Preparation method and application of porcine pseudorabies virus multi-epitope polypeptide

Abstract

The invention discloses a preparation method and application of porcine pseudorabies virus multi-epitope polypeptide, and belongs to the technical field of veterinary biological products. The multi-epitope polypeptide comprises a cytotoxic T cell epitope, a helper T cell epitope and a linear B cell epitope; the cell epitopes are screened from key immunogenic proteins of the pseudorabies virus by using an immunoinformatics method. The porcine pseudorabies virus multi-epitope polypeptide designed by the invention is high in safety, has better antigenicity and physicochemical properties, can be efficiently expressed, and can be recognized by a pseudorabies virus specific antibody. By applying the porcine pseudorabies virus multi-epitope polypeptide, high-level specific antibodies and cellular immune response can be continuously induced to generate, strong protective efficacy is generated, and mice can be protected from death when infected with a lethal dose of porcine pseudorabies virus. Therefore, the multi-epitope polypeptide disclosed by the invention can be used as an alternative antigen of a porcine pseudorabies virus vaccine, the use of active virus is avoided, and a tool is provided for eradication of porcine pseudorabies virus.

Description

Technical Field

[0001] The present invention relates to the technical field of veterinary biological products, and in particular to a preparation method of a multi-epitope polypeptide of porcine pseudorabies virus and an application thereof. Background Art

[0002] Pseudorabies (PR) is an acute infectious disease caused by pseudorabies virus (PRV) in porcine animals and wild animals. Pigs are the natural hosts of the virus, which mainly causes mass mortality of newborn piglets, reproductive disorders in breeding pigs, and growth retardation in fattening pigs. PRV infection can cause viral encephalitis, neurological symptoms and latent infection. Pigs of any age can establish latent infection in the body after surviving PRV, making the virus persist in the pig herd for a long time and difficult to eliminate. Since the discovery of PR, the disease has shown a global epidemic trend and has been widely spread in Chinese pig herds in recent years. The disease can also infect humans, especially the emergence of PRV variants has brought great troubles to the development of my country's breeding industry, posing a serious threat to the safety of pig herds and human health.

[0003] The envelope of pseudorabies virus in swine contains 12 glycoproteins, including gB, gC, gD, gE, gG, gH, gI, gJ, gK, gL, gM, and gN, of which gB, gC, gD, gE, and gI are related to the virulence of the virus. The gB protein is an important neutralizing antigen of PRV, which can stimulate the body to produce specific humoral and cellular immune responses, and plays an indispensable role in the invasion of host cells by the virus and the fusion of cell membranes with the envelope. The gC protein is an adsorption protein of the virus, which promotes the rapid and effective adhesion of the virus to the cell surface. The gD protein is one of the main immunogenic proteins of the virus, which can induce the body to produce neutralizing antibodies, mediate the specific binding between the virus and the target cells, and is an essential substance in the process of the virus invading the target cells.

[0004] Vaccine immunization is an important means of preventing and controlling pseudorabies in pigs. Gene-deficient live vaccines and inactivated vaccines of various strains have been developed at home and abroad. Common PRV classic vaccine strains include Bartha-K61 strain and Bucharest strain. Bartha strain is the most widely used pseudorabies vaccine for pigs. Many countries have achieved the purification of pseudorabies using Bartha strain vaccine, but it can only provide partial protection against mutant strains that have appeared in China. Since the emergence of mutant strains, domestic research institutions have used newly isolated pseudorabies mutant strains to develop gene-deficient vaccines, but there have always been safety risks. Therefore, there is an urgent need to develop a new type of safe and effective vaccine that will not produce latent infection for the eradication of pseudorabies virus in pigs.

[0005] Multi-epitope peptide vaccines are designed based on multiple protective antigenic epitopes using the reverse vaccinology method. They have been widely studied in recent years because they are cheaper, safer, and can rationally design epitopes to improve effectiveness. The dominant epitopes in multi-epitope vaccines are screened using immunoinformatics methods, and are rationally designed to improve stability and achieve the best immune effect. However, there is currently no successfully developed and maturely applied multi-epitope peptide vaccine for pseudorabies virus in pigs. Therefore, it is necessary to develop a multi-epitope peptide vaccine to achieve safe and effective prevention and treatment of pseudorabies in pigs. Summary of the invention

[0006] In view of the above prior art, the purpose of the present invention is to provide a multi-epitope polypeptide of porcine pseudorabies virus and its application. The present invention finds that the multi-epitope polypeptide has good physicochemical properties and tertiary structure, can produce cellular and humoral immune responses, and has good immunogenicity and protection.

[0007] To achieve the above object, the present invention adopts the following technical solution:

[0008] In a first aspect of the present invention, a multi-epitope polypeptide of porcine pseudorabies virus is provided, wherein the multi-epitope polypeptide is any of the following:

[0009] (1) The amino acid sequence of the polypeptide shown in SEQ ID NO.1;

[0010] (2) A polypeptide having the same function as that of SEQ ID NO. 1, wherein one or more amino acid residues are replaced or deleted;

[0011] (3) A protein obtained by connecting a tag to the N-terminus and / or C-terminus of the polypeptide described in (1) or (2).

[0012] The present invention also provides a gene encoding a poly-epitope polypeptide of porcine pseudorabies virus, and the nucleotide sequence of the gene is shown in SEQ ID NO.2.

[0013] The second aspect of the present invention provides a method for preparing a multi-epitope polypeptide of porcine pseudorabies virus.

[0014] The third aspect of the present invention provides a multi-epitope polypeptide vaccine for pseudorabies virus of porcine, wherein the multi-epitope vaccine comprises a multi-epitope vaccine for pseudorabies virus of porcine.

[0015] Preferably, the multi-epitope vaccine further comprises a pharmaceutically acceptable adjuvant.

[0016] Beneficial effects of the present invention:

[0017] (1) Simple design and good immunogenicity: The present invention uses the cytotoxic T lymphocyte (CTL), helper T lymphocyte (HTL) and linear B cell epitopes (LBL) of the three major glycoproteins (gB, gC and gD) of PRV that we predicted. Epitopes with high antigenicity, sensitization, toxicity and sequence conservation are selected and connected using appropriate adjuvants and linkers. The multi-epitope polypeptide construct is evaluated by various computer methods and then a 3D structure is generated. Molecular docking and molecular dynamics simulation are used to elucidate the interaction between the multi-epitope polypeptide construct and Toll-like receptors (TLRs).

[0018] (2) Ability to achieve protective immunity against PRV infection: The multi-epitope vaccine prepared based on the multi-epitope peptide of pseudorabies virus can induce the body to produce high levels of neutralizing antibodies after two immunizations, and the proportion of CD4+T cells increased significantly, indicating that it can induce the body to produce cellular immunity. In addition, the mouse challenge experiment showed that the protection rate of mice immunized with the multi-epitope vaccine was as high as 100% during the experimental period, and the viral load in the body was extremely low. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 : Combination of multi-epitope peptides.

[0020] Figure 2 : Secondary structure diagram of multi-epitope polypeptide.

[0021] Figure 3 : The tertiary structure diagram and structure refinement of multi-epitope polypeptides. A is the tertiary structure model of the multi-epitope polypeptide, B is the Ramachandran diagram of the tertiary structure after refinement, and C is the Z value of the tertiary structure after refinement.

[0022] Figure 4 : Conformational B-cell epitopes of multi-epitope polypeptides.

[0023] Figure 5 : Docking complex of multi-epitope peptide and TLR. A is the docking result of multi-epitope peptide and TLR2, and B is the docking result of multi-epitope vaccine and TLR4.

[0024] Figure 6 : Molecular dynamics simulation results of the docking complex of multi-epitope peptides and TLRs. A is the RMSD during the molecular dynamics simulation, B is the change in Rg during the molecular dynamics simulation, C is the RMSF during the molecular dynamics simulation, D is the molecular dynamics simulation Gibbs free energy morphology of the docking complex of multi-epitope peptides and TLR2, and E is the molecular dynamics simulation Gibbs free energy morphology of the docking complex of multi-epitope peptides and TLR4.

[0025] Figure 7: Recombinant expression plasmid of multi-epitope polypeptide pET-28a-PRV-MEV.

[0026] Figure 8 : The structure of mRNA encoding multi-epitope polypeptides.

[0027] Fig. 9 : Expression and identification of multi-epitope polypeptides. A is the result of SDS-PAGE Coomassie blue staining, B is the result of Western blot using His tag antibody, and C is the result of PRV antibody positive pig serum.

[0028] Fig.10 : Specific IgG antibody level test results.

[0029] Fig.11 : Neutralizing antibody levels.

[0030] Fig.12 :Mouse spleen CD4 + T cells and CD8 + T cell ratio.

[0031] Fig.13 : Survival curve of mice challenged with virus.

[0032] Fig.14 : Detection of viral load in organs of challenged mice. DETAILED DESCRIPTION

[0033] It should be noted that the following detailed descriptions are illustrative and are intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present application belongs.

[0034] In order to enable those skilled in the art to more clearly understand the technical solution of the present application, the technical solution of the present application will be described in detail below in conjunction with specific embodiments.

[0035] The experimental materials used in the examples of the present invention that are not specifically described are all conventional experimental materials in the art and can be purchased through commercial channels. If the specific experimental conditions and methods are not specified in the examples of the present invention, conventional conditions are usually followed, such as J. Sambrook et al., ed., Molecular Cloning Experiment Guide (3rd Edition), Science Press, 2002; DL Spector et al., ed., Cell Experiment Guide, Science Press, 2001; or the conditions recommended by the manufacturer.

[0036] Example 1: Design of multi-epitope polypeptides and construction of plasmids.

[0037] In this example, epitope screening was performed on the three main glycoproteins of pseudorabies virus gB, gC and gD as targets. NetMHCpan (v4.1) tool (https: / / services.healthtech.dtu.dk / services / NetMHCpan-4.1 / ) was used to predict CTL epitopes, and NetMHCIIpan 2.1 server (https: / / services.healthtech.dtu.dk / services / NetMHCIIpan-2.1 / ) was used to predict HTL epitopes, and then BepiPred 3.0 server (https: / / services.healthtech.dtu.dk / services / BepiPred-3.0 / ) and ABCpred server (http: / / crdd.osdd.net / raghava / abcpred / ) were used to predict LBL epitopes. After that, further screening was performed based on antigenicity, allergenicity and toxicity. In addition, HTL epitopes were further screened based on whether they could produce IFN-γ, IL-4 or IL-10 to obtain 6 CTL, HTL and LBL epitopes each. The servers used for screening are shown in Table 1.

[0038] Table 1: Cellular epitope screening server

[0039]

[0040] The amino acid sequences, antigenicity, allergenicity and toxicity of the 6 selected CTL epitopes are shown in Table 2, the amino acid sequences, antigenicity, allergenicity, toxicity and cytokine induction of the 6 selected HTL epitopes are shown in Table 3, and the amino acid sequences, antigenicity, allergenicity and toxicity of the 6 selected CTL epitopes are shown in Table 2. Among them, an antigenicity score greater than 0.4 indicates a high antigenicity.

[0041] Table 2: CTL epitope status

[0042]

[0043] Table 3: HTL epitope status

[0044]

[0045] Table 4: LBL epitope status

[0046]

[0047] Then, the selected epitopes are connected using a suitable Linker. The specific connection method is as follows: Figure 1The multi-epitope polypeptide sequence is shown in SEQ ID NO.1;

[0048] Next, the designed multi-epitope peptides were evaluated and analyzed:

[0049] (1) Analysis of physicochemical properties: The ProtParam tool (https: / / web.expasy.org / protparam / ) was used to predict the physicochemical properties of the multi-epitope peptide. The instability index was 29.15 and the aliphatic index (AI) value of thermal stability was 77.86, indicating that the multi-epitope peptide remained stable at normal mammalian body temperature. The GRAVY value was -0.258, indicating that the vaccine protein was hydrophilic. The predicted half-life of the multi-epitope peptide was 30 hours in mammalian reticulocytes and greater than 10 hours in Escherichia coli cells. These results indicate that large-scale production and purification can be achieved using the E. coli cell culture system.

[0050] (2) Antigenicity and allergenicity analysis: The prediction results using Vaxijen 2.0 and AllerTOP v2.0 showed that the multi-epitope peptide had high antigenicity (0.5462) and was non-allergenic.

[0051] (3) Secondary structure prediction: PSIPRED v4.0 (http: / / bioinf.cs.ucl.ac.uk / psipred / ) was used to predict the secondary structure of multi-epitope peptides. Figure 2 As shown, the results showed that it contained 33% α-helix, 19% β-sheet, and 48% random coil.

[0052] (4) Tertiary structure analysis: The tertiary structure of the multi-epitope peptide was predicted using the Colabfold v1.5.3 server (https: / / colab.research.google.com / github / sokrypton / ColabFold / blob / main / AlphaFold2.ipynb). The most credible model was selected and refined using the GalaxyRedine server (https: / / galaxy.seoklab.org / ). The refined structure is shown in the figure. Figure 3 A, Ramachandran diagram is generated by PROCHECK server (https: / / saves.mbi.ucla.edu / ), the result is as follows Figure 3B, indicating that 96.2% of the residues are located in the allowed region and 0.3% of the amino acid residues are located in the unallowed region, which meets the quality requirements. The ProSA-web server (https: / / prosa.services.came.sbg.ac.at / prosa.php) predicted a Z value of -4.29, which is within the range of natural proteins of similar size ( Figure 3 C).

[0053] (5) Prediction of conformational B cell epitopes: The ElliPro (http: / / tools.iedb.org / ellipro / ) server was used to predict the conformational epitopes of multi-epitope peptides, and the conformational epitopes with higher scores were visualized using jmol. Figure 4 (Spherical structure representation).

[0054] (6) Analysis of the interaction between multi-epitope peptides and TLRs: The multi-epitope peptide constructs were docked with the predicted crystal structures of TLR2 and TLR4 using the HawkDock server. The best docked complex with the lowest binding free energy was selected and visualized using pyMOL ( Figure 5 A. Figure 5 B). The analysis results of the PDBsum server showed that 5 hydrogen bonds, 5 salt bridges and 89 non-covalent bonds were formed between the multi-epitope peptide and TLR2, and 5 hydrogen bonds, 2 salt bridges and 114 non-covalent bonds were formed between the vaccine and TLR4.

[0055] (7) Molecular dynamics simulation analysis of the docking complex of multi-epitope peptides and TLRs: In order to evaluate the conformational stability and interaction deviation of the multi-epitope peptide and TLR complex, a 50 ns molecular dynamics simulation was performed using Gromacs 2020.6. During the MD simulation, the conformational fluctuations of the multi-epitope peptide TLR complex were examined by the root mean square deviation (RMSD) value. The average RMSD values ​​of the peptide-TLR2 complex and the peptide-TLR4 complex were and Although the peptide-TLR2 complexes presented relatively high RMSD, each system showed conformational stability during the simulation ( Figure 6 A), indicating that the multi-epitope peptide and TLR have a good match and can form a stable complex. The compactness of the protein was characterized by calculating the radius of gyration (Rg). The results of Rg showed that the Rg of the peptide-TLR2 complex and the peptide-TLR4 complex decreased significantly during the entire MD simulation, indicating that their structures are stable and compact ( Figure 6 B) The average Rg values ​​of the peptide-TLR2 complex and the peptide-TLR4 complex are and In addition, the root mean square fluctuation (RMSF) was used to evaluate the flexibility of individual residues of each docked complex. Both complexes showed large fluctuations in only a few residues ( Figure 6 C), indicating that the flexibility of the complex has increased. The average RMSF of the peptide-TLR2 complex and the peptide-TLR4 complex are and Finally, we performed PCA and drew FEI diagrams to evaluate the conformation and system stability of the multi-epitope peptide-TLR complex. The results showed that the peptide-TLR4 complex had more low-energy basins than the peptide-TLR2 complex ( Figure 6 D. Figure 6 E), indicating that the structure of the system is more stable.

[0056] (8) Codon optimization and gene synthesis: The sequence of the multi-epitope polypeptide is optimized according to the Escherichia coli codons. The optimized nucleotide sequence encoding the multi-epitope polypeptide is shown in SEQ ID No. 2;

[0057] The optimized sequence was cloned into the MCS of the vector to construct the pET-28a-Vaccine expression plasmid, such as Figure 7 As shown. RNAfold was used to predict the secondary structure of the multi-epitope vaccine mRNA, and no hairpin loop was detected at the start site ( Figure 8 ). The minimum free energy (ΔG) of the vaccine mRNA is -482.09 kcal / mol, indicating that the optimized mRNA sequence of the multi-epitope polypeptide is stable. The optimized sequence was subjected to gene synthesis to construct a recombinant expression plasmid.

[0058] Example 2: Expression, purification and verification of multi-epitope polypeptides

[0059] The constructed recombinant expression plasmid of porcine pseudorabies virus multi-epitope polypeptide was transformed into Escherichia coli BL21 (DE3) competent cells, and a single clone was picked for expression. IPTG was added to a final concentration of 1 mM when OD600 was 0.6. After inducing expression for 4 hours, the cells were collected by centrifugation, washed with PBS, resuspended in lysis buffer, and then disrupted using a low-temperature ultra-high pressure continuous flow cell disruptor.

[0060] Then, Ni Sepharose 6Fast Flow filler was used to purify the multi-epitope peptides through AKTApure protein purifier, and the purified protein was dialyzed and desalted using a dialysis bag (MWCO=10k), and then SDS-PAGE Coomassie Brilliant Blue staining was performed for identification ( Fig. 9 A) and Western blot ( Fig. 9 B, 9C) identification, it can be seen that the protein has a single target band at 50 kDa.

[0061] Example 3: Evaluation of the immune effect of multi-epitope vaccines

[0062] BABL / c female mice aged 4-6 weeks were randomly divided into 3 groups, namely, multi-epitope peptide group (MEP), multi-epitope peptide + adjuvant group (MEP + Adj) and PBS control group (Control), with 6 mice in each group. The vaccine was prepared by emulsification with ISA201 VG adjuvant at a mass ratio of 1:1. Immunization was performed by subcutaneous injection on days 0 and 14, and the amount of immune protein was 100 μg. The 14-day serum was collected for use, and the mice were euthanized by anesthesia followed by bleeding on day 28, and the serum was collected and the spleen was aseptically separated.

[0063] Antibody level detection: The recombinant multi-epitope peptide was used as the coating antigen for antibody detection by ELISA, and the serum was diluted 1:6400 to evaluate the humoral immune response. Fig.10 As shown, it can be seen that the mouse serum IgG level increased significantly on the 28th day compared with 14th day, and the MEP+Adj antibody level was significantly higher than that of the MEP group, indicating that the immunogenicity of the multi-epitope vaccine was significantly increased when adjuvants were used.

[0064] Neutralizing antibody detection: The neutralizing antibody level was further detected using the porcine pseudorabies virus Ea strain (Genbank access No. KU315430.1). The 28-day serum was heat-inactivated at 56°C for 30 minutes and then diluted 2-fold. The serum was mixed with 100 TCID 50 After incubation with PRV for 1 h, the cells were inoculated into PK-15 cells in a 96-well plate and then incubated at 37°C in 5% CO2 for 4-5 days, and the cytopathic effect (CPE) was recorded. The neutralizing antibody titer was calculated based on the Reed–Muench method. Fig.11 It can be seen that MEP+Adj can produce higher levels of neutralizing antibodies.

[0065] Detection of spleen lymphocyte level: The spleen of the mouse was ground to obtain a single cell suspension, and then the red blood cells were lysed with red blood cell lysis buffer, washed with D-Hanks buffer and resuspended in staining buffer for cell staining. The staining antibodies were FITC-CD4, PE-CD8a, and APC-CD3. After incubation in the dark for 30 minutes, the cells were washed three times with staining buffer and the proportion of CD4+T cells and CD8a+T cells was detected by flow cytometry. Results ( Fig.12 ) showed that compared with the control group, the cellular immunity of the multi-epitope vaccine prepared with multi-epitope polypeptides was more inclined to helper T cell immunity, with more CD4+T cells than CD8+T cells.

[0066] Example 4: Relative protection rate of multi-epitope vaccine after PRV infection

[0067] BABL / c female mice aged 4-6 weeks were randomly divided into 3 groups, namely, multi-epitope peptide group (MEP), multi-epitope peptide + adjuvant group (MEP+Adj) and PBS control group (Control), with 5 mice in each group. Immunization was performed by subcutaneous injection on days 0 and 14, and the amount of immune protein was 100μg. On the 29th day of immunization, the mice were subjected to PRV infection test (mice are an ideal animal model for basic research on PRV), and each mouse was intraperitoneally injected with 100LD50 of virus solution, and the incidence and mortality of the mice were recorded. After 10 days, the surviving mice were euthanized, and the brain and lungs were removed to detect the viral load. The survival of the infected mice within 10 days is as follows. Fig.13 As shown in the figure, it can be seen that all mice in the control group died, only one mouse survived in the multi-epitope polypeptide without adjuvant, and all mice in the multi-epitope polypeptide + adjuvant group survived, with a relative protection efficiency of 100%. The viral load test results of PRV in the brain and lungs ( Fig.14 ) It can also be seen that the viral load of the multi-epitope polypeptide + adjuvant group is the lowest. The above results indicate that the multi-epitope vaccine prepared with PRV multi-epitope polypeptide can provide immune protection against PRV infection.

[0068] In summary, the present invention uses immunoinformatics to design a multi-epitope polypeptide of porcine pseudorabies virus, which has good antigenicity and physicochemical properties. The polypeptide can be efficiently expressed in Escherichia coli and can be recognized by pseudorabies virus-specific antibodies. The vaccine prepared using the polypeptide can continuously induce the production of high levels of specific antibodies and cellular immune responses, produce strong protective efficacy, and can protect mice from death when infected with a lethal dose of porcine pseudorabies virus, providing a technical basis for the purification of porcine pseudorabies virus and providing guidance for the development of other new vaccines.

[0069] The above description is only the preferred embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A multi-epitope polypeptide of porcine pseudorabies virus, characterized in that: The epitope peptide sequence is shown as SEQ ID NO.

1.

2. The nucleotide encoded by the multi-epitope polypeptide of pseudorabies virus according to claim 1, characterized in that: The nucleotide sequence of the gene is shown in SEQ ID NO.

2.

3. A recombinant strain, characterized in that: The recombinant strain is used for recombinantly expressing the porcine pseudorabies virus multi-epitope polypeptide according to claim 1.

4. Use of the multi-epitope peptide according to claim 1 in the preparation of drugs for the prevention and treatment of swine herpesvirus.

5. The use according to claim 4, characterized in that: Application in the preparation of porcine pseudorabies virus vaccine or porcine pseudorabies virus antibody.

6. A porcine pseudorabies virus vaccine, characterized in that: The porcine pseudorabies virus vaccine contains the porcine pseudorabies virus multi-epitope polypeptide according to claim 1.

7. Porcine pseudorabies virus antibody, characterized in that: An antibody prepared by the porcine pseudorabies virus multi-epitope vaccine according to claim 1.

8. The porcine pseudorabies virus multi-epitope vaccine according to claim 6, characterized in that: The multi-epitope vaccine also includes a pharmaceutically acceptable adjuvant.

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