A PRRSV multi-epitope recombinant protein containing an internal adjuvant and its application in the preparation of porcine reproductive and respiratory syndrome virus vaccine

Abstract

The present invention relates to the field of biopharmaceutical technology. The present invention provides a porcine reproductive and respiratory syndrome virus recombinant protein, and a PRRSV multi-epitope recombinant protein containing an intramolecular adjuvant comprising the porcine reproductive and respiratory syndrome virus recombinant protein. The present invention also provides the use of the PRRSV multi-epitope recombinant protein containing an intramolecular adjuvant in the preparation of a porcine reproductive and respiratory syndrome virus vaccine. The present invention uses bioinformatics analysis to screen epitopes, uses the neutralizing epitopes of each protein to prepare a PRRSV recombinant protein, and then introduces an intramolecular adjuvant to further improve the immune level and enhance the immunogenicity of the vaccine.

Description

Technical Field

[0001] The present invention relates to the technical field of biopharmaceuticals, and in particular to a PRRSV multi-epitope recombinant protein containing an intramolecular adjuvant and application thereof in preparing a porcine reproductive and respiratory syndrome virus vaccine. Background Art

[0002] Porcine reproductive and respiratory syndrome virus (PRRSV) is the most common swine pathogen in China's pig herds and a major disease that affects the healthy and sustainable development of the pig industry. Although several vaccines have been used for more than ten years, they are unable to meet demand due to rapid virus mutation, low intramuscular cellular / mucosal immunity, and potential ADE.

[0003] The GP5 protein of PRRSV is the main protective antigen targeted by neutralizing antibodies induced by PRRSV vaccination or previous infection. The GP5 protein has at least two antigenic regions: an extracellular region (27aa-41aa) and one at the C-terminus (180aa-197aa). Zhang (Construction of porcine reproductive and respiratory syndrome virus with GP5 protein mutants atputative glycosylation sites) et al. found that the glycosylation of PRRSV GP5 protein plays a role in host immune function and affects the relationship between the virus and specific neutralizing antibodies. For the PRRSV GP4 protein, Wang Aiping et al. (Preliminary screening of B cell epitopes of porcine reproductive and respiratory syndrome virus GP4 protein) initially screened out a GP4 B cell epitope peptide (50-67aa) containing the core region of the neutralizing epitope, which has great research value. GP2, GP3 and GP4 are believed to combine to form a complex located on the surface of the envelope, which is necessary for the production of infectious virus particles. The PRRSV M protein is a hydrophobic structural protein that plays an extremely important role in the process of viral self-assembly and budding. The M protein can aggregate with the GP5 protein to form a dimer. This heterodimer plays an important role in the process of virus infection of alveolar macrophages, which is of great significance for the formation of infectious virus particles.

[0004] In summary, the functions and neutralizing epitopes of the main proteins of PRRSV have been studied extensively. Although multiple subunit vaccines can induce immunity and produce neutralizing antibodies, the intensity of induced cellular immunity, humoral immunity and neutralizing titer have not reached the ideal level, and there is a risk of causing ADE effect under sub-neutralization conditions. Therefore, it is very necessary to obtain a porcine reproductive and respiratory syndrome virus vaccine with a higher immune level that can cope with the potential ADE effect. Summary of the invention

[0005] The purpose of the present invention is to provide a PRRSV multi-epitope recombinant protein containing an intramolecular adjuvant and the application of the protein in the preparation of a porcine reproductive and respiratory syndrome virus vaccine.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The invention provides a porcine reproductive and respiratory syndrome virus recombinant protein, comprising a porcine reproductive and respiratory syndrome virus recombinant protein 1 and a porcine reproductive and respiratory syndrome virus recombinant protein 2.

[0008] Preferably, the amino acid sequence of the porcine reproductive and respiratory syndrome virus recombinant protein 1 is shown in SEQ ID NO:13; the amino acid sequence of the porcine reproductive and respiratory syndrome virus recombinant protein 2 is shown in SEQ ID NO:24.

[0009] The invention provides a PRRSV multi-epitope recombinant protein containing an intramolecular adjuvant, comprising the porcine reproductive and respiratory syndrome virus recombinant protein and the intramolecular adjuvant.

[0010] Preferably, the internal adjuvant comprises any one of a p28T4 vector carrying Tufisn and T4-Fd, a p28GCN vector carrying Tufisn and GCN4, and a p28Fe vector carrying Tufisn and ferritin.

[0011] Preferably, the amino acid sequence of Tufisn is shown in SEQ ID NO:25; the amino acid sequence of the polymer peptide T4-Fd is shown in SEQ ID NO:26; the amino acid sequence of the polymer peptide GCN4 is shown in SEQ ID NO:27; and the amino acid sequence of ferritin is shown in SEQ ID NO:28.

[0012] The present invention also provides the use of the PRRSV multi-epitope recombinant protein containing an intramolecular adjuvant in the preparation of a porcine reproductive and respiratory syndrome virus vaccine.

[0013] The present invention also provides a porcine reproductive and respiratory syndrome virus subunit vaccine, comprising the PRRSV multi-epitope recombinant protein containing an intramolecular adjuvant and a pharmaceutically acceptable adjuvant.

[0014] By adopting the above technical solution, the present invention has the following beneficial effects:

[0015] The technical solution of the present invention adopts bioinformatics analysis means and based on the glycan masking theory, reverse thinking adopts the prokaryotic system to avoid glycosylation, finds non-glycosylated conserved neutralizing epitopes, and combines the neutralizing epitopes of each screened protein in a reasonable manner to obtain PRRSV recombinant protein; then the intramolecular adjuvant is introduced to enhance the neutralizing epitope antibody level, further improve the immune level, and enhance the immunogenicity of the vaccine.

[0016] Ferritin nanoparticles in the technical solution of the present invention can enhance the immunogenicity of the vaccine as an intramolecular adjuvant, regulate the type of immune response, enhance antigen presentation and processing, and improve the stability and durability of the vaccine. These effects help to improve the immune effect and protective effect of the vaccine, and help to improve the ability of the cellular and humoral immune response of mice. DETAILED DESCRIPTION

[0017] The invention provides a porcine reproductive and respiratory syndrome virus recombinant protein, comprising a porcine reproductive and respiratory syndrome virus recombinant protein 1 and a porcine reproductive and respiratory syndrome virus recombinant protein 2.

[0018] In the present invention, the composition of the porcine reproductive and respiratory syndrome virus recombinant protein 1 includes HTL and CTL epitopes of GP5 protein and M protein, the amino acid sequence of the GP5 protein HTL epitope is shown in SEQ ID NO:1, and the specific sequence is SSSHLQLIY; the amino acid sequence of the M protein HTL epitope is shown in SEQ ID NO:2, and the specific sequence is IFLNCAFTF. The GP5 protein selects 4 different CTL epitopes, and the amino acid sequences of each epitope are shown in SEQ ID NO:3-SEQ ID NO:6, wherein the specific sequence of SEQ ID NO:3 is MSSTYQYIYNLTICELNGTDWLSNHA, the specific sequence of SEQ ID NO:4 is LAALICFVIRLAKNC, the specific sequence of SEQ ID NO:5 is KGRLYRWRSPVIIEK, and the specific sequence of SEQ ID NO:6 is GLITVSAAGY. The M protein selected 6 CTL epitopes, and the amino acid sequences of each epitope are shown in SEQ ID NO:7-SEQ ID NO:12, wherein the specific sequence of SEQ ID NO:7 is TWKFITSRCRLCLLGRKYIL, the specific sequence of SEQ ID NO:8 is STNKVALTM, the specific sequence of SEQ ID NO:9 is FSITYTPVM, the specific sequence of SEQ ID NO:10 is YSAIETWKF, the specific sequence of SEQ ID NO:11 is AVKQGVVNL, and the specific sequence of SEQ ID NO:12 is MDAHHVKSAAGLHSIPASGNRAYAVRKPGLTSVNGTLVPGLRSL.

[0019] In the present invention, the amino acid sequence of the porcine reproductive and respiratory syndrome virus recombinant protein 1 is shown in SEQ ID NO: 13, and the specific sequence is

[0020] MVGRKKRRQRRRPPQGPGPAAYSSSHLQLIYAAYIFLNCAFTFHEYGAEALERAGMSSTYQYIYNLTICELNGTDWLSNHAGPGPGLAALICFVIRLAKNCGPGPGKGRLYRWRSPVIIEKGPGPGGLITVSAAGYGPGPGTWKFITSRCRLCLLGRKYILGPGPGSTNKVALTMGPGPGFSITYTPVMGPGPGYSAIETWKFGPGPGAVKQGVVNLGPGPGMDAHHVKSAAGLHSIPASGNRAYAVRKPGLTSVNGTLVPGLRSLGSSIQYRLFSQVRSGKWKKLFSQVRSGKWFVKKWAVETFVIFPVLTHIVSYKKFSQVDRELVKKSHIQLIYNLKKHFQSIYNKKHLQIYNKKHLDLIYNKKGSSLDDFCYDSTAPQKVLLAFSITYAKKFRSTNKVAKKVRRPGSTTVNGTLVKKGRRAVKRGVVNLVKHEYGAEALERAGKLGGGGSLE。

[0021] In the present invention, the composition of the porcine reproductive and respiratory syndrome virus recombinant protein 2 includes HTL and CTL epitopes of GP2 protein, GP3 protein and GP4 protein, and the GP3 protein selects 2 HTL epitopes, and the amino acid sequence of each epitope is shown in SEQ ID NO: 14 and SEQ ID NO: 15, wherein the specific sequence of SEQ ID NO: 14 is IFLCCGFLY, and the specific sequence of SEQ ID NO: 15 is ISAVFQTYY. The GP4 protein selects 2 HTL epitopes, and the amino acid sequence of each epitope is shown in SEQ ID NO: 16 and SEQ ID NO: 17, wherein the specific sequence of SEQ ID NO: 16 is LMLSSCLFY, and the specific sequence of SEQ ID NO: 17 is RTAIGTPVY. The amino acid sequence of the HTL epitope of the GP2 protein is shown in SEQ ID NO: 18, and the specific sequence is SFSEWFAPRFSVRALP. The GP3 protein selected two CTL epitopes, and the amino acid sequences of each epitope are shown in SEQ ID NO:19 and SEQ ID NO:20, wherein the specific sequence of SEQ ID NO:19 is FSFELLVNY, and the specific sequence of SEQ ID NO:20 is ITAVYQTYY. The GP4 protein selected two CTL epitopes, and the amino acid sequences of each epitope are shown in SEQ ID NO:21 and SEQ ID NO:22, wherein the specific sequence of SEQ ID NO:21 is MAASFLFLL, and the specific sequence of SEQ ID NO:22 is RTAIGTPVY. The amino acid sequence of the CTL epitope of the GP2 protein is shown in SEQ ID NO:23, and the specific sequence is FPTPGTRPKL.

[0022] In the present invention, the amino acid sequence of the porcine reproductive and respiratory syndrome virus recombinant protein 2 is shown in SEQ ID NO: 24, and the specific sequence is MVGRKKRRQRRRPPQGPGPGSAYYSFSEWFAPRFSVRALPAAYIFLCCGFLYAAYISAVFQTYYAAYLMLSSCLFYAAYRTAIGTPVYHEYGAEALERAGFPTPGTRPKLGPGPGFSFELLVNYGPGPGITAVYQTYYGPGPGMAASFLFLLGPGPGRTAIGTPVYHEYGAEALERAGKKSQAGYWSFFSEWLAPRFSVRKKHSGQAAWKQVVSEATLTKLSRLDVVKKLCPTRQAAAEILEPGKSKKRRFIDWKKRQSGLNKKGVSAAQEKISFGHEYGAEALERAGKLGGGGSLE.

[0023] The present invention also provides a PRRSV multi-epitope recombinant protein containing an intramolecular adjuvant, comprising the porcine reproductive and respiratory syndrome virus recombinant protein and the intramolecular adjuvant.

[0024] In the present invention, the internal adjuvant includes any one of a p28T4 vector carrying Tufisn and T4-Fd, a p28GCN vector carrying Tufisn and GCN4, and a p28Fe vector carrying Tufisn and ferritin.

[0025] In the present invention, the vector p28T4 is obtained by synthesizing Tufisn and T4-Fd sequences onto a PET28a vector, the amino acid sequence of Tufisn is shown in SEQ ID NO: 25, and the specific sequence is TKPR; the amino acid sequence of the polymer peptide T4-Fd is shown in SEQ ID NO: 26, and the specific sequence is MQALQEAGYIPEAPRDGQAYVRKDGEWVLLSTFLSPA.

[0026] In the present invention, the vector p28GCN is obtained by synthesizing GCN4 and Tufisn sequences onto a PET28a vector, and the amino acid sequence of the polymer peptide GCN4 is shown in SEQ ID NO: 27, and the specific sequence is MKQIEDKIEEILSKIYHIENEIARIKKLIGEV.

[0027] In the present invention, the vector p28Fe vector is obtained by synthesizing ferritin and Tufisn sequences onto a PET28a vector, and the amino acid sequence of the ferritin is shown in SEQ ID NO: 28, and the specific sequence is MLSKDIIKLLNEQVNKEMNSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIVFLNENNVPVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDHATFNFLQWYVAEQHEEEVLFKDILDKIELIGNENHGLYLADQYVKGIAKSRKS.

[0028] The present invention also provides the use of the PRRSV multi-epitope recombinant protein containing an intramolecular adjuvant in the preparation of a porcine reproductive and respiratory syndrome virus vaccine.

[0029] The present invention also provides a porcine reproductive and respiratory syndrome virus subunit vaccine, comprising the PRRSV multi-epitope recombinant protein containing an intramolecular adjuvant and a pharmaceutically acceptable adjuvant.

[0030] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0031] Example 1

[0032] (1) Identification of candidate vaccine strains

[0033] The amino acid sequences of GP2 protein, GP3 protein, GP4 protein, GP5 protein and M protein were collected from the NCBI database in the fasta file format and used as templates. The sequence conservation was analyzed using MEGA 7.0 software, and the resulting comparative data set was then visualized and analyzed for conservation using the WebLogo server.

[0034] (2) Immunoinformatics design

[0035] Epitope prediction and screening: ABCpred server was used to predict candidate linear B lymphocyte epitopes of proteins. MHC-II epitopes were predicted using NetMHCIIpan-4.0 server. The potential for epitope combination with MHC-II was evaluated by EpiTOP server. Prediction was performed by IFN epitope server and IL4pred server. MHC-I was combined with NetMHCpan4.1 server and IEDB server for CTL epitope prediction. All predicted epitopes were determined by IEDB server. Non-toxic immunogenicity test was performed by ToxinPred server.

[0036] Identification of protective epitopes and assessment of antigenic epitope conservation: Dominant epitopes were assessed by the conservation analysis tool on the IEDB server.

[0037] Concatenation of candidate epitopes: Sequences collected from literature and predicted sequences are concatenated with candidate vaccines using flexible linkers to extend the duration of protein action in vivo, effectively avoiding the introduction of new binding epitopes. GGPPG is used for CTL epitopes, AAY is used for HTL epitopes, and KK is used for B lymphocyte epitopes. In order to improve the immunogenicity of candidate vaccines, intramolecular adjuvants are added and connected to the N-terminus of the construct through an EAAAK linker, and a Flynn-sensitive linker (RVRR) is used for cross-protein connection.

[0038] By adjusting the number of repeated copies and the construction position, the neutralizing epitopes selected from known literature were designed to enhance antigenicity.

[0039] Construction of candidate vaccines and prediction of antigenicity, sensitization, solubility and physicochemical properties: The final antigenicity analysis of the candidate vaccine was performed using the ANTIGENpro server and the VaxiJen v2.0 server; the AllerTOPv2.0 server was used for the final allergenicity analysis of the candidate vaccine; and the SOLpro server was used for the final solubility analysis of the candidate vaccine. The physicochemical properties of the candidate vaccine were analyzed using the ExPASy server, and several candidate combinations were selected.

[0040] Secondary and tertiary structure modeling, refinement and disulfide engineering of candidate vaccines: The secondary structure prediction of the final candidate vaccine was completed using PSIPRED4.0 server and SOPMA server. The tertiary structure prediction of the final candidate vaccine was completed using trRosetta server. The structure was optimized using Galaxy-WEB server. Ramachandran plots were generated by EBI-PDBSum server to evaluate the quality of the model before and after optimization. Disulfide engineering of the candidate vaccine was performed using DisulfidebyDesign32.2 server. Global detection of the model was performed using ProSA-Web server.

[0041] Tertiary epitope prediction and protein docking analysis: The tertiary structure of the vaccine protein was predicted by ElliPro server. The interaction between TLR3 as receptor and protein vaccine as ligand was predicted using PyDockWEB server. LigPLot+ was used to determine the amino acid residues with hydrogen bonds and hydrophobic interactions after docking.

[0042] Example 2

[0043] 1. Synthesis, expression and purification of porcine reproductive and respiratory syndrome virus recombinant protein

[0044] According to the above screening, the dominant HTL and CTL epitopes of GP2 protein, GP3 protein, GP4 protein, GP5 protein and M protein were obtained, and then the porcine reproductive and respiratory syndrome virus recombinant protein 1 (abbreviated as GP5MHCN) composed of the dominant HTL and CTL epitopes of GP5 protein and M protein, and the porcine reproductive and respiratory syndrome virus recombinant protein 2 (abbreviated as GP234HCN) composed of the dominant HTL and CTL epitopes of GP2 protein, GP3 protein and GP4 protein were designed. The specific sequences are shown in Table 1.

[0045] Table 1 Recombinant protein of porcine reproductive and respiratory syndrome virus

[0046]

[0047] The above designed sequence forms were sent to BGI for synthesis, and BGI connected the synthesized sequence to pET28a to prepare the corresponding recombinant plasmids (dry powder). The recombinant plasmid was centrifuged at 5000rpm / min for 5min, then taken into the clean bench and dissolved by adding 40μL of double distilled water. Take the competent cells of Escherichia coli BL21 (DE3) in the -80℃ refrigerator and place them in ice for 10min, add 3μL of recombinant plasmid to the competent cells, place them in ice for 30min, heat shock them in a 42℃ water bath for 90s, and immediately place them in ice for 2min. Then add 750μL of LB liquid medium pre-warmed at 37℃ to the EP tube and shake and culture at 37℃ for 1h. 5000rpm / min, centrifuge for 5min, discard 800μL of supernatant, resuspend the remaining bacteria and apply them to the plate containing kanamycin, and invert the plate in a 37℃ incubator for overnight culture.

[0048] The next day, monoclonal colonies were picked and added to 5 mL of LB liquid medium containing kanamycin to verify their expression. Different gradients of induction temperature (16°C, 28°C, 37°C), different gradients of induction time (4h, 6h, 8h, 10h), and different gradients of inducer addition (0.2mmol / L, 0.4mmol / L, 0.6mmol / L, 0.8mmol / L, 1mmol / L) were set to explore the conditions for inducing the expression of recombinant proteins.

[0049] The results showed that the optimal induction temperature was 37℃, the optimal induction time was 6h, and the optimal inducer concentration was 0.4mmol / L.

[0050] The verified strain was fermented in 2.4 L of bacterial liquid in a shake flask, and the bacteria were harvested: centrifuged at 10,000 r / min for 10 min, the precipitate was broken with high-salt lysis buffer 1 (composed of NaCl, Tris-HCL, EDTA, glycerol and NP40) (3s, 3s, 30min), and then centrifuged at 10,000 r / min for 10 min. The supernatant was discarded, and the precipitate was broken with ordinary lysis buffer 2 (composed of Tris-HCL and Triton-X100) (3s, 3s, 30min), and then centrifuged at 10,000 r / min for 10 min. The precipitate was suspended with 2 mL of 8 M urea and dropped into 40 mL of 8 M urea for sedimentation for 30 min to allow the protein to better integrate into 8 M urea and then broken (3s, 3s, 30min), and then centrifuged at 10,000 r / min for 10 min. The precipitate was collected and washed with inclusion body washing solution to obtain inclusion body protein solution, and then the inclusion body protein solution was centrifuged at 10000r / min for 10min, and the supernatant protein was filtered with a 0.22μm filter membrane to remove significant precipitate and prevent clogging of the column filter membrane; the filtered supernatant was purified with Ni-NTA resin at 4°C, and after combination, gradient elution was performed with eluent containing different concentrations of imidazole (100, 300, 500mmol / L), and the optimal elution concentration was 300mmol / L. The purified protein after elution was collected and stored at -20°C.

[0051] 2. Dialysis refolding of porcine reproductive and respiratory syndrome virus recombinant protein

[0052] Cut the dialysis bag into a suitable size, put it into a beaker, add appropriate amount of pure water, boil it in a boiling water bath for 10 minutes, and take it out to cool.

[0053] Take 50mL of the above filtered inclusion body protein solution and add refolding solution (20mmol / LTris-HCl, 500mmol / LNaCl, 45mL / L glycerol, 1g / L arginine) and dithiothioethanol respectively, and put it into the pretreated dialysis bag for refolding. The volume of the refolding solution added is 10% of the volume of the inclusion body protein solution, and the volume of dithiothiothioethanol added is 0.5% of the volume of the inclusion body protein solution. After confirming that there is no leakage in the seal, place it at 4°C for refolding procedure, and stir the dialyzate with a magnetic stirrer. The refolding procedure is:

[0054] 1) Dialyze with 10 times the volume of external solution A for 1 hour (the external solution A is 8M urea diluted 100 times with 2 times the volume of dialysate), and then add 3 times the volume of external solution water for 1 hour;

[0055] 2) Change the volume of external solution B by 100 times and dialyze for 4 hours (the external solution B is the refolding solution diluted 1000 times);

[0056] 3) The external solution C was replaced with 100 times the volume and dialyzed for 4 h (the external solution C was a 10 mM CB solution with a pH of 9.5, and the CB solution was prepared by diluting the volume to 1 L with 0.318 g of sodium carbonate and 0.586 g of sodium bicarbonate).

[0057] The renatured protein was centrifuged at 5000 r / min for 10 min, and the supernatant was retained. Then, the protein before and after renaturation was subjected to 12% SDS-PAGE electrophoresis to determine its renaturation status.

[0058] The results showed that the target band existed after renaturation, and its size was consistent with that before renaturation, and no other impurity bands were generated.

[0059] 3. Identification and stability study of porcine reproductive and respiratory syndrome virus recombinant protein

[0060] The purified protein was extracted with Triton X-114 to remove endotoxins, and the endotoxin removal effect was evaluated. The initial protein endotoxin content was higher than 100 EU / mL and was less than 10 EU / mL after two extractions.

[0061] Western-blot method was used to verify whether it was a recombinant protein, and Dot-ELISA method was used to identify the immunogenicity of the protein by reacting the antigen with PRRSV positive serum. The results showed that both had positive reactions.

[0062] Long-term stability experiments were conducted to investigate the storage stability of recombinant antigens at different temperatures (-80°C, -20°C, 4°C or 37°C for accelerated destruction). The results confirmed that the protein was most stable at -80°C, followed by -20°C, and could only be stably stored for two weeks at 4°C.

[0063] Example 3

[0064] 1. Vaccine preparation and mouse immunization

[0065] The renatured protein was quantitatively set to 200ug / mL and mixed with 201 adjuvant at a ratio of 1:1. After mixing, a homogenizer was used to emulsify for 5 minutes. After centrifugation, no stratification occurred, and the vaccine preparation was obtained. Balb / c mice (purchased from Henan Sikebeisi Biotechnology Co., Ltd.) were selected for immunization. The mice were selected to be over two months old. The mice were immunized subcutaneously. Three points were selected for immunization, including subcutaneous injection in the neck and subcutaneous injection in the hind legs. Each mouse received a total of 200μL, including 100μL in the neck and 50μL in the inner side of the hind legs. The immunization was performed three times, with an interval of 14 days each time. Blood was collected from the tail vein one week after each immunization, and blood was collected from the orbits two weeks after the three immunizations. The corresponding immunization times when the titer was 1:100,000 were the common immunization endpoints. If the titer did not meet the requirements, enhanced immunization was performed. Blood was collected from the tail vein one week after immunization. The operation was the same as the previous three times. Enhanced immunization was performed until the titer was reached, and a maximum of 5 immunizations were performed.

[0066] (II) Humoral Immunity Testing

[0067] The blood of the immunized mice was collected, and the serum obtained was separated and tested for specific antibody IgM and IgG indicators according to the ELISA method. The results are shown in Table 2.

[0068] Table 2 Serum titers of different porcine reproductive and respiratory syndrome virus recombinant proteins in humoral immunity

[0069] Protein name Potency GP5MHCN 1：128000 GP234HCN 1：100000

[0070] The results showed that after immunization of the above mice, the optimal serum titer was 128,000.

[0071] (III) Evaluation of cellular immune effects

[0072] After blood was drawn from the eyeball, the mice were killed by cervical dislocation, and the spleen tissue of the mice was dissected to prepare spleen single cell suspension, which was lysed with red blood cell lysis buffer and then stimulated and cultured (1×10 6 cells / mL), cell surface staining, fixation, membrane permeabilization, cell activation stimulation, extracellular factor staining after blocking the antibody Fc-terminal receptor, fixation, membrane permeabilization and washing, resuspended and then flow cytometry detection, detection of CD3, CD4, CD8 indicators.

[0073] The results showed that CD3, CD4 and CD8 all increased.

[0074] (IV) Safety evaluation

[0075] The mice were observed after immunization, and their appearance, behavior, spirit, and food intake were observed every day during the immunization period. No abnormalities were found.

[0076] Three days after the last immunization, histopathological examination was performed: the mice were killed by cervical dislocation, and the lungs, hearts, spleens, kidneys, etc. were taken out on a sterile operating table. No pathological changes such as edema and congestion were observed with the naked eye.

[0077] Then, the above tissues were rinsed twice with PBS and fixed in 10% formalin solution. The fixed tissues were paraffin-embedded for 24 hours, cut into 4 μm slices under a sliding microtome, and dehydrated step by step with different concentrations of ethanol. The obtained slices were stained with hematoxylin-eosin (HE), and the inflammatory cell infiltration of the tissues was observed under an optical microscope. The results showed that no inflammatory cell infiltration was observed in any tissue.

[0078] (V) In vitro neutralization test evaluation

[0079] (1) Orbital blood was collected from mice 42 days after immunization and centrifuged at 12000 rpm / min for 2 min. 200 μL of each sample was incubated in 56°C water or metal bath for 30 min. The serum was diluted with DMEM containing double antibodies at the dilution ratios of 1:2, 1:4, 1:8, 1:16, 1:32, 1:64, 1:128, and 1:256. 250 μL of each dilution was prepared.

[0080] (2) Dilute the virus (purchased from Pleco) to 200 TCID50 using DMEM with double antibody. - 5 TCID50 / mL, after diluting 500 times, take 250 μL and add it to the diluted serum.

[0081] (3) Vortex the diluted virus solution and treated serum evenly (250 μL diluted serum + 250 μL diluted virus) and place in a 37°C incubator for 1.5 hours.

[0082] (4) The culture medium of the 96-well plate with Marc145 cells (purchased from Pleco) was removed, and the plates were washed once with 1×PBS. 100 μL of serum-virus mixture was added to each well, and the plates were cultured in a 37°C, 5% CO2 incubator for 1.5 hours. The culture medium was then discarded, and the plates were washed once with 1×PBS. 100 μL of DMEM containing 2% FBS+dual antibodies was added to each well. The plates were cultured for 4-6 days, and the cell pathological changes were observed and the results were recorded. The neutralization index was calculated according to the Reed-Muench method.

[0083] (5) Note: After inoculating cells, 0.1 TCID50 should not cause any cell pathology, while 100 TCID50 must cause cell pathology;

[0084] The blank control cells should always maintain good morphology and characteristics. The results are shown in Table 3.

[0085] Table 3 Neutralization titers of different porcine reproductive and respiratory syndrome virus recombinant proteins in cell experiments

[0086] Protein name Cell assay neutralization titer GP5MHCN 1：4 GP234HCN 1：2

[0087] Example 4

[0088] The sequences of the dominant components screened out in Example 1 were sent to BGI, which synthesized the sequences of the dominant components into PET28a vectors, respectively, and the corresponding new vectors obtained were used as intramolecular adjuvants, as shown in Table 4.

[0089] Table 4 Intramolecular adjuvant

[0090] Serial number Advantageous ingredients New carrier Remark 1 Tufisn, T4-Fd p28T4 Aggregate 2 Tufisn, GCN4 p28GCN Aggregate 3 Tufisn, ferritin p28Fe Ferritin particles

[0091] Table 5 Sequences of the main dominant components in intramolecular adjuvants

[0092]

[0093] The two restriction sites of BamHI and HindIII were selected on the above-mentioned intramolecular adjuvants to subclone and synthesize the above-mentioned optimal sequence porcine reproductive and respiratory syndrome virus recombinant protein 1, so as to obtain the PRRSV multi-epitope recombinant protein containing intramolecular adjuvant. Then the same vaccine preparation and mouse immunization were carried out, and the in vitro cell virus neutralization experiment was evaluated.

[0094] Table 6 The potency of PRRSV multi-epitope recombinant proteins containing different internal adjuvants

[0095]

[0096] The results showed that when the internal adjuvant was p28Fe, the neutralization titer in the cell experiment was relatively the highest, which means that the immunogenicity of the PRRSV multi-epitope recombinant protein containing the internal adjuvant p28Fe was relatively the best.

[0097] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A porcine reproductive and respiratory syndrome virus recombinant protein, characterized in that: Including porcine reproductive and respiratory syndrome virus recombinant protein 1 and porcine reproductive and respiratory syndrome virus recombinant protein 2; The amino acid sequence of the porcine reproductive and respiratory syndrome virus recombinant protein 1 is shown in SEQ ID NO: 13; The amino acid sequence of the porcine reproductive and respiratory syndrome virus recombinant protein 2 is shown in SEQ ID NO:

24.

2. A PRRSV multi-epitope recombinant protein containing an internal adjuvant, characterized in that: The invention comprises the porcine reproductive and respiratory syndrome virus recombinant protein as claimed in claim 1 and an intramolecular adjuvant.

3. The PRRSV multi-epitope recombinant protein containing an internal adjuvant according to claim 2, characterized in that: The internal adjuvant includes any one of a p28T4 vector carrying Tufisn and T4-Fd, a p28GCN vector carrying Tufisn and GCN4, and a p28Fe vector carrying Tufisn and ferritin.

4. The PRRSV multi-epitope recombinant protein containing an internal adjuvant according to claim 3, characterized in that: The amino acid sequence of Tufisn is shown in SEQ ID NO:25; the amino acid sequence of the polymer peptide T4-Fd is shown in SEQ ID NO:26; the amino acid sequence of the polymer peptide GCN4 is shown in SEQ ID NO:27; and the amino acid sequence of ferritin is shown in SEQ ID NO:

28.

5. Use of the PRRSV multi-epitope recombinant protein containing an intramolecular adjuvant according to claim 2 in the preparation of a porcine reproductive and respiratory syndrome virus vaccine.

6. A porcine reproductive and respiratory syndrome virus subunit vaccine, characterized in that: It comprises the PRRSV multi-epitope recombinant protein containing an internal adjuvant as claimed in claim 2 and a pharmaceutically acceptable adjuvant.

Images