0038] Figure 3 Purification and immunoblotting analysis of SVA VP2 recombinant protein, where M is a protein molecular weight marker, 1 is an unpurified recombinant SVA VP2 protein, 2 is a purified recombinant SVA VP2 protein, 3 is a Western blotting analysis of recombinant SVA VP2 protein and rabbit negative serum, 4 is a Western blotting analysis of recombinant SVA VP2 protein and rabbit anti-SVA positive serum; 5 is a Western blotting analysis of recombinant SVA VP2 protein and anti-His antibody;

[0039] Figure 4 Indirect ELISA test results for serum antibodies, where A is the IgG antibody level and B is the serum titer;

[0040] Figure 5 Mouse serum neutralization titer results;

[0041] Figure 6 Results of SVA VP2 immune lymphocyte subset analysis;

[0042] Figure 7 Results of SVA VP2 immune lymphocyte proliferation analysis;

[0043] Figure 8 Results of SVA VP2 immune cytokine level analysis. DETAILED DESCRIPTION

[0044] The above scheme is further described below in conjunction with specific examples. It should be understood that these examples are used to illustrate the present invention and are not limited to the scope of the present invention. The implementation conditions adopted in the examples can be further adjusted according to the conditions of the specific manufacturer, and the unspecified implementation conditions are usually the conditions in conventional experiments.

[0045] The experiments described in the following examples were performed with biosafety permits and foot-and-mouth disease laboratory activity permits:

[0046] In accordance with the relevant requirements of biosafety level 3 laboratories (BSL-3) and foot-and-mouth disease-related biosafety, the Lanzhou Veterinary Research Institute of the Chinese Academy of Agricultural Sciences has obtained permission from the Ministry of Agriculture to conduct research on highly pathogenic SVA pathogens and animals through the Biosafety Committee of the Lanzhou Veterinary Research Institute, the Laboratory Animal Ethics Committee, the Biosafety Committee of the Chinese Academy of Agricultural Sciences, the Laboratory Animal Ethics Committee of the Lanzhou Veterinary Research Institute, and the Biosafety Committee of the Lanzhou Veterinary Research Institute. The research has been filed with the Ministry of Agriculture and Rural Affairs and meets the national biosafety level requirements.

[0047] Senecavirus type A SVA / CH-FJ-2017 was obtained from the National Senecavirus Reference Laboratory.

[0048] Hind III and BamH I restriction endonucleases were purchased from NEB (USA); pTF16 (Cm+) chaperone protein plasmid was stored in our laboratory; competent cell preparation kit was purchased from Sangon Biotechnology Co., Ltd. (Shanghai, China); IPTG, L-arabinose, goat anti-mouse IgG-HRP antibody, goat anti-swine IgG-HRP antibody, mouse anti-His Tag monoclonal antibody, and Freund's adjuvant (complete and incomplete adjuvant) were purchased from Sigma (USA); ECL color development kit was purchased from Yazyme Biopharmaceutical Technology Co., Ltd. (Shanghai, China); Ni Sepharose HP and AKTA pure150 purification instrument were purchased from GE (USA); mouse lymphocyte separation medium, mouse IFN-γ, IL-2, IL-4 and IL-10 ELISA detection kits were purchased from Beijing Dakowei Biotechnology Co., Ltd. (Beijing, China); RPMI 1640 and DMEM culture media were purchased from Gibco (USA); other reagents were domestic analytical grade. Escherichia coli BL21 (DE3) competent cells were purchased from Quanshijin Biotechnology Co., Ltd. (Beijing, China); fetal bovine serum (FBS) was purchased from Gibco (USA); porcine anti-SVA positive serum was prepared by our laboratory; BALB / c mice were purchased from the Experimental Animal Center of Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences.

[0049] Plasmid pTF16 (expressing molecular chaperone protein Trigger factor) was constructed as follows:

[0050] (1) The molecular chaperone gene fragment transition plasmid pMV-tig was synthesized by Beijing Liuhe BGI Gene Technology Co., Ltd.; the transition plasmid pMV-tig was digested by enzymes, and the DNA was purified to recover the target fragment;

[0051] (2) Construction of molecular chaperone plasmid:

[0052] Molecular chaperone expression plasmid pTF16: The vector plasmid pACYC184 was digested with enzymes and the DNA recombination kit from NEB was used. HiFi DNA Assembly Master, the DNA fragment of the recovered plasmid pMV-tig was seamlessly connected with the vector fragment by homologous recombination DNA to construct the molecular chaperone expression plasmid pTF16.

[0053] The present invention takes type A Senecavirus SVA / CH-FJ-2017 as an example, but is not limited to type A Senecavirus SVA / CH-FJ-2017. Other type A Senecavirus structural proteins VP2 can be soluble expressed by referring to the method described in the present invention.

[0054] The term "vaccine" refers to a biological preparation that is capable of providing a protective response in the animal to which the vaccine has been delivered and that is incapable of causing severe disease.

[0055] The vaccine of the present invention further optionally comprises one or more adjuvants, excipients, carriers and diluents. The adjuvant can be any suitable adjuvant, chemical immune adjuvants such as aluminum hydroxide, Freund's adjuvant, mineral oil, Span, etc.; microbial immune adjuvants such as mycobacteria, BCC, lipopolysaccharide, muramyl dipeptide, murine peptide, fat-soluble wax D, short Corynebacterium; plant immune adjuvants are mostly polysaccharides extracted from plants or large fungi, such as tuckahoe polysaccharide, safflower polysaccharide, Chinese herbal medicine, etc. And biochemical immune adjuvants such as thymosin, transfer factor, interleukin, etc. Preferred adjuvants can be nano adjuvants, biological adjuvants, interleukins, interferons, etc.

[0056] The vaccine of the present invention can also be used in combination vaccines, such as in combination with other vaccines for pigs, but the focus is on live attenuated vaccines, especially the integration of viral genes, such as bivalent vaccines, trivalent vaccines, etc.

[0057] The administration of the vaccine of the present invention can adopt convenient routes, such as intramuscular injection, intranasal, oral, subcutaneous, transdermal and vaginal routes. The attenuated vaccine of the present invention preferably adopts intramuscular injection. The vaccine can be administered after the initial immunization-boosting scheme. For example, after the first vaccination, the subject can receive a second booster administration after a period of time (e.g., about 7,14,21 or 28 days). Usually, the dosage of booster administration is the same or lower than the dosage of the initial immunization administration. In addition, a third booster immunization can also be performed, such as 2-3 months, 6 months or one year after immunization.

[0058] GraphPad Prism 8.0 software was used to analyze and plot the test data. Data are expressed as mean ± standard error of the mean (SE). NS = P> 0.05: no significant difference between the groups; * P<0.05, ** P < 0.01 and *** P<0.001: There is a significant statistical difference among the groups.

[0059] Example 1 Soluble Expression of Recombinant Senecavirus Type A Structural Protein VP2

[0060] 1. Construction and identification of recombinant expression plasmid

[0061] With the gene sequence of the structural protein VP2 of the SVA / CH-FJ-2017 strain as a reference, the VP2 gene codons were optimized for Escherichia coli. Xi'an Qingke Biotechnology Co., Ltd. was commissioned to synthesize the SVA VP2 gene (852 bp, the gene sequence is shown in SEQID NO.1) and ligated to the Escherichia coli expression vector pET28a(+) to construct the recombinant expression plasmid pET28a-SVA-VP2, which was verified by double enzyme digestion with Hind III and BamH I and identified by gene sequencing.

[0062] The constructed recombinant plasmid pET-28a-SVA-VP2 was identified by double digestion with Hind III and BamH I. Figure 1 As shown, two specific bands of approximately 5300 bp and 850 bp were generated, the size of the enzyme digestion product was consistent with the theoretical value, and sequencing showed that the SVA VP2 gene was correctly inserted.

[0063] 2. Expression and identification of recombinant SVA VP2 protein

[0064] According to the plasmid transformation steps, the recombinant plasmid pET-28a-SVA-VP2 was transformed into BL21 (DE3) competent cells and plated on a plate containing kanamycin (Kanamycin). + ) on solid LB medium at 37°C for 12 h, and three positive monoclonal colonies were selected and inoculated into 5 mL of kanamycin (Kanamycin + ) in liquid LB medium and culture at 37°C and 220 r / min for 2 to 4 h. 600When the value reached about 0.6-0.8, the induction was carried out by comparing the temperature, IPTG concentration and time. After induction culture, the cells were collected by centrifugation at 4500r / min for 30min and resuspended in PBS. The resuspended cells were ultrasonically broken, and the supernatant and precipitate were separated by high-speed centrifugation at 4℃ and 12000r / min for 10min. The precipitate was dissolved again with an equal volume of 8M urea to prepare the sample, and the solubility of the recombinant SVA VP2 protein expression was preliminarily analyzed by SDS-PAGE.

[0065] The results are as follows Figure 2 As shown in the figure, under the conditions of 16°C, 1mmol / L IPTG (final concentration), and 12h of culture, the SVA VP2 positive recombinant expression bacteria were induced. The induced recombinant expression strain observed a specific protein band at about 37kD, but it was mostly concentrated in the precipitate, mainly expressed in inclusion bodies ( Figure 2 (shown in lane 6).

[0066] 3. Expression and purification of recombinant SVAVP2 protein

[0067] (1) Preparation of molecular chaperone co-expression strains

[0068] According to the plasmid transformation procedure, the synthetic molecular chaperone pTF16 (Cm + ) plasmid was transformed into BL21(DE3) competent cells and plated with chloramphenicol (Cm + ) on solid LB medium at 37°C for 12 h, pick three positive monoclonal colonies and incubate them in 5 mL of chloramphenicol (Cm + ) in liquid LB medium, cultured at 37°C and 220r / min for 12h, collected the cells to extract the plasmid, and prepared pTF16-BL21(DE3) competent cells using a competent preparation kit. Then, the recombinant plasmid pET-28a-SVA-VP2 (2μL) was transferred into pTF16-BL21(DE3) competent cells (50μL) and spread on a plate containing kanamycin and chloramphenicol (Kanamycin-28a-SVA-VP2). + / Cm + ) on LB solid medium and cultured at 37°C for 12 h. Three positive monoclonal colonies were picked again and plated in 5 ml Kan + / Cm + The cells were cultured in double-resistance LB liquid medium at 37°C for 12 h, and the plasmids were extracted and sequenced for identification.

[0069] (2) Expression and purification of recombinant SVA VP2 protein

[0070] The above bacterial solution was expanded 100 times and cultured in Kan + / Cm +Incubate in double resistance LB medium at 37°C and 220 rpm for 2-4 h. 600 When the value reached about 0.6-0.8, after comparing the temperature, inducer concentration and induction time, L-arabinose with a final mass concentration of 2g / L and IPTG with a final concentration of 1mmol / L were added to the culture to induce the expression of molecular chaperones TF16 and SVA VP2 proteins, respectively, to set up controls; the cells were collected by centrifugation at 4500r / min for 30min and resuspended in PBS; the resuspended cells were broken by homogenizer, and the supernatant and precipitate were separated after breaking at 4℃ and 12000r / min for 10min. The precipitate was dissolved again with an equal volume of 8M urea to prepare samples for SDS-PAGE analysis. The supernatant of the identified expressed bacterial solution was filtered through a 0.22μm filter membrane, selectively bound to a Ni Sepharose HP purification column, and eluted with a linear gradient elution buffer (50mM NaCl, 500mM Tris-HCl, 20mM-500mM imidazole, pH 8.0) to collect the target protein.

[0071] (3) Western blotting

[0072] The purified recombinant SVA VP2 protein was subjected to SDS-PAGE electrophoresis and transferred to NC membrane, blocked with 5% skim milk at room temperature for 2 h, and eluted 5 times with PBST; anti-His monoclonal antibody (1:5000 dilution) and porcine anti-SVA positive serum (1:5000 dilution) were used as primary antibodies, incubated at room temperature for 1 h, eluted 5 times with PBST, and then goat anti-mouse IgG-HRP antibody (1:10000 dilution) and goat anti-porcine IgG-HRP antibody (1:10000 dilution) were used as secondary antibodies, incubated at room temperature for 1 h, eluted 5 times with PBST, and developed with ECL.

[0073] Using the molecular chaperone co-expression strain, under the conditions of 16°C, 2g / L L-arabinose (final concentration), 1mmol / L IPTG (final concentration), and culturing for 12h, the induced recombinant expression strain showed two clear protein bands at about 50kD (TF16) and 37kD, which were the target bands of TF16 and VP2 proteins, respectively. The VP2 protein was clearly expressed in the supernatant (see Figure 2 middle lane 9).

[0074] Under non-denaturing conditions, the recombinant VP2 protein in the supernatant of bacterial lysate was purified using nickel ion affinity chromatography filler Ni Sepharose HP. The SDS-PAGE identification results are shown in Figure 3As shown, the purity of the purified VP2 antigen protein is about 80%. Western-blot results show that both His Tag monoclonal antibody and SVA positive serum can detect the purified recombinant SVA VP2 protein, indicating that the recombinant SVA VP2 protein prepared by the present invention has good immunoreactivity.

[0075] Example 2 Immunogenicity Analysis of Recombinant SVA VP2 Protein

[0076] 1. Mouse Immunization

[0077] Female healthy BALB / c mice aged 6 to 8 weeks were selected and divided into 2 groups, 5 mice in each group; PBS control group and VP2 immunization group (recombinant SVA VP2 protein prepared by the method described in Example 1) were immunized twice by subcutaneous multi-point injection on the back, with an interval of 14 days. The dose of recombinant SVAVP2 protein for the first immunization was 50 μg (emulsified with Freund's incomplete adjuvant), the injection dose was 100 μL, and PBS was the same. The dose of recombinant SVA VP2 protein for the second immunization was 50 μg (emulsified with Freund's complete adjuvant), the injection dose was 100 μL, and PBS was the same. Blood was collected and serum was separated at 0, 7, 14, 21, and 28 days respectively.

[0078] 2. Indirect ELISA test

[0079] The level of specific IgG antibodies in the serum of immunized mice was determined by indirect ELISA. The recombinant SVA VP2 protein prepared in Example 1 was used as the coating antigen, and the antigen was diluted with 50 mmol / L carbonate buffer, and 100 μL / well was coated on the ELISA enzyme-labeled plate (100 ng / well), and allowed to stand at 4°C for 12 hours, the liquid was discarded, and eluted 3 times with PBST. 200 μL / well of 5% skimmed milk powder was blocked at 37°C for 2 hours, the liquid was discarded, and eluted 3 times with PBST. The serum of the mouse to be tested was diluted 1:1 000, 100 μL / well, incubated at 37°C for 1 hour, the liquid was discarded, and eluted 3 times with PBST. The goat anti-mouse IgG-HRP anti-antibody was diluted 1:10000, 100 μL / well, incubated at 37°C for 1 hour, the liquid was discarded, and eluted 3 times with PBST. 50 μL / well of TMB substrate colorimetric solution was used for color development, and 50 μL / well of stop solution was used for termination. OD was measured on the microplate reader 450 According to the formula P / N = (detection hole OD 450 Value-Blank well OD 450 value) / (OD of negative control well) 450 Value-Blank well OD 450 value)>2.1, and the P / N value was calculated to analyze the specific antibody titer.

[0080] The sera of mice at 0, 7, 14, 21 and 28 days were tested by indirect ELISA. Figure 4As shown in Figure 2, mice were able to produce high titer specific IgG antibody levels on the 14th day after immunization (P < 0.001), and the antibody level was maintained until 28 days after immunization ( Figure 4 The maximum serum titer was calculated by referring to the P / N calculation formula. The results showed that the highest titer of mouse immune serum could reach 1∶32 000 ( Figure 4 (shown in B).

[0081] 3. Neutralizing antibody detection

[0082] The serum to be tested was inactivated at 56°C for 30 minutes, and DMEM medium without FBS was added to the 96-well plate at 50 μL / well. Inactivated serum was added at 50 μL / well in the first row, and the serum was diluted from 1:4 to 1:512 in a continuous ratio. After mixing in the last row, 50 μL was discarded. Each dilution was repeated 3 times. Then 200 TCID 50 Virus solution (50 μL / well) was incubated at 37°C, 5% CO2 for 1 h, and then 100 μL of IBRS-2 cell suspension (1×10 5 / well), and set up virus control group and cell control group at the same time, cultured at 37°C, 5% CO2 for 48-72h, and calculated the neutralizing antibody titer of the serum according to the Reed-Muench method.

[0083] Neutralization test results Figure 5 As shown, neutralizing antibodies in the recombinant SVA VP2 protein immune serum could be detected 14 days after the first immunization, and could reach 1:128 28 days after the second immunization, which was significantly higher than the PBS control group (<1:4).

[0084] 4.CD4 + and CD8 + T lymphocyte subset detection

[0085] According to the instructions of mouse lymphocyte separation solution, spleen lymphocytes were separated, (cell number) cells were taken and centrifuged at 1500r / min for 3min in a 1.5mL EP tube, the supernatant was discarded, and the cell pellet was resuspended in PBS. Then 6μL of antibody (2μL CD3e-FITC, 2μL CD4-APC, 2μL CD8a-PE) was added to each cell suspension, mixed, ice-bathed for 30min away from light, centrifuged at 1500r / min for 5min, supernatant was discarded, washed three times with PBS, and finally 200μL of PBS was added to resuspend the cells, the voltage of the flow cytometer was adjusted for the blank group, and the flow cytometer was used for detection.

[0086] Detection of CD4 by flow cytometry + / CD3 + and CD8 + / CD3 +The percentage of T lymphocytes. Each group counted 1×10 4 The average value of each group was taken. The test results were as follows: Figure 6 As shown, compared with the control group, the CD4 + / CD3 + The percentage of T lymphocytes was 34.11%, which was significantly higher than that of the control group (P<0.001). + / CD3 + The percentage of T lymphocytes was 13.24%, which was significantly increased compared with the PBS control group (P<0.001).

[0087] 5. Lymphocyte proliferation assay

[0088] Mouse lymphocyte suspension was inoculated into 96-well cell culture plates, 100 μL / well (2.5×10 5 Cells / well), and cell-free 1640 medium was used as a blank control. 50 μL of 1640 complete medium, recombinant SVAVP2 protein (10 μg / mL), and ConA protein (10 μg / mL) were added to each group of cells as a negative control group, a specific antigen stimulation group, and a positive control group. Each group had 6 replicate wells. After culturing at 37°C and 5% CO2 for 60 h, 10 μL of CCK8 solution (5 μg / mL) was added to each well, and incubated at 37°C and 5% CO2 for 3.5 h. The OD was read by an enzyme reader. 450 The stimulation index (SI) was calculated.

[0089] The CCK-8 method was used to measure the lymphocyte proliferation ability of immunized mice. Figure 7 As shown, the proliferation SI of lymphocytes in the test group and the control group stimulated by Con A protein were 3.63 and 3.81 (P>0.05). Under the stimulation of recombinant SVA VP2 protein, the SI of lymphocytes in the immunization group was significantly higher than that in the control group (P<0.01), indicating that the recombinant SVA VP2 protein prepared by the present invention can effectively stimulate the proliferation of spleen lymphocytes.

[0090] 6. Cytokine Level Detection

[0091] Mouse lymphocyte suspension was inoculated into 24-well plates at 0.5 mL / well (1×10 6Cells / well), cell-free 1640 medium as blank control. 250 μL of 1640 complete medium and SVA VP2 protein (10 μg / mL) were added to each well as negative control group and specific antigen stimulation group, respectively, and incubated at 37°C for 60 h. The culture supernatant was collected from each well, and the levels of IL-2, IL-4, IL-10 and IFN-γ were detected by ELISA.

[0092] The levels of IFN-γ, IL-2, IL-4, and IL-10 secreted by lymphocytes of each group of mice were detected by ELISA detection kits. The results are shown in Figure 8 As shown, the expression level of IFN-γ in the recombinant SVA VP2 protein immunization group was significantly higher than that in the PBS group (P<0.001); the expression level of IL-2 was significantly higher than that in the PBS group (P<0.001); the expression level of IL-4 was significantly higher than that in the PBS group (P<0.001); the expression level of IL-10 was significantly higher than that in the PBS group (P<0.001). This shows that the recombinant SVA VP2 protein prepared by the present invention can not only induce the expression and secretion of Th1 type cytokines IFN-γ and IL-2, but also induce the expression and secretion of Th2 type cytokines IL-4 and IL-10.

[0093] The above results indicate that the recombinant SVA VP2 protein prepared by the present invention can induce a strong humoral immune response and a cellular immune response in the body and has good immunogenicity in animals.

[0094] In summary, the present invention successfully prepared a soluble recombinant SVA VP2 protein using the molecular chaperone TF16. Animal immunization experiments have shown that the recombinant protein has good immunogenicity and can induce the body to produce strong humoral and cellular immune responses, providing important experimental materials and theoretical basis for the further development of new SVA subunit vaccines and diagnostic preparations.

[0095] The above examples are only for illustrating the technical concept and features of the present invention, and their purpose is to enable people familiar with the technology to understand the content of the present invention and implement it accordingly, and they cannot be used to limit the protection scope of the present invention. Any equivalent changes or modifications made according to the spirit of the present invention should be included in the protection scope of the present invention.

Claims

1. Application of molecular chaperone protein Trigger factor in promoting the expression of Seneca virus type A structural protein VP2.

2. A genetically engineered bacterium that solublely expresses the structural protein VP2 of type A Seneca virus, characterized in that: The genetically engineered bacteria contain a plasmid expressing the structural protein VP2 of type A Seneca virus and a plasmid expressing the molecular chaperone Trigger factor.

3. A method for soluble expression of recombinant Senecavirus type A structural protein VP2, characterized in that: The method comprises: fermenting and culturing a genetically engineered bacterium that simultaneously expresses the A-type Seneca virus structural protein VP2 and the molecular chaperone protein Trigger factor, and inducing expression to obtain the recombinant A-type Seneca virus structural protein VP2.

4. The method according to claim 3, characterized in that The method comprises the following steps: Preparation of genetically engineered bacteria: Preparation of genetically engineered bacteria that simultaneously express the structural protein VP2 of type A Seneca virus and the molecular chaperone protein Trigger factor; Induced expression: The obtained genetically engineered bacteria were fermented and cultured, and L-arabinose and IPTG were added to induce expression to obtain the recombinant A-type Seneca virus structural protein VP2.

5. The method according to claim 4, characterized in that The preparation of the genetically engineered bacteria comprises the following steps: (1) The codons of the Senecavirus A structural protein VP2 gene were optimized in E. coli, the SVA VP2 gene was synthesized and ligated into an E. coli expression vector to construct a recombinant expression plasmid; (2) Transforming the molecular chaperone pTF16 plasmid into Escherichia coli competent cells, and screening and preparing competent cells; (3) The recombinant plasmid described in step (1) is transferred into the competent cells of step (2), and a genetically engineered bacterium that simultaneously expresses the structural protein VP2 of type A Seneca virus and the molecular chaperone protein Trigger factor is obtained by screening.

6. The method according to claim 5, characterized in that The Escherichia coli include BL21(DE3), BL21(DE3)pLysS, B834(DE3), BLR(DE3), JM109, XL1Blue, ER2566, Rosetta, and GI698.

7. The method according to claim 6, characterized in that The preparation of the genetically engineered bacteria is as follows: (1) synthesizing the SVA VP2 gene sequence shown in SEQ ID NO.1 and ligating it into the Escherichia coli expression vector pET28a(+) to construct the recombinant expression plasmid pET28a-SVA-VP2; (2) Synthesis of the molecular chaperone pTF16 (Cm + ) plasmid, transformed into BL21(DE3) competent cells, cultured in a medium containing chloramphenicol, and picked positive clones to obtain pTF16-BL21(DE3) competent cells; (3) The recombinant plasmid pET-28a-SVA-VP2 described in step (1) is transferred into the pTF16-BL21 (DE3) competent cells described in step (2), cultured in a medium containing kanamycin and chloramphenicol, and positive clones are picked to obtain genetically engineered bacteria that simultaneously express the type A Seneca virus structural protein VP2 and the molecular chaperone protein Trigger factor.

8. The method according to claim 4, characterized in that The induced expression is: The genetically engineered bacteria that simultaneously express the type A Seneca virus structural protein VP2 and the molecular chaperone protein Trigger factor are fermented and cultured, and L-arabinose with a final concentration of 2 g / L and IPTG with a final concentration of 1 mmol / L are added to the culture to induce expression; The bacteria were collected by centrifugation, resuspended in PBS, broken, and centrifuged to obtain the supernatant, and the recombinant Seneca virus type A structural protein VP2 was purified.

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