Porcine reproductive and respiratory syndrome virus GP5 protein antigen epitope peptide and application thereof

Abstract

The invention aims to provide a porcine reproductive and respiratory syndrome virus GP5 protein antigen epitope peptide, the amino acid sequence of which is as shown in SEQ ID NO.1. The amino acid sequence of capsid protein of the porcine reproductive and respiratory syndrome virus GP5 protein is used as a material for the first time, the antigen epitope of M is identified, and the antigen epitope has good antigenicity and can be used for preparing the antigen epitope of the porcine reproductive and respiratory syndrome virus GP5 protein. The acquisition of the antigen epitope enriches a porcine reproductive and respiratory syndrome virus antigen epitope database, and lays a foundation for research and development of porcine reproductive and respiratory syndrome detection reagents and kits, preparation of monoclonal antibodies, and safe and differentiable diagnosis epitopes, vaccines and other products with the antigen epitopes as materials.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, in particular to a porcine reproductive and respiratory syndrome virus GP5 protein antigen epitope peptide and application thereof. Background Art

[0002] Porcine reproductive and respiratory syndrome (PRRS) is an infectious disease mainly caused by porcine reproductive and respiratory syndrome virus (PRRSV), characterized by abortion in pregnant sows, respiratory damage in piglets and immunosuppression. It is listed as a Class II infectious disease in China. PRRSV is one of the important diseases affecting the global pig industry and is still widely present in pig herds around the world. The main prevention and control measures for this disease are prevention first. The main vaccines on the market are inactivated vaccines and live attenuated vaccines, but these two vaccines have some insurmountable shortcomings in terms of effectiveness and safety. So far, there is no specific treatment for this disease.

[0003] PRRSV is a single-stranded positive-strand RNA virus. At the 10th International Virus Conference, the virus was classified as Arteriviridae, Arterivirus genus, and is wrapped in an envelope. According to the different genotypes, it is divided into two types, one is PRRSV-1 (European type) and the other is PRRSV-2 (North American type). The PRRSV genome is about 15kb in length, with open reading frames such as ORF1a / ORF1b / ORF2a / ORF2b / ORF5a / ORF3-ORF7. ORF1a / ORF1b, accounting for 80% of the whole genome, is responsible for encoding two polyprotein precursors pp1a and pp1ab. ORF2-7 only accounts for 20% of the viral genome and encodes 8 viral structural proteins, corresponding to GP2a, E, GP3, GP4, GP5, GP5a, M, and N proteins. Among them, N is the nucleocapsid protein, and the remaining structural proteins are all envelope proteins. GP5 and M are the main envelope proteins, and GP2a, E, GP3, GP4, and GP5a are secondary envelope proteins. Among them, the main structural proteins GP5 and M are bound by disulfide bonds to form heterodimers. They are considered to be the main structural proteins on the viral envelope and play an important role in antibody-mediated virus neutralization reactions.

[0004] Screening for proteins with good immunogenicity encoded by the PRRSV genome is of great significance for the study of the pathogenicity of PRRSV and the development of detection kits. In addition, the development of detection reagents and kits, monoclonal antibody preparation, differential diagnosis epitope vaccines and other prevention and control products based on antigenic epitopes will generate huge economic benefits and have important social benefits. GP5 protein has been reported to have neutralizing activity and is one of the main antigens studied in genetic engineering vaccines. It can induce neutralizing antibodies for linear epitopes and conformational epitopes. The study by D.Liu et al. identified 12 B cell epitopes of GP3 and GP5, and used immunoinformatics methods to predict, screen, design and construct candidate vaccines that fused B cell epitopes, CTL and htl dominant PRRSV strain GP3 and GP5 protein protective epitopes.

[0005] Therefore, finding suitable GP5 protein antigen epitope peptides with immunogenicity is the current key research direction. Summary of the invention

[0006] The purpose of the present invention is to provide a porcine reproductive and respiratory syndrome virus GP5 protein antigen epitope peptide, the amino acid sequence of which from C-terminus to N-terminus is shown in SEQ ID NO.1.

[0007] Another object of the present invention is to provide a GP5 protein antigen, wherein the antigen epitope peptide is coupled with a carrier protein, and preferably, the carrier protein is keyhole limpet hemocyanin or bovine serum albumin.

[0008] Another object of the present invention is to provide the use of the GP5 protein antigen epitope peptide or the GP5 protein antigen as an immunogen in preparing anti-GP5 protein antibodies.

[0009] Another object of the present invention is to provide use of the antibody in detecting GP5 protein or preparing a product for detecting GP5 protein.

[0010] Another object of the present invention is to provide the use of GP5 protein antigen epitope peptide or GP5 protein antigen or the antibody in the following (a) or (b): (a) Products for the detection of diseases related to GP5 protein expression for non-diagnostic and therapeutic purposes; (b) preparing products for treating and / or preventing diseases associated with abnormal GP5 protein; Another object of the present invention is to provide a product, the active ingredient of which is a GP5 protein antigen epitope peptide or a GP5 protein antigen or the antibody described above; The product has at least one of the following functions: (A) Detection of diseases related to GP5 protein expression for non-diagnostic and non-therapeutic purposes; (B) Treating and / or preventing diseases associated with abnormal GP5 protein.

[0011] Another object of the present invention is to provide a nucleic acid molecule encoding an antigenic epitope peptide of the GP5 protein.

[0012] Another object of the present invention is to provide an expression cassette, a recombinant vector or a recombinant cell line containing the nucleic acid molecule.

[0013] Another object of the present invention is to provide a separated antigen-antibody complex, characterized in that the antigen forming the complex contains the above-mentioned antigen epitope peptide or the above-mentioned expression box, the antigen epitope peptide forms an antigen epitope, and the antibody specifically binds to the antigen epitope.

[0014] Another object of the present invention is to provide a vaccine, wherein the vaccine comprises the above antigen epitope peptide, the above expression cassette, the above nucleic acid molecule, the above recombinant vector or the above recombinant cell line. Furthermore, the vaccine also comprises an immunologically acceptable carrier and / or adjuvant.

[0015] Another object of the present invention is to provide an indirect ELISA test kit for porcine reproductive and respiratory syndrome virus antibody, wherein the kit contains the above-mentioned antigen epitope peptide. Furthermore, the kit also contains a diluent, a washing solution, a blocking solution, an HRP-labeled goat anti-swine IgG antibody, a color developing solution, and a stop solution.

[0016] The beneficial effects are: The present invention uses the amino acid sequence of the capsid protein of the porcine reproductive and respiratory syndrome virus GP5 protein as material for the first time, and utilizes the combination of phage display technology, immunoprecipitation technology, and second-generation sequencing technology to identify the antigenic epitope of M, which has good antigenicity. The acquisition of this antigenic epitope enriches the porcine reproductive and respiratory syndrome virus antigenic epitope database, and lays a foundation for the research and development of porcine reproductive and respiratory syndrome detection reagents and their kits, monoclonal antibody preparation, safe and identifiable diagnostic epitopes, vaccines and other products using antigenic epitopes as materials. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 is a diagram of the serological test results of the antigenic epitope peptide of the present invention; Figure 2 This is a diagram showing the results of three rounds of biological panning of the phage library with porcine reproductive and respiratory syndrome positive serum; Figure 3 This is a preliminary analysis of a single plaque agarose gel electrophoresis showing the fragment length results; Figure 4 This is a graph showing the ability of phage displaying the GP5 antigen epitope peptide to bind to PRRSV positive serum. DETAILED DESCRIPTION

[0018] The technical solutions in the embodiments of the invention are described clearly and completely below. Obviously, the described embodiments are part of the embodiments of the invention, not all of the embodiments. The detailed description of the embodiments of the invention provided below is not intended to limit the scope of the claimed invention, but merely represents selected embodiments of the invention. Based on the embodiments in the invention, all other embodiments obtained by ordinary method personnel in the field without creative work are within the scope of protection of the invention.

[0019] In the following examples, unless otherwise specified, experimental methods without specific conditions are usually carried out under conventional conditions, and the reagents used in the examples are all commercially available.

[0020] The present invention uses the amino acid sequence of the GP5 protein of porcine reproductive and respiratory syndrome virus as a material, displays the truncated GP5 protein sequence on it through the T7 phage display technology, constitutes a phage display library, and uses the immunoprecipitation and sequencing of the positive serum of pigs infected with porcine reproductive and respiratory syndrome virus and the above T7 phage display library to identify it as an antigen dominant epitope. Subsequently, through the bio-panning of the phage library and the positive serum of pigs infected with porcine reproductive and respiratory syndrome virus and the picking of a single plaque, a T7 phage displaying the polypeptide sequence shown in SEQ ID NO.1 is obtained, and the B cell antigen epitope of the GP5 protein of porcine reproductive and respiratory syndrome virus is determined for the first time.

[0021] After a lot of theoretical research and experimental exploration, the present invention finally screened out an antigen epitope peptide with good antigenicity whose amino acid sequence is shown in SEQ ID NO.1.

[0022] The preparation method of the GP5 antigen epitope peptide of the present invention can be prepared by chemical synthesis. The synthesis process is as follows: (1) The amino acid chain fragment of the GP5 antigen epitope peptide is as follows: DGSAATPVTKVSAEQWGHP, The order is from C-terminus to N-terminus. Weigh n equivalents of resin and put them into the reactor, add DCM to swell for half an hour; then remove DCM, add 2n equivalents of the first amino acid at the C-terminus in the sequence, add 2n equivalents of DIEA, and then add DMF and DCM to pass nitrogen bubbling reaction for 60 minutes; wherein, the amount of DMF and DCM added can make the resin fully agitated. After the reaction is completed, add 5n equivalents of methanol to react, and remove the reaction liquid after half an hour of reaction to obtain the first reactant, and rinse the first reactant with DMF and methanol. Next, add 2n equivalents of the second amino acid, 2n equivalents of HBTU and DIEA to the reactor containing the first reactant, pass nitrogen bubbling reaction for half an hour, remove the reaction liquid, obtain the second reactant, and rinse the second reactant with DMF and methanol; use ninhydrin to detect the second reactant. According to the same operation, connect the amino acids of the composed GP5 antigen epitope peptide in sequence.

[0023] (2) After the amino acid is connected, the end-capping is performed with pyridine and acetic anhydride, and the end-capped reactant is washed; then a decapping liquid is added to the end-capped reactant to remove the Fmoc protecting group, and the decapping reactant is washed, and then detected with ninhydrin. The decapping reactant is blown dry with nitrogen, removed from the reactor, poured into a flask, and then the cutting liquid added to the flask is reacted. The ratio of the cutting liquid to the decapping reactant is 10 mL of the cutting liquid per gram of the decapping reactant. The cutting liquid includes 95% TFA, 2% ethanedithiol, 2% triisopropylsilane and 1% water in terms of mass percentage. The reaction is shaken, solid-liquid separation is performed, and the filtrate is collected. Then ether is added to the filtrate to precipitate, and the precipitate is collected and washed after solid-liquid separation to obtain a crude product. The crude product is purified to the required purity by high performance liquid chromatography. The purified collected liquid is concentrated and dried into powder, which is the GP5 antigen epitope peptide.

[0024] The fusion protein prepared by coupling the GP5 antigen epitope peptide of the present invention with a carrier protein can be used to immunize animals to prepare specific GP5 antibodies.

[0025] The present invention also provides human GP5 antibodies and humans. The antibodies can be prepared by immunizing animals with the fusion protein of the present invention. The preparation method can adopt conventional techniques in the art.

[0026] Example 1: Identification of porcine reproductive and respiratory syndrome virus GP5 antigenic epitope by phage immunoprecipitation sequencing.

[0027] Dilute the Swine-Virscan library to 3.5 × 10 using PBS. 9pfu / mL, add 1 mL of the diluted Swine-Virscan library to each 1.5 mL centrifuge tube, then add a 1:100 serum dilution containing 2 μg IgG, rotate and mix at 4°C for 18 h; wash the protein A / G magnetic beads three times with 0.02% PBST; then add 20 μL of protein A / G magnetic beads to each centrifuge tube and incubate for 4 h; transfer the magnetic beads to a 96-well PCR plate and wash three times with 200 μL IP washing solution; construct a second-generation sequencing library and sequence. Data analysis revealed that the phage members were enriched in the library. By comparing the GP5 protein sequences of each strain, it was found that the strain where the sequence shown in SEQ ID NO: 1 is located is relatively conservative in the sequences of each strain, such as Figure 1 As shown, it has good potential for ASFV serological detection.

[0028] Example 2: Bio-panning of phage library and porcine reproductive and respiratory syndrome positive serum and single plaque picking.

[0029] Through three rounds of biological panning of the phage library with porcine reproductive and respiratory syndrome positive serum, with three replicates in each round, a phage library with affinity to porcine reproductive and respiratory syndrome positive serum was finally obtained. The retention rate was calculated by titer detection of the library after each round of panning. The results showed that the average retention rate in the first round was about 0.79%, the average retention rate in the second round was about 6.34%, and the average retention rate in the third round was about 9.12%. Figure 2 As shown in the figure, as the number of library panning increases, the retention rate gradually increases, proving that the affinity between the serum and the panning library gradually increases. Subsequently, 384 plaques were randomly selected from the library after the third round of biological panning, and then added to the BLT5403 Escherichia coli in the logarithmic growth phase for amplification. After obtaining the amplified product of a single plaque, PCR amplification was first performed, and the fragment length was preliminarily analyzed by agarose gel electrophoresis. The results are shown in the figure. Figure 3 As shown, only 1 case did not match the expected fragment length, and another case was not successfully picked and amplified. The second-generation sequencing library was constructed and sequenced for the 384 phages. After filtering out 15 samples with low counts through sequencing results analysis, the maximum counts in each sample (i.e.: the theoretical counts of a single plaque display fragment) was greater than 90% of the total counts, which was considered a successful identification. Finally, 355 phages were successfully identified, of which 259 phages displaying different fragments were identified, and 46 displayed ASFV protein fragments. Subsequently, the above 42 phages were secondary identified by Sanger sequencing, and finally a phage that correctly displayed the GP5 antigen epitope as shown in SEQ ID NO.1 was successfully obtained.

[0030] Example 3: Dot ELISA verifies the affinity activity of GP5 epitope to porcine reproductive and respiratory syndrome positive serum.

[0031] The phage displaying the T cell epitope of GP5 as shown in SEQ ID NO: 1 was diluted to 10 8 pfu / μL, and then diluted 4-fold for 5 times to dilute the control phage without exogenous fragment display to 10 8 pfu / μL, the above phages were coated onto NC membranes, and after being completely dried, they were used to verify the affinity with the positive serum of porcine reproductive and respiratory syndrome. Figure 4 The results showed that the phage displaying the GP5 epitope as shown in SEQ ID NO.1 showed a dose-dependent binding ability with ASFV positive serum, and had no binding ability with the control phage displaying no exogenous fragments, and had the ability to neutralize porcine reproductive and respiratory syndrome virus.

[0032] In this embodiment, the antigen preparation method can be artificial synthesis, separation or other biological methods; the antibody preparation method is: artificial synthesis according to the antibody sequence, biological method or combination of biological and chemical methods, etc. The specific method can be based on the method in the prior art, as long as the sequence can be guaranteed to be consistent with the antibody sequence in the above embodiment.

[0033] In the present invention, the above-mentioned antigenic epitope (synthetic peptide) and single antigenic epitope recombinant protein with immune activity obtained for the first time for GP5 protein, the obtained antigenic epitope and the designed recombinant antigen can not only be used for the development of porcine reproductive and respiratory syndrome virus infection serological detection reagents and kits (ELISA, WB, colloidal gold chromatography test strips, etc.), but also contribute to the development of porcine reproductive and respiratory syndrome epitope vaccines and any vaccines related to the epitope (epitope monovalent / multivalent vaccines, synthetic peptide vaccines, in series with other pathogenic antigenic epitopes or antigens, etc.), and also lay a material foundation for the preparation of porcine reproductive and respiratory syndrome antigenic epitope-specific monoclonal antibodies; in addition, the research results can also be used for the study of viral immune mechanisms, to explore and analyze viral infection and vaccine immune mechanisms, and to achieve the purpose of preventing and controlling porcine reproductive and respiratory syndrome.

[0034] The antigen epitope peptide sequence and its related subsidiary subjects, antibody sequence and its related subsidiary subjects etc. given in the present invention all belong to the inventive points of the present invention.

[0035] The above is only a preferred embodiment of the invention and is not intended to limit the invention. For those skilled in the art, the invention may have various modifications and changes. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the invention shall be included in the protection scope of the invention.

Claims

1. A porcine reproductive and respiratory syndrome virus GP5 protein antigen epitope peptide, characterized in that: Its amino acid sequence is shown in SEQ ID NO.

1.

2. A GP5 protein antigen, characterized in that: The antigen epitope peptide is coupled with a carrier protein.

3. Use of the GP5 protein antigen epitope peptide according to claim 1 or the GP5 protein antigen according to claim 2 as an immunogen in preparing antibodies against GP5 protein.

4. Use of the antibody according to claim 3 in detecting GP5 protein or preparing a product for detecting GP5 protein.

5. Use of the GP5 protein antigen epitope peptide according to claim 1 or the GP5 protein antigen according to claim 2 or the antibody according to claim 3 in the following (a) or (b): (a) Products for the detection of diseases related to GP5 protein expression for non-diagnostic and therapeutic purposes; (b) preparing products for treating and / or preventing diseases associated with abnormal GP5 protein.

6. A product characterized by: The active ingredient is the GP5 protein antigen epitope peptide as claimed in claim 1 or the GP5 protein antigen as claimed in claim 2 or the antibody as claimed in claim 3; The product has at least one of the following functions: (A) Detection of diseases related to GP5 protein expression; (B) Treating and / or preventing diseases associated with abnormal GP5 protein.

7. A nucleic acid molecule encoding the GP5 protein antigen epitope peptide as claimed in claim 1.

8. Another object of the present invention is to provide an expression cassette, a recombinant vector or a recombinant cell line containing the nucleic acid molecule according to claim 8.

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