Porcine reproductive and respiratory syndrome virus gp5 protein antigen epitope peptide and application thereof

Abstract

The application aims to provide a porcine reproductive and respiratory syndrome virus GP5 protein antigen epitope peptide, which has an amino acid sequence shown as SEQ ID NO. 1, and is first identified from the amino acid sequence of a porcine reproductive and respiratory syndrome virus GP5 protein capsid protein, and has good antigenicity. The antigen epitope is obtained, which enriches a porcine reproductive and respiratory syndrome virus antigen epitope database, and lays a foundation for developing a porcine reproductive and respiratory syndrome detection reagent and kit, a monoclonal antibody preparation, a safe and identifiable diagnostic epitope, and a vaccine and other products.

Description

TECHNICAL FIELD

[0001] The application relates to the technical field of biotechnology, and particularly relates to a porcine reproductive and respiratory syndrome virus GP5 protein antigen epitope peptide and application thereof. BACKGROUND

[0002] Porcine reproductive and respiratory syndrome (PRRS) is mainly caused by porcine reproductive and respiratory syndrome virus (PRRSV) and is an infectious disease characterized by abortion of pregnant sows, respiratory system damage of piglets and immunosuppression, which is listed as a class II infectious disease. PRRSV is one of the important diseases affecting the global pig industry, and currently still widely exists in the global pig population. The main prevention and control measures for the disease are mainly prevention, and the current market vaccines are inactivated vaccines and attenuated live vaccines, but the two vaccines have some insurmountable shortcomings in effectiveness and safety. So far, there is no specific treatment drug for the disease.

[0003] PRRSV is a single-stranded RNA virus, and the virus is classified into arterivirus family arterivirus genus in the tenth international virus conference. The virus is wrapped by an envelope. According to the different genotypes, the virus 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 15 kb in length, and has ORF1a / ORF1b / ORF2a / ORF2b / ORF5a / ORF3-ORF7 open reading frames. ORF1a / ORF1b accounts for 80% of the whole genome, and is responsible for encoding pp1a and pp1ab two polyprotein precursors. ORF2-7 accounts for only 20% of the viral genome, and encodes eight virus structural proteins, which correspond to GP2a, E, GP3, GP4, GP5, GP5a, M and N proteins in turn. The N is a nucleocapsid protein, and the remaining structural proteins are envelope proteins, GP5 and M are main envelope proteins, and GP2a, E, GP3, GP4 and GP5a are secondary envelope proteins. Among them, the main structural proteins GP5 and M form a heterodimer through a disulfide bond and are considered to be the main structural proteins on the virus envelope, and play an important role in antibody-mediated virus neutralization reaction.

[0004] Screening of PRRSV genome coding protein with good immunogenicity has important significance for pathogenicity research and development of detection kit of PRRSV, and research and development of detection reagent and kit, preparation of monoclonal antibody and differential diagnosis epitope vaccine and other prevention and control products with antigen epitope as material will produce huge economic benefits and important social benefits. GP5 protein is reported to have neutralizing activity, and is one of the main antigens for genetic engineering vaccine research, which can induce neutralizing antibodies of linear epitope and conformational epitope. D. Liu et al. identified 12 B cell epitopes of GP3 and GP5, and screened, designed and constructed a candidate vaccine of fusion B cell epitope, CTL and htl dominant protective epitope of GP3 and GP5 protein of PRRSV strain by using immunoinformatics method.

[0005] Therefore, searching for suitable GP5 protein antigen epitope peptide with immunogenicity is the current research direction. SUMMARY

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

[0007] The present application also has another purpose to provide a GP5 protein antigen, and the antigen epitope peptide is coupled with a carrier protein to form the GP5 protein antigen, preferably, the carrier protein is keyhole limpet hemocyanin or bovine serum albumin.

[0008] The present application also has another purpose to provide application of the GP5 protein antigen epitope peptide or the GP5 protein antigen as an immunogen in preparation of an antibody against GP5 protein.

[0009] The present application also has another purpose to provide application of the antibody in detection of GP5 protein or preparation of a product for detecting GP5 protein.

[0010] The present application also has another purpose to provide application of the GP5 protein antigen epitope peptide or the GP5 protein antigen or the antibody in (a) or (b) as follows:

[0011] (a) preparation of a product for detecting GP5 protein expression related diseases for non-diagnostic and therapeutic purposes;

[0012] (b) preparation of a product for treating and / or preventing GP5 protein abnormality related diseases;

[0013] The present application also has another purpose to provide a product, and the active ingredient thereof is the GP5 protein antigen epitope peptide or the GP5 protein antigen or the antibody;

[0014] The product has at least one of the following functions:

[0015] (A) detecting diseases related to GP5 protein expression for non-diagnostic and therapeutic purposes;

[0016] (B) treating and / or preventing diseases related to GP5 protein abnormalities.

[0017] It is another object of the present application to provide a nucleic acid molecule encoding the antigenic epitope peptide of GP5 protein.

[0018] It is another object of the present application to provide an expression cassette, a recombinant vector or a recombinant cell line containing the nucleic acid molecule.

[0019] It is another object of the present application to provide an isolated antigen-antibody complex, characterized in that the antigen forming the complex contains the antigenic epitope peptide or the expression cassette described above, the antigenic epitope peptide forms an antigenic epitope, and the antibody specifically binds to the antigenic epitope.

[0020] It is another object of the present application to provide a vaccine containing the antigenic epitope peptide, the expression cassette, the nucleic acid molecule, the recombinant vector or the recombinant cell line described above. Further, the vaccine further comprises an immunologically acceptable carrier and / or adjuvant.

[0021] It is another object of the present application to provide a porcine reproductive and respiratory syndrome virus antibody indirect ELISA detection kit, which contains the antigenic epitope peptide described above. Further, the kit further contains a diluent, a washing solution, a blocking solution, an HRP-labeled goat anti-swine IgG antibody, a coloring solution and a termination solution.

[0022] The beneficial effects are that:

[0023] The present application first uses the amino acid sequence of GP5 protein capsid protein of porcine reproductive and respiratory syndrome virus as a material, and identifies the antigenic epitope of M by combining phage display technology, immunoprecipitation technology and second-generation sequencing technology. The antigenic epitope has good antigenicity. The acquisition of the antigenic epitope enriches the database of porcine reproductive and respiratory syndrome virus antigenic epitopes, and lays a foundation for developing porcine reproductive and respiratory syndrome detection reagents and kits, monoclonal antibody preparation, safe and identifiable diagnostic epitopes and vaccines and other products using antigenic epitopes as materials. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 is a serological detection result diagram of the antigenic epitope peptide of the present application;

[0025] Figure 2 is a three-round biological panning result diagram of a phage library and porcine reproductive and respiratory syndrome positive serum;

[0026] Figure 3Figure 1 is a single plaque agarose gel electrophoresis preliminary analysis display fragment length results figure;

[0027] Figure 4 Figure 2 is a result figure of the binding ability of the phage displaying the GP5 antigen epitope peptide to the PRRSV positive serum. DETAILED DESCRIPTION

[0028] The technical solutions in the embodiments of the application will be described clearly and completely below. Obviously, the described embodiments are part of, rather than all, the embodiments of the application. The detailed description of the embodiments of the application provided below is not intended to limit the scope of the claimed application, but merely represents selected embodiments of the application. Based on the embodiments of the application, all other embodiments obtained by those of ordinary skill in the art without creative work fall within the scope of the claimed application.

[0029] In the following examples, unless otherwise specified, the experimental methods not marked with specific conditions are generally implemented according to conventional conditions, and the reagents used in the examples are commercially available.

[0030] The application takes the amino acid sequence of the GP5 protein of the porcine reproductive and respiratory syndrome virus as material, displays the sequence containing the truncated GP5 protein on a T7 phage through T7 phage display technology, and forms a phage display library. Sequencing after immunoprecipitation of the above T7 phage display library with the PRRSV positive serum of the pig infected with the porcine reproductive and respiratory syndrome virus identifies it as an antigen dominant epitope. Subsequently, through biopanning of the phage library with the PRRSV positive serum of the pig infected with the porcine reproductive and respiratory syndrome virus and single plaque picking, the T7 phage displaying the polypeptide sequence as shown in SEQ ID NO. 1 is obtained, and the B cell antigen epitope of the GP5 protein of the porcine reproductive and respiratory syndrome virus is first determined.

[0031] The application is finally screened to obtain the antigen epitope peptide with good antigenicity, and the amino acid sequence of the antigen epitope peptide is as shown in SEQ ID NO. 1.

[0032] The preparation method of the GP5 antigen epitope peptide of the application can be a chemical synthesis method. The synthesis process is as follows:

[0033] (1) The antigen epitope peptide amino acid chain fragment of GP5 is as shown below: DGSAATPVTKVSAEQWGHP,

[0034] The sequence is from C-terminal to N-terminal. Weigh n equivalent of resin into the reactor, and add DCM to swell for half an hour; then remove DCM, add 2n equivalent of the first amino acid at C-terminal in the sequence, add 2n equivalent of DIEA, and then add DMF and DCM to bubble nitrogen for 60 minutes; wherein the amount of DMF and DCM added can make the resin be fully stirred. After the reaction is completed, 5n equivalent of methanol is added to react for half an hour, and then the reaction solution is removed to obtain the first reactant, which is washed with DMF and methanol. Then, 2n equivalent of the second amino acid, 2n equivalent of HBTU and DIEA are added into the reactor containing the first reactant, and nitrogen is bubbled for half an hour, and then the reaction solution is removed to obtain the second reactant, which is washed with DMF and methanol; the second reactant is detected by ninhydrin. The amino acids constituting the GP5 antigen epitope peptide are sequentially connected according to the same operation.

[0035] (2) After the amino acids are connected, the reactant after capping is washed; then the capped reactant is added with a decapping liquid to remove the Fmoc protective group, and the reactant after decapping is washed and detected by ninhydrin. The reactant after decapping is blown dry by nitrogen, taken out of the reactor, and poured into a flask, and then a cleavage liquid is added into the flask to react, the ratio of the cleavage liquid to the reactant after decapping is 10 mL of the cleavage liquid per gram of the reactant after decapping, and the cleavage liquid includes 95% of TFA, 2% of ethanedithiol, 2% of triisopropylsilane and 1% of water in mass percentage; the mixture is shaken, and then solid-liquid separation is performed to collect the filtrate. Then, the filtrate is added with ether to precipitate a precipitate, and the precipitate is collected and washed after solid-liquid separation to obtain a crude product. The crude product is purified to a required purity by high performance liquid chromatography. The purified liquid is concentrated and dried into powder, which is the GP5 antigen epitope peptide.

[0036] The fusion protein prepared by coupling the GP5 antigen epitope peptide of the application with a carrier protein can be used to immunize animals to prepare specific GP5 antibodies.

[0037] The application also provides human GP5 antibodies and humans, wherein the antibodies can be respectively prepared by immunizing animals with the fusion protein of the application, and the preparation method can adopt conventional techniques in the art.

[0038] Example 1: Identification of GP5 antigen epitope of porcine reproductive and respiratory syndrome virus by phage immunoprecipitation sequencing.

[0039] The Swine-Virscan library is diluted to 3.5x10 9pfu / mL, 1 mL of the diluted Swine-Virscan library was added to each 1.5 mL centrifuge tube, followed by the addition of 1:100 serum dilution containing 2 μg IgG, and incubated at 4 °C for 18 h with rotation; the protein A / G magnetic beads were washed three times with 0.02% PBST; then 20 μL of protein A / G magnetic beads were added to each centrifuge tube, and incubated for 4 h; the magnetic beads were transferred to a 96-well PCR plate, and washed three times with 200 μL of IP wash solution; the second-generation sequencing library was constructed and sequenced. Data analysis obtained the enriched phage members in the library. By comparing the sequences of the GP5 protein of each strain, it was found that the strain with the sequence shown in SEQ ID NO: 1 was relatively conserved in each strain sequence, and had good potential for ASFV serological detection, as shown in Figure 1 .

[0040] Example 2, biopanning of phage library with porcine reproductive and respiratory syndrome positive serum and single phage plaque picking.

[0041] Through three rounds of biopanning of the phage library with porcine reproductive and respiratory syndrome positive serum, each round of biopanning was repeated three times, and a phage library with affinity to porcine reproductive and respiratory syndrome positive serum was finally obtained. The retention rate was calculated by detecting the titer of the library after each round of biopanning, and the results showed that the average retention rate of the first round was about 0.79%, the average retention rate of the second round was about 6.34%, and the average retention rate of the third round was about 9.12%, as shown in Figure 2 . Since the retention rate gradually increased with the increase of the number of biopanning of the library, it was proved that the affinity between the serum and the biopanned library gradually increased. Then 384 phage plaques were randomly picked from the library after the third round of biopanning, and then added to the logarithmic growth phase of BLT5403 E. coli for amplification. After obtaining the amplification product of the single phage plaque, PCR amplification was first performed, and the length of the displayed fragment was analyzed by agarose gel electrophoresis. The results are shown in Figure 3 . Only 1 case did not match the expected fragment length, and another 1 case was not successfully picked and amplified. The second-generation sequencing library was constructed and sequenced for the 384 phages, and through the analysis of the sequencing results, 15 samples with low counts were filtered out. The maximum value of counts in each sample (i.e. the counts of the theoretically single phage displayed fragment) was greater than 90% of the total counts, which was determined as successful identification. Finally, 355 phages were successfully identified, of which 259 phages displayed different fragments, and 46 phages displayed ASFV protein fragments. Then the above 42 phages were identified by sanger sequencing for the second time, and finally the phage correctly displaying the GP5 antigen epitope shown in SEQ ID NO. 1 was successfully obtained.

[0042] Example 3, Dot ELISA verification of the affinity activity of GP5 epitope and porcine reproductive and respiratory syndrome positive serum.

[0043] The phages displaying the T cell epitope of GP5 shown as SEQ ID NO: 1 were diluted to 10 8 pfu / μL, and then diluted 5 times successively by 4 times, the control phages without foreign fragment display were diluted to 10 8 pfu / μL, the above phages were coated on NC membrane, and after being completely dried, were used for affinity verification with porcine reproductive and respiratory syndrome positive serum. As Figure 4 The results show that the phages displaying the GP5 epitope shown as SEQ ID NO. 1 have dose-dependent binding ability with ASFV positive serum, and have no binding ability with the control phages without foreign fragment display, and have the ability to neutralize porcine reproductive and respiratory syndrome virus.

[0044] In the embodiment, the preparation method of the antigen can be artificial synthesis, isolation or other biological methods; the preparation method of the antibody is that the antibody sequence is artificially synthesized, biological methods or biological and chemical methods, etc. The specific method can be the method in the prior art, as long as the sequence is consistent with the antibody sequence in the above embodiment.

[0045] In the application, the above antigen epitope (synthetic peptide) and single antigen epitope recombinant protein with immunocompetence are obtained for the first time for GP5 protein. The obtained antigen epitope and designed recombinant antigen can be used not only for the development of reagents and kits (ELISA, WB, colloidal gold chromatography test strip, etc.) for serological detection of porcine reproductive and respiratory syndrome virus infection, but also for the development of porcine reproductive and respiratory syndrome epitope vaccine and any vaccine related to the epitope (epitope monovalent / multivalent vaccine, synthetic peptide vaccine, and other pathogenic antigen epitope or antigen in series, etc.), and lays a material foundation for the preparation of porcine reproductive and respiratory syndrome antigen epitope specific monoclonal antibody; in addition, the research results can also be used for virus immune mechanism research, exploration and analysis of virus infection and vaccine immune mechanism, and realization of the purpose of preventing and controlling porcine reproductive and respiratory syndrome.

[0046] The antigen epitope peptide sequence and the related accessory subject matter, the antibody sequence and the related accessory subject matter, etc. given in the application are the points of the application.

[0047] The above only describes the preferred embodiments of the application, and is not used to limit the application. For those skilled in the art, the application can have various modifications and changes. Any modification, equivalent replacement, improvement, etc. within the spirit and principle of the application shall be included in the protection scope of the application.

Claims

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

1.

2. The use of the GP5 protein antigenic epitope peptide as described in claim 1 in the preparation of products for detecting porcine reproductive and respiratory syndrome virus infection.

3. A product for detecting porcine reproductive and respiratory syndrome, characterized in that: Its active ingredient is the GP5 protein antigenic epitope peptide as described in claim 1.

4. A nucleic acid molecule encoding the GP5 protein antigenic epitope peptide as described in claim 1.

5. The expression cassette, recombinant vector, or recombinant cell line of the nucleic acid molecule as described in claim 4.

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