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

Abstract

The invention provides a porcine reproductive and respiratory syndrome virus M protein antigen epitope peptide and application thereof, and the amino acid sequence is shown as SEQ ID NO.1 from the C terminal to the N terminal. The amino acid sequence of the capsid protein of the porcine reproductive and respiratory syndrome virus M protein is used as a material for the first time; the antigen epitope of M is identified by combining a phage display technology, an immunoprecipitation technology and a next-generation sequencing technology, the antigen epitope has good antigenicity, and the acquisition of the antigen epitope enriches a porcine reproductive and respiratory syndrome virus antigen epitope database; a foundation is laid for research and development of porcine reproductive and respiratory syndrome detection reagents and kits, monoclonal antibody preparation, safety, identification and diagnosis of epitopes, vaccines and other products in urgent need of strategic prevention and control by taking 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 M protein antigen epitope peptide and application thereof. Background Art

[0002] Porcine Reproductive and Respiratory Syndrome (PRRS), commonly known as Blue Ear Disease, can cause reproductive disorders in sows and severe respiratory diseases in piglets. It is a swine infectious disease that seriously affects economic benefits. The main prevention and control measures for this disease are prevention-oriented. Both inactivated vaccines and live attenuated vaccines have some insurmountable shortcomings in terms of effectiveness and safety. So far, there is no specific therapeutic drug for this disease.

[0003] Porcine reproductive and respiratory syndrome is mainly caused by porcine reproductive and respiratory syndrome virus (PRRSV). PRRSV is a single-stranded positive-strand RNA virus that is not segmented. The virus belongs to the genus Arterivirus of the family Arteriviridae. PRRSV can grow on porcine alveolar macrophages (PAM cells), and cultured on MARC-145 cells can cause obvious cytopathic effects (CPE) such as cell rounding, aggregation and disintegration. The RNA of PRRSV has a cap structure (5'-Cap) at the 5' end and a polyadenine nucleotide tail structure (3'-polyA) at the 3' end. It contains a total of 9 open reading frames (ORFs), namely 1a, 1b, 2a, 2b, 3, 4, 5, 6, and 7. There are overlapping regions between adjacent ORFs. The total length of the PRRSV genome is about 15kb, 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, encoding 8 viral structural proteins, the main structural proteins include GP5 protein (envelope glycoprotein), M protein (matrix protein), N protein (nucleocapsid protein), and the secondary structural proteins are GP2, GP3, GP4, etc. GP3 is co-encoded by ORF2-7, GP4 is ORF3-7, GP5 is ORF5-7, M is ORF6-7, and N is ORF7, thus forming a typical 3' nested structure. 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, and differential diagnosis epitope vaccines based on antigenic epitopes will generate huge economic and social benefits.

[0004] GP5 and M protein form heterodimers through disulfide bonds and are considered to be the main structural proteins on the viral envelope, playing an important role in antibody-mediated virus and response. M protein is the most conserved protein on PRRSV strains from different sources and has good immunogenicity. It is reported to have neutralizing activity and is one of the main antigens studied in genetic engineering vaccines. C. Liang et al. used the ELISPOT method to separate cells from peripheral blood mononuclear cells of large white pigs immunized with PRRSV, screened and identified 9 immune linear epitopes, and confirmed that three of them can effectively stimulate the secretion of IFN-γ, providing new ideas for the research and development of vaccines.

[0005] In summary, finding suitable immunogenic M protein antigen epitope peptides has become a key research direction. Summary of the invention

[0006] The purpose of the present invention is to provide a porcine reproductive and respiratory syndrome virus M 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 an M protein antigen, wherein the antigen epitope peptide is coupled to 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 M protein antigen epitope peptide or the M protein antigen as an immunogen in preparing anti-M protein antibodies.

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

[0010] Another object of the present invention is to provide the use of M protein antigen epitope peptide or M protein antigen or said antibody in the following (a) or (b): (a) Products for the detection of diseases related to M protein expression for non-diagnostic and therapeutic purposes; (b) preparing products for treating and / or preventing diseases associated with abnormal M protein.

[0011] Another object of the present invention is to provide a product, the active ingredient of which is an M protein antigen epitope peptide or an M protein antigen or said antibody; The product has at least one of the following functions: (A) Detection of diseases related to M protein expression for non-diagnostic and non-therapeutic purposes; (B) Treating and / or preventing diseases associated with abnormal M protein.

[0012] Another object of the present invention is to provide a nucleic acid molecule encoding an M protein antigen epitope peptide.

[0013] 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.

[0014] 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.

[0015] 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.

[0016] 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.

[0017] The beneficial effects are: The present invention uses the amino acid sequence of the capsid protein of the porcine reproductive and respiratory syndrome virus M protein as material for the first time, and utilizes a 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 urgently needed strategic prevention and control products such as porcine reproductive and respiratory syndrome detection reagents and their kits, monoclonal antibody preparation, and safe, identifiable diagnostic epitopes and vaccines using antigenic epitopes as materials. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] 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 M antigen epitope peptide to bind to PRRSV positive serum. DETAILED DESCRIPTION

[0019] 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.

[0020] 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.

[0021] The present invention uses the amino acid sequence of the M protein of porcine reproductive and respiratory syndrome virus as a material, displays the truncated M 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 SEQID NO.1 is obtained, and the B cell antigen epitope of the M protein of porcine reproductive and respiratory syndrome virus is determined for the first time.

[0022] 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.

[0023] The preparation method of the M 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 antigen epitope peptide of M is as follows: GFHPITANDNHAFVVRRPGSTTVNGTLVPGLKSLVLGGKRAVKQG, in order from C-terminus to N-terminus.

[0024] 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, then add DMF and DCM, and introduce nitrogen bubbling for 60 minutes; wherein, the amount of DMF and DCM added can fully agitate the resin. 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, introduce nitrogen bubbling 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 M antigen epitope peptide in sequence.

[0025] (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 and 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 M antigen epitope peptide.

[0026] The fusion protein prepared by coupling the M antigen epitope peptide of the present invention with a carrier protein can be used to immunize animals to prepare specific M antibodies.

[0027] The present invention also provides human M 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.

[0028] Example 1: Identification of porcine reproductive and respiratory syndrome virus M antigen epitopes by phage immunoprecipitation sequencing.

[0029] 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 M 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.

[0030] Example 2: Bio-panning of phage library and porcine reproductive and respiratory syndrome positive serum and single plaque picking.

[0031] 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 M antigen epitope as shown in SEQ ID NO.1 was successfully obtained.

[0032] Example 3: Dot ELISA verifies the affinity activity of M epitope to porcine reproductive and respiratory syndrome positive serum.

[0033] The phage displaying the T cell epitope of M 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 M 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.

[0034] The antigens described in the present invention can be prepared by artificial synthesis, separation or other biological methods.

[0035] The method for preparing the antibody described in the present invention 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.

[0036] 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 the M 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.

[0037] 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.

[0038] 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 M protein antigen epitope peptide, characterized in that: The amino acid sequence from C-terminus to N-terminus is shown in SEQ ID NO.

1.

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

3. Use of the M protein antigen epitope peptide according to claim 1 or the M protein antigen according to claim 2 as an immunogen for preparing anti-M protein antibodies.

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

5. Use of the M protein antigen epitope peptide according to claim 1 or the M 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 M protein expression for non-diagnostic and therapeutic purposes; (b) preparing products for treating and / or preventing diseases associated with abnormal M protein.

6. A product characterized by: The active ingredient is the M protein antigen epitope peptide as claimed in claim 1 or the M 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 M protein expression; (B) Treating and / or preventing diseases associated with abnormal M protein.

7. A nucleic acid molecule encoding the M protein antigen epitope peptide according to claim 1.

8. An expression cassette, a recombinant vector or a recombinant cell line containing the nucleic acid molecule according to claim 8.

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