Preparation and application of specific monoclonal antibody of GP5 protein of porcine reproductive and respiratory syndrome virus NADC30-like virus

Abstract

The invention discloses a porcine reproductive and respiratory syndrome virus (PRRSV) NADC30-like virus GP5 protein antigen epitope peptide, the antigen epitope peptide is a peptide fragment of porcine reproductive and respiratory syndrome virus GP5 protein 155-156 or / and 196-200, and the amino acid sequence of the antigen epitope peptide is 155WR156 or / and 196QWGRP200. The antigen epitope peptide and the monoclonal antibody provided by the invention are used for detecting the NADC30-like PRRSV and detecting the neutralizing antibody of the virus, replace the traditional method for detecting the neutralizing antibody through cell culture, and provide a new tool for researching the PRRSV and preventing and controlling the PRRSV.

Description

Technical Field

[0001] The invention belongs to the field of medicine and biological technology, and specifically relates to the preparation and application of a monoclonal antibody specific to the GP5 protein of porcine reproductive and respiratory syndrome virus (PRRSV) NADC30-like virus. Background Art

[0002] Monoclonal antibody technology is an important technology in modern life science research. Monoclonal antibodies have many applications in disease prevention, clinical treatment, disease diagnosis and detection. At the same time, monoclonal antibodies are also widely developed and used as research tools in the field of biological research. Monoclonal antibodies have many advantages such as high purity, strong specificity, good repeatability, and continuous unlimited supply. With the continuous development and improvement of monoclonal antibody technology, monoclonal antibodies currently play a pivotal role in various fields such as medicine, agriculture, food, and environment. Porcine reproductive and respiratory syndrome (PRRS), also known as "blue ear disease", is one of the most important economic diseases in the global pig industry. It is caused by PRRS virus (PRRSV) and can cause sow reproductive disorders and piglet respiratory damage. It is divided into European species (PRRSV-I) and American species (PRRSV-II). Porcine reproductive and respiratory syndrome is widely present in the world. The disease appears in a short time, spreads quickly, occurs in a wide range, and has great harm. Therefore, prevention and control of PRRSV epidemics is an urgent problem to be solved.

[0003] PRRSV is an enveloped single-stranded positive-sense RNA virus belonging to the order Nidovirales, the family Arteriviridae, and the genus Arterivirus. PRRSV virus particles are spherical, with a diameter of about 50-65nm, and a nucleocapsid diameter of about 30-35nm. It contains a single-stranded positive-sense genomic RNA of about 15.3kb, which is the longest among the four members of Arterivirus. The PRRSV genome contains 12 open reading frames (ORFs): ORF1a, ORF1a', TF, ORF1b, ORF2a, ORF2b, ORF3, ORF4, ORF5a, and ORF5-ORF7. Among them, ORF5 encodes the GP5 envelope protein, which interacts with the M envelope protein encoded by ORF6 to form a heterodimer and bind to the virus particle, which is necessary for the formation of virus particles. The molecular weight of the GP5 protein is about 25kDa and consists of about 200 amino acids. GP5 is one of the most variable regions in the structural proteins of the PRRSV genome and is therefore often used in phylogenetic analysis. The GP5 protein contains two B cell antigen epitopes: a non-neutralizing epitope A( 27 VLVNA 31 ) and a neutralizing epitope B ( 36 SHLQLIYNL 44). Epitope B induces the body to produce specific neutralizing antibodies against viral infection. In contrast, epitope A inhibits the body's recognition of epitope B, thereby delaying the production of neutralizing antibodies. GP5 is the main protective antigen that induces the production of neutralizing antibodies by PRRSV. After the neutralizing antibodies appear in the animal body, the antiserum mainly recognizes the GP5 protein, making the development of GP5 protein play an important role in the diagnosis and prevention of PRRS.

[0004] Enzyme-linked immunosorbent assay (ELISA) has the characteristics of strong specificity, high sensitivity and low cost, and is currently a popular PRRSV antibody detection method. Based on the purified GP5 protein, an indirect ELISA antibody detection method for PRRSV GP5 has been established. However, its antigen preparation requires a long purification time. The present invention uses cell fusion hybridoma technology to prepare PRRSV NADC30-like virus GP5 protein-specific monoclonal antibodies, and the obtained NADC30-like PRRSV GP5 protein monoclonal antibodies are successfully used in various immunological detection tests, and an epitope peptide indirect ELISA method for detecting PRRSV neutralizing antibodies is established and optimized, which can be used for the development of subsequent commercial kits. Summary of the invention

[0005] The present invention aims at the technical problem to be solved, overcomes the deficiencies of the prior art and provides a NADC30-like PRRSV GP5 protein antigen epitope peptide and a monoclonal antibody thereof and application thereof.

[0006] One of the purposes of the present invention is to provide an antigenic epitope peptide of a NADC30-like PRRSV GP5 protein, wherein the antigenic epitope peptide is a peptide segment 155 to 156 or / and a peptide segment 196 to 200 of the PRRSV GP5 protein, and its amino acid sequence is 155 WR 156 or / and 196 QUR 200 .

[0007] The second object of the present invention is to provide the use of the above-mentioned antigen epitope peptide in detecting NADC30-like PRRSV specific neutralizing antibodies.

[0008] The third object of the present invention is to provide a monoclonal antibody against the NADC30-like PRRSV GP5 protein, wherein the monoclonal antibody is specific to the above-mentioned antigen epitope peptide.

[0009] A method for preparing an anti-NADC30-like PRRSV GP5 protein monoclonal antibody comprises the following steps:

[0010] 1) Connect the PRRSV GP5 expression gene to the intermediate expression vector pENTER4-MCS-2HA to obtain the recombinant expression plasmid pENTER4-GP5-2HA;

[0011] 2) Perform site-specific LR recombination reaction between pENTER4-GP5-2HA and target expression vector pDEST527 to obtain recombinant expression plasmid pDEST527-GP5-2HA;

[0012] 3) Transform pDEST527-GP5-2HA into DE3 / BL21 competent Escherichia coli, induce expression, and obtain PRRSVGP5 protein;

[0013] 4) Immunizing mice with PRRSV GP5 protein, taking spleen cells from the immunized mice and fusing them with myeloma cells SP2 / 0 to obtain hybridoma cells;

[0014] 5) After the hybridoma cells are subcloned, they are injected intraperitoneally to obtain specific monoclonal antibodies against the PRRSV GP5 protein.

[0015] The present invention prepared three monoclonal antibodies by prokaryotic expression of NADC30-like PRRSV GP5 and identified two new antigenic epitopes. 155 WR 156 " or / and " 196 QUR 200 ". At the same time, three monoclonal antibodies specific for the above antigenic epitopes were used in a variety of immunological tests, including but not limited to ELISA, WB, and IFA. Then, based on the two identified antigenic epitopes, an indirect ELISA method that can be used to detect PRRSV antibodies was explored and optimized. The antigenic epitope peptides and monoclonal antibodies provided by the present invention are used for the detection of NADC30-like PRRSV, and have the characteristics of good specificity and high accuracy, providing an effective tool for studying the biological function of NADC30-like PRRSV GP5 protein.

[0016] A fourth object of the present invention is to provide the use of the above-mentioned NADC30-like PRRSV GP5 protein monoclonal antibody in immunological detection.

[0017] The above applications include ELISA testing, WB testing, and IFA testing.

[0018] Finally, the present invention also provides the use of the antigen epitope peptide and monoclonal antibody in diagnosing or detecting NADC30-like PRRSV. The above-mentioned application includes any one or more of the following:

[0019] (1) The antigen epitope peptide and the monoclonal antibody are used in the preparation of a kit for diagnosing or detecting NADC30-like PRRSV;

[0020] (2) Use of the antigen epitope peptide and the monoclonal antibody in the preparation of a drug for diagnosing or detecting NADC30-like PRRSV infection;

[0021] (3) Use of the antigen epitope peptide and the monoclonal antibody in the preparation of reagents for diagnosing or detecting NADC30-like PRRSV infection.

[0022] The present invention establishes an indirect ELISA method for detecting NADC30-like PRRSV neutralizing antibodies based on NADC30-like PRRSV monoclonal antibodies and antigenic epitope peptides thereof.

[0023] The indirect ELISA detection method comprises the following steps:

[0024] 1) Mix the NADC30-like PRRSV GP5 protein epitope peptide 155WR156 or / and 196QWGRP200 in a ratio of 1:3 and coat the ELISA plate at a coating concentration of 0.5 ng / μL. Add 100 μL to each ELISA well, incubate overnight at 4°C, block with 5% BSA for 2 hours, and then wash;

[0025] 2) Add the serum to be tested, add 100 μL of PRRSV-positive pig serum diluted 1:10 with PBS buffer to each well, and incubate for 90 minutes;

[0026] 3) Add enzyme-labeled secondary antibody, add 100 μL of porcine enzyme-labeled antibody diluted 1:10000 with PBS buffer to each well and incubate for 75 minutes;

[0027] 4) Add color developing solution, color development for 15 min, and add stop solution to terminate the reaction;

[0028] 5) The OD450 value was measured using an enzyme marker. When the OD450 value of the sample was greater than 0.240, the sample was judged to be positive.

[0029] Beneficial effects of the present invention:

[0030] The specific monoclonal antibody of the present invention against the structural protein GP5 of NADC30-like PRRSV is a specific and useful tool for PRRSV research, and can be successfully applied to various immunological tests, including enzyme-linked immunosorbent assay (ELISA), protein blotting (WB) and immunofluorescence (IFA) experiments, and can also be applied to the functional study of NADC30-like PRRSV. At the same time, the present invention uses a prokaryotic expression system to obtain recombinant NADC30-like PRRSV GP5 protein to prepare its specific monoclonal antibody, identify the monoclonal antibody to recognize a new antigen epitope, establish and optimize the PRRSV serological antibody detection method, which can be used to detect NADC30-like PRRSV neutralizing antibodies, has important application prospects, and provides an effective tool for PRRSV detection and prevention and control. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 The results of SDS-PAGE / Western blotting identification of the induced expressed GP5 recombinant protein and the purified GP5 protein in the present invention. In the figure, M: pre-stained marker; 1: pDEST527 empty load without induction of the whole bacteria; 2: pDEST527-GP5 recombinant protein without induction of the whole bacteria; 3: pDEST527-GP5 recombinant protein induced for 12h at 16℃ for the whole bacteria; 4: pDEST527-GP5 recombinant protein induced for 24h at 16℃ for the whole bacteria; 5: pDEST527-GP5 recombinant protein induced for 12h at 37℃ for the whole bacteria; 6: pDEST527-GP5 recombinant protein induced for 12h at 16℃ for the supernatant ; 7: supernatant of pDEST527-GP5 recombinant protein induced at 16℃ for 24h; 8: supernatant of pDEST527-GP5 recombinant protein induced at 37℃ for 12h; 9: precipitate of pDEST527-GP5 recombinant protein induced at 16℃ for 12h; 10: precipitate of pDEST527-GP5 recombinant protein induced at 16℃ for 24h; 11: precipitate of pDEST527-GP5 recombinant protein induced at 37℃ for 12h; 12: purified pDEST527-GP5 recombinant protein.

[0032] Figure 2 This is the Western blotting identification result of the NADC30-like PRRSV GP5 monoclonal antibody in the present invention.

[0033] Figure 3 This is the IFA detection result of the monoclonal antibody against NADC30-like PRRSV GP5 protein after 293T cells were transfected with pEGFP-N1-GP5 in the present invention.

[0034] Figure 4This is the IFA detection result of the monoclonal antibody against the NADC30-like PRRSV GP5 protein after PRRSV infects 3D4 / 21-CD163 cells in the present invention.

[0035] Figure 5 This is the subclass identification result of the NADC30-like PRRSV GP5 protein monoclonal antibody in the present invention.

[0036] Figure 6 Schematic diagram of the antigenic epitope recognized by the NADC30-like PRRSV GP5 protein monoclonal antibody of the present invention and the Western blotting identification results.

[0037] Figure 7 The results are Western blotting results of the NADC30-like PRRSV GP5 protein monoclonal antibody of the present invention recognizing different strains of PRRSV.

[0038] Figure 8 The figure is a schematic diagram of the sequence comparison of GP5 proteins of different PRRSV strains in the present invention.

[0039] Fig. 9 This is the endpoint neutralization test result of the monoclonal antibody against the NADC30-like PRRSV GP5 protein in the present invention.

[0040] Fig.10 The results are as follows: identification of antigen epitope peptide combination of NADC30-like PRRSV GP5 protein monoclonal antibody and optimization of indirect ELISA conditions in the present invention.

[0041] Fig.11 It is the specificity and sensitivity test result of indirect ELISA in the present invention.

[0042] Fig.12 It is the repeatability test result of indirect ELISA in the present invention. DETAILED DESCRIPTION

[0043] The technical solution of the present invention is further described in detail below in conjunction with the embodiments: This embodiment is implemented on the premise of the technical solution of the present invention, and a detailed implementation method and a specific operation process are given, but the protection rights of the present invention are not limited to the following embodiments. The materials and reagents mentioned in the present invention are all available for purchase or free of charge from domestic and foreign channels, and are not described one by one here.

[0044] Example 1: PRRSV NADC30-like GP5 gene cloning and expression, protein purification and preparation of monoclonal antibodies

[0045] Using the cDNA reverse transcribed from RNA in cells infected with the NADC30-like PRRSV SD17-38 virus strain preserved in the laboratory of the College of Veterinary Medicine of Yangzhou University as a template, cloning and detection PCR primers were designed (see Table 1). Based on the bioinformatics analysis of the PRRSV GP5 protein, the predicted transmembrane region and signal peptide sequence were removed, and two primers were designed for segmented amplification. The corresponding DNA sequence was homologously recombined into the intermediate expression vector pENTER4-MCS-2HA to obtain the recombinant expression plasmid pENTER4-GP5-2HA. The successfully obtained intermediate vector pENTER4-GP5-2HA was subjected to site-specific LR recombination reaction with the target expression vector pDEST527 to obtain the recombinant expression plasmid pDEST527-GP5-2HA. The above recombinant expression plasmid was transformed into DE3 / BL21 competent Escherichia coli (E.coli) to explore the optimal induction conditions for prokaryotic protein expression: the best induction expression conditions were 1mM IPTG inducer concentration and 12h culture at 37℃. The supernatant and precipitate analysis of the bacterial lysate showed that the protein was mainly expressed in the inclusion body (see Figure 1 AC, SDS-PAGE Coomassie Brilliant Blue staining and Western blotting analysis were performed on the GP5 protein expressed under different induction conditions. The results showed that the expression level of GP5 protein was the highest at 37°C and induced culture for 12h, and it was highly expressed in bacterial lysis precipitates). After the bacteria were fully broken by ultrasound, the precipitate was collected by high-speed centrifugation, washed, and then resuspended in a PBS solution containing 8M urea and incubated at 4°C overnight. The overnight suspension was broken by an ultrasonic disruptor for 30min, placed in a dialysis bag, and placed in PBS dialysis fluid containing 6M, 4M, 2M, and 1M urea at 4°C for gradient dialysis. After the dialysis was completed, the dialysis bag was removed and concentrated with PEG20000. The liquid in the bag was then removed and a 30μL sample was taken for SDS-PAGE Coomassie Brilliant Blue staining analysis. The results showed that the purified GP5 protein band was relatively single, and the protein concentration was about 1μg / μL, which met the requirements of later immunization (see Figure 1 D. The purified inclusion body GP5 protein was subjected to SDS-PAGE Coomassie Brilliant Blue staining detection. The results are shown in the figure. The purified GP5 protein band is relatively single, and the protein concentration is 1 μg / uL, which meets the later immune requirements).

[0046] Table 1. GP5 amplification primer sequences and truncated protein gene PCR amplification primer sequences

[0047]

[0048]

[0049] Note: F and R represent upstream and downstream primers, respectively. The lowercase letters represent restriction sites in the cloning primers. The start codon is marked in bold.

[0050] BALB / c mice aged 6-8 weeks were taken and subcutaneously immunized with purified GP5 protein. Two weeks later, after intraperitoneal injection of Shutai 50 (10×) anesthesia, the mice were fixed on the operating table in the left lateral position, the left abdomen was fully exposed, the hair was shaved in the spleen area, and the skin was disinfected with iodine and 75% ethanol. A small incision was made 0.5 cm below the left rib, and the abdominal skin, muscle and peritoneum were cut open in turn to expose the abdominal mesentery. The root of the mesentery was clamped to pull part of the spleen out of the abdominal cavity. 50 μg of purified GP5 protein was slowly injected along the long axis of the spleen with a 1mL syringe. After the injection, the spleen was returned to the abdominal cavity, and the peritoneum and skin were sutured. 7 days after immunization, the mouse spleen cells were taken and fused with mouse myeloma SP2 / 0 cells (additional information after the specific operation steps) to obtain hybridoma cell clones. Among them, there were 3 ELISA-positive cell clones. Western blotting and IFA detection showed that the corresponding supernatants obtained from the 3 hybridoma cell clones could react specifically with the GP5 protein expressed by transfected eukaryotic cells and viruses.

[0051] The three identified hybridoma cell lines were subcloned three times by limiting dilution. 8-12 week old BALB / c mice were injected intraperitoneally with Freund's incomplete adjuvant (0.2-0.3 mL / mouse). Three days later, subcloned hybridoma cells (1-3×10 6 Cells / 0.3mL / mouse) were injected intraperitoneally, and the degree of abdominal distension of the mouse was observed after 5 days to determine the ascites production in the mouse. If the mouse abdomen is obviously swollen and the mouse abdomen is tight when touched, the ascites can be collected with a 16-gauge needle. The ascites can be collected 2-3 times in a row. The collected ascites is centrifuged at 2000rpm for 5min to remove the top layer of fat tissue and red blood cells mixed in the ascites, and the supernatant is collected. After aliquoting, it is frozen in a -80℃ refrigerator. In this way, we obtained 3 strains of PRRSV GP5 monoclonal antibodies.

[0052] Example 2: Identification of monoclonal antibodies against PRRSV NADC30-like GP5 protein

[0053] (1) Monoclonal antibody Western blotting identification

[0054] After ELISA screening, three positive cell clones were obtained ( Figure 2 A), the titers of ascites obtained from the three positive cell clones were 409600, 819200 and 819200 ( Figure 2 B). 293T cells were plated in 12-well cell culture plates (2×10 5When the cell density reached 70%-80%, pCAGGS-GP5-2HA plasmid and its empty vector plasmid pCAGGS-2HA were transfected with Lip2000 at 1 μg / well. Cells were collected 24 hours after transfection, lysed with cell lysis buffer, and Western blotting was performed using the prepared monoclonal antibody as the primary antibody for cell lysate protein detection. The experimental results are shown in Figure 2 As shown in C, three monoclonal antibodies (3A8, 11E6 and 12D1) can specifically recognize exogenous GP5.

[0055] At the same time, 3D4 / 21-CD163 cells were plated in 12-well cell culture plates (2×10 5 The cells were infected with the SD17-38 virus stored in our laboratory. After 72 hours, the cells were collected and lysed with cell lysis buffer. The cell lysate protein was detected by Western blotting using the prepared monoclonal antibody as the primary antibody. The experimental results are shown in Figure 2 As shown in D, three monoclonal antibodies (3A8, 11E6 and 12D1) can specifically recognize endogenous GP5.

[0056] (2) Monoclonal antibody IFA identification

[0057] 293T cells were plated in 12-well cell culture plates (2×10 5 When the cell density reached 70%-80%, pEGFP-N1-GP5 plasmid and its empty vector plasmid pEGFP-N1 were transfected with Lip2000 at 1 μg / well. IFA experiments were performed 24 hours after transfection. Immunofluorescence was used to detect the reactions of 3A8, 11E6 and 12D1 monoclonal antibodies and 293T cells transfected with pEGFP-N1-GP5. The experimental results are shown in Figure 3 As shown, 3A8 (A), 11E6 (B) and 12D1 (C) clones can specifically react with exogenous GP5.

[0058] At the same time, 3D4 / 21-CD163 cells were plated in 12-well cell culture plates (2×10 5 The cells were infected with SD17-38 virus stored in our laboratory, and the IFA experiment was performed 48 hours later. The reaction of 3A8, 11E6 and 12D1 monoclonal antibodies and 3D4 / 21-CD163 cells infected with PRRSV was detected by immunofluorescence. The experimental results are shown in Figure 4 As shown, 3A8 (A), 11E6 (B) and 12D1 (C) clones were able to specifically react with endogenous GP5.

[0059] The above experimental results showed that the three monoclonal antibodies (3A8, 11E6 and 12D1) can recognize exogenous and endogenous GP5.

[0060] (3) Identification of monoclonal antibody subclasses

[0061] The three monoclonal antibodies obtained were identified by using the monoclonal antibody subclass identification kit of Servi. For specific methods, see the additional information. After the end, the results can be directly observed with the naked eye. The enzyme-labeled secondary antibody corresponding to the dark blue well is the subclass of the monoclonal antibody to be tested. In addition, the stop solution was added to terminate the reaction, and the OD was read using an enzyme reader. 450 Value (see Figure 5 The results showed that among the three monoclonal antibodies (3A8, 11E6 and 12D1), the subclass of 3A8 monoclonal antibody was IgG1, the subclass of 11E6 monoclonal antibody was IgG2b, and the subclass of 12D1 monoclonal antibody was IgG2a.

[0062] (4) Identification of monoclonal antibody antigen epitopes

[0063] The minimal epitope recognized by the monoclonal antibody was identified by Western blotting. First, the GP5 protein was divided into three segments (1-60aa, 61-130aa, 131-200aa), and the fragment coding sequences were connected to the eukaryotic expression vector pEGFP-N1. After sequencing verification, the plasmids were extracted and transfected into 293T cells respectively. After 24 hours, the cells were collected and lysate was added. Western blotting was used to verify the truncated protein fragments that the monoclonal antibody can recognize. According to the monoclonal antibody reaction, the N segment and C terminus were shortened respectively to construct N and C terminal truncated expression fragments, and the vector was pEGFP-N1 (the length and amino acid position of the truncated recombinant GP5 protein are shown in Figure 6 ). The fragment was truncated until it could not be recognized by the corresponding monoclonal antibody, and the key amino acid sites at the N-terminus and C-terminus that reacted with the monoclonal antibody were determined. Based on this, it can be clearly determined that the minimum fragment recognized by the GP5 monoclonal antibody is the antigenic epitope of the GP5 monoclonal antibody (see Figure 6 In the figure, blue indicates reaction with monoclonal antibody, and black indicates no reaction with monoclonal antibody). The results showed that among the three monoclonal antibodies (3A8, 11E6 and 12D1), the antigen epitope recognized by 3A8 monoclonal antibody was " 155 WR 156 The antigen epitopes recognized by the two monoclonal antibodies 11E6 and 12D1 are 196 QUR 200 ”.

[0064] (5) Identification and conservation analysis of the reactivity of PRRSV NADC30-like GP5 monoclonal antibody with different PRRSV strains

[0065] Cell samples infected with porcine respiratory reproductive syndrome virus PRRSV-1 (SD1291, HLJB1) and PRRSV-2 (SD17-38 (lineage 1), Anheal-1 (lineage 1), R98 (lineage 5), CH-1R (lineage 8), XJ17-5 (lineage 8), JSTZ1712-12 (lineage 8)) were collected and analyzed by Western blotting with three GP5 monoclonal antibodies to determine the cross-reactivity of the three GP5 monoclonal antibodies with different PRRSV strains (see Figure 7 ). The results showed that among the three monoclonal antibodies (3A8, 11E6 and 12D1), 3A8 monoclonal antibody could recognize the GP5 protein of all identified PRRSV-2 strains; 11E6 and 12D1 monoclonal antibodies recognized the GP5 protein of PRRSV-2 (SD17-38 (lineage 1), Anheal-1 (lineage 1), R98 (lineage 5), CH-1R (lineage 8)) strains. The sequence conservation of GP5 proteins of different PRRSV strains was analyzed by comparing the sequences (see Figure 8 ). Through sequence comparison, it was found that 155 WR 156 "The sequence is relatively conservative in the sequences of GP5 proteins of different PRRSV strains. 196 QUR 200 The sequence is also partially conserved in the sequences of GP5 proteins of different PRRSV strains.

[0066] (6) Analysis of neutralization resistance of PRRSV NADC30-like GP5 monoclonal antibody

[0067] The ascites of the three monoclonal antibodies were purified (see Fig. 9 A), collected porcine respiratory reproductive syndrome virus PRRSV-1 (HLJB1) and PRRSV-2 (SD17-38 (lineage 1), Anheal-1 (lineage 1), R98 (lineage5), XJ17-5 (lineage 8)) virus samples, and used three GP5 purified monoclonal antibodies for endpoint neutralization assay analysis (see Fig. 9The results showed that among the three GP5 monoclonal antibodies (3A8, 11E6 and 12D1), 11E6 and 12D1 had certain neutralizing activity against SD17-38, that is, the tested antibodies could protect 50% of tissue culture cells from cytopathic effect (CPE) when diluted at 1:148 and 1:165, respectively, while 3A8 and four N protein monoclonal antibodies had no neutralizing activity against SD17-38. The three GP5 monoclonal antibodies and four N protein monoclonal antibodies had no neutralizing activity against Anheal-1 (lineage 1), R98 (lineage 5) and XJ17-5 (lineage 8).

[0068] Example 3: Establishment, optimization and application of indirect ELISA detection method based on PRRSV NADC30-like GP5 antigen epitope peptide

[0069] (1) Selection of optimal epitope peptide

[0070] Two synthesized epitope peptides P1 (153-158aa) and P2 (196-200aa) were coated individually or in different combinations and ratios (P1, P1:P2=3:1, P1:P2=2:1, P1:P2=1:1, P1:P2=1:2, P1:P2=1:3, P2). Each combination was coated at a concentration of 1 ng / μL and 2 ng / μL (100 μL / well) per well, and the best combination of epitope peptides was screened out according to the P / N value (see Fig.10 The results showed that the best combination of antigen epitope peptides was P1:P2=1:3.

[0071] (2) Optimization of optimal epitope peptide coating concentration and optimal serum dilution

[0072] According to the best combination of epitope peptides determined by the above experimental results, the epitope peptides were gradient coated, and the serum was gradient diluted for checkerboard cross test. The epitope peptides were set at 0.25ng / μL, 0.5ng / μL, 1ng / μL, 2ng / μL, 4ng / μL, and 8ng / μL for overnight incubation and coating at 4°C. The blocking and washing operations are the same as the ELISA operation above. PRRSV-positive pig serum was used as the primary antibody, and the dilution ratio of positive pig serum in each column from left to right was 1:10, 1:20, 1:40, 1:80, 1:160, 1:320, 1:640, and 1:1280. At the same time, negative pig serum was used as a negative control, and antibody diluent was added to the last column as a blank control. The secondary antibody used was a porcine enzyme-labeled antibody, and the subsequent color development operation was the same as above. Finally, 2M sulfuric acid was added to terminate the reaction, and the OD was read using an enzyme reader. 450By calculating the P / N value, the antigen peptide coating concentration and serum dilution corresponding to the maximum P / N value were selected as the optimal ELISA conditions ( Fig.10 B). The results showed that the optimal coating concentration of the antigen epitope peptide for the indirect ELISA method was 0.5 ng / μL, and the optimal serum dilution was 1:10.

[0073] (3) Optimization of optimal epitope peptide coating and blocking conditions

[0074] Based on the above experimental results, the epitope peptide coating conditions and blocking conditions were further optimized. The epitope peptide coating conditions were set to three conditions: 4°C overnight, 37°C 1 h, 37°C 2 h, and the blocking conditions were set to five conditions: no blocking and 2% bovine serum albumin (BSA), 5% bovine serum albumin (BSA), 2% skim milk powder (SMP), 5% skim milk powder (SMP), all blocked at 37°C for 2 h. Other conditions were the same as above. OD was read by microplate reader 450 numerical value, calculate the P / N value, and determine the condition with the largest P / N value as the optimal condition ( Fig.10 C). The results showed that the best coating condition for the antigen epitope peptide of the indirect ELISA method was incubation at 4°C overnight, and the best blocking condition was 2% BSA at 37°C for 2 h.

[0075] (4) Optimization of the optimal primary antibody incubation time

[0076] Based on the above experimental results, the optimal primary antibody incubation time was optimized. The primary antibody pig serum incubation was set at 37°C for 45 min, 60 min, 75 min, 90 min, 105 min, and 120 min. Other conditions were the same as above. The OD was read by an ELISA reader. 450 The P / N value was calculated and the optimal primary antibody incubation time was determined to be the largest P / N value ( Fig.10 The results showed that the optimal reaction time of the primary antibody in the indirect ELISA method was 90 min.

[0077] (5) Optimization of the optimal secondary antibody incubation time

[0078] Based on the above experimental results, the optimal secondary antibody incubation time was optimized. The detection antibody was incubated at 37°C for 45 min, 60 min, 75 min, 90 min, 105 min, and 120 min. Other conditions were the same as above. The OD was read by an enzyme-labeled instrument. 450 The P / N value was calculated and the optimal secondary antibody incubation time was determined to be the largest P / N value ( Fig.10 The results showed that the optimal reaction time of the secondary antibody in the indirect ELISA method was 75 min.

[0079] (6) Optimization of the optimal color development time

[0080] Based on the above experimental results, the optimal color development time was optimized. The color development time was set to 5 min, 10 min, 15 min, 20 min, and 25 min. Other conditions were the same as above. The OD was read by an enzyme reader. 450 The P / N value was calculated and the best color development incubation time was determined to be the largest P / N value ( Fig.10 The results showed that the optimal color development time of the indirect ELISA method was 15 min.

[0081] (7) Determination of sample critical value

[0082] The optimal antigen epitope peptide combination, optimal coating concentration, optimal blocking conditions, optimal primary and secondary antibody incubation time, and optimal color development time were determined through the above experiments. Subsequently, the optimal conditions of the indirect ELISA after optimization were used to conduct experiments and determine the critical value of the established indirect ELISA method. The antigen epitope peptide was coated on a 96-well ELISA plate at the optimal concentration, and 100 μL was added to each well. 30 negative pig sera were used as the primary antibody, and 100 μL was added to each well. Other operations were the same as the optimal experimental conditions. The OD was read by an ELISA reader. 450 Numerical value, average calculation and error SD, according to the statistical formula Get two critical values and (Table 2) The results showed that the critical value of the indirect ELISA method was 0.227-0.240. When the OD450 value of the sample was greater than 0.240, the sample was judged to be positive, when the OD450 value of the sample was less than 0.227, the sample was judged to be negative, and when the OD450 value of the sample was 0.227-0.240, the sample was judged to be suspicious.

[0083] Table 2. Determination of cut-off values ​​for epitope peptide indirect ELISA

[0084]

[0085]

[0086] (8) Specificity detection

[0087] The experiment was carried out according to the above optimized conditions, and the established indirect ELISA detection method was specifically tested. After coating the antigen peptide, it was blocked, and the primary and secondary antibodies were incubated for color development, and finally the reaction was terminated. Inactivated PRRSV, ASFV, SIV, PEDV positive pig serum and negative pig serum were used as primary antibodies. The OD was read by an enzyme reader. 450 The specificity of the indirect ELISA detection method was determined by Fig.11 The results showed that the indirect ELISA method had a specific reaction only with PRRSV serum, but had no reaction with ASFV, SIV, and PEDV serum.

[0088] (9) Sensitivity testing

[0089] The experiment was carried out according to the above optimized conditions, and the sensitivity of the established indirect ELISA detection method was tested. After the antigen peptide was coated, it was blocked, and the primary antibody and secondary antibody were incubated for color development, and finally the reaction was terminated. The pig serum was diluted in a gradient of 1:10 to 1:1280 as the primary antibody, and the OD was read by an enzyme reader. 450 The minimum detection limit was determined according to the critical value, and the sensitivity of the indirect ELISA detection method was evaluated ( Fig.11 The results showed that the indirect ELISA method could still detect positive results when the pig serum dilution ratio was 1:640, which showed high sensitivity.

[0090] (10) Repeatability test

[0091] The experiment was carried out according to the above optimized conditions, and the established indirect ELISA detection method was tested for repeatability. The 96-well ELISA plates coated in the same batch were used for intra-batch repeatability testing, and the 96-well ELISA plates coated in different batches were used for inter-batch repeatability testing. Five PRRSV positive samples and one negative sample were taken, and three replicate wells were set for each sample, and the established indirect ELISA detection method was used for testing. The OD was read by an ELISA reader. 450 The coefficient of variation was calculated to evaluate the stability of the indirect ELISA detection method ( Fig.12 The results showed that the intra-batch coefficient of variation (CV) was between 1.4% and 3.3%, and the inter-batch coefficient of variation (CV) was between 4.3% and 8.4%, both less than 10%, indicating that the indirect ELISA method has good repeatability.

[0092] (11) Clinical sample testing

[0093] 81 clinical samples were taken and tested using the established indirect ELISA test method and the IDEXX antibody test standard kit. The samples were statistically analyzed to evaluate the compliance rate of the indirect ELISA test method and the IDEXX test method (Table 3). The results showed that 31 samples were positive and 50 samples were negative by the IDEXX method. 19 samples were positive and 62 samples were negative by the indirect ELISA method. The compliance rate between the two was 82.7%.

[0094] Table 3. Indirect ELISA compliance test

[0095]

[0096] Note: * represents positive samples that can only be detected by epitope peptide indirect ELISA, # represents positive samples that can only be detected by IDEXX antibody test kit Additional information: Specific operation of cell fusion, method for identifying monoclonal antibody subclasses Indirect ELISA method

[0097] Cell fusion

[0098] ① 1×10 8 Splenocytes and 1-2×10 7 Myeloma cells SP2 / 0 were mixed in a 50 mL fusion tube, serum-free DMEM medium was added to 30 mL, and the mixture was thoroughly mixed.

[0099] ② Centrifuge at 1000rpm for 5-10min and remove the supernatant (the precipitate at the bottom of the tube is red spleen cells in the lower layer and white SP2 / 0 cells in the upper layer, and the pressure volume of the two is roughly the same).

[0100] ③ Hold the tube in your palm, rotate and tap the bottom of the fusion tube to make the cell clumps loose and homogeneous.

[0101] ④ Use a 1 mL pipette to slowly add 1 mL of PEG1500 preheated at 37°C within 60 seconds, rotating the centrifuge tube while adding to fully mix the PEG and cells.

[0102] ⑤ Use a pipette to add 10-20 mL of serum-free DMEM medium preheated to 37°C in a slow and then fast manner within 90 seconds to dilute and terminate the effect of PEG1500. Let stand at 37°C for 10 minutes.

[0103] ⑥ Centrifuge at 1000rpm for 5-10min and discard the supernatant.

[0104] ⑦ Add 40 mL of HAT culture medium and gently pipette to mix the precipitated cells.

[0105] ⑧ Use a dispenser to dispense into a 96-well cell culture plate containing peritoneal feeder cells, 100 μL per well; then place the culture plate in a 37°C, 5% CO2 incubator for culture.

[0106] ⑨ 7-10 days after fusion (hybridoma cell clones are very obvious), 1 / 2 of the culture medium can be replaced with fresh HAT culture medium.

[0107] ⑩Observe the growth of hybridoma cells. When the cells exceed 1 / 10 of the bottom area of ​​the well and the supernatant turns yellow, aspirate the supernatant for ELISA antibody detection.

[0108] Methods for identification of monoclonal antibody subclasses

[0109] ① Return the kit to room temperature and use pure water to prepare the cleaning solution to the working concentration (1 part concentrated cleaning solution plus 19 parts pure water).

[0110] ② Take out the ELISA plate, set up 6 wells for each monoclonal antibody sample to be tested, and set up 6 wells for positive control and positive control respectively.

[0111] ③ First add 50 μL / well of sample diluent to the test wells of the sample to be tested, then add 50 μL of cell culture supernatant to the test wells containing sample diluent, and add 6 wells for each sample. Add universal positive control solution and negative control solution to the positive control wells and 100 μL to each well. Apply sealing film and incubate at 37℃ for 30 minutes.

[0112] ④Discard the liquid in the plate, wash 5 times and pat dry. Add 100 μL of each of the 6 enzyme-labeled secondary antibodies to the 6 sample detection wells, and add the same enzyme-labeled secondary antibodies to the 6 wells of the universal positive control. Apply the sealing film and incubate at 37℃ for 30 minutes.

[0113] ⑤Discard the liquid in the plate, wash 5 times and pat dry. Add 50μL of colorimetric solution A and colorimetric solution B to each well, replace a new sealing film, and cover the plate in the dark at 37℃ for 20 minutes.

[0114] ⑥ After the reaction is finished, the results can be observed directly with the naked eye. The enzyme-labeled secondary antibody corresponding to the dark blue well is the subclass of the monoclonal antibody to be tested. In addition, the reaction stop solution (50 μL per well) is added to stop the reaction and the OD is measured with an enzyme-labeled instrument. 450 Numeric value.

[0115] ELISA operation method

[0116] ① Antigen epitope peptide coating: After mixing and diluting the two synthesized antigen epitope peptides P1 (153-158aa) and P2 (196-200aa) with the coating solution, 100 μL was added to each well of the ELISA plate and incubated at 4°C overnight.

[0117] ② Washing: Wash 3-6 times with PBST, 5 minutes each time, and pat dry on absorbent paper.

[0118] ③ Blocking: add 200 μL / well of blocking solution (PBS solution containing 2% BSA), incubate at 37°C for 2 h, and wash as above.

[0119] ④ Primary antibody incubation: Add 1:10 diluted inactivated PRRSV positive serum or negative serum (100 μL / well), incubate at 37°C for 1 hour, and wash as above.

[0120] ⑤ Secondary antibody incubation: Add 1:10000 diluted HRP porcine enzyme-labeled antibody (100 μL / well), incubate at 37°C for 1 h, and wash as above.

[0121] ⑥ Color development: Add commercial TMB color development solution (50 μL / well), react at 37°C in the dark for 15 min, add 2M H2SO4 (50 μL / well) to terminate the reaction, and read the OD using an enzyme reader 450 Numeric value.

[0122] The above description is only a specific implementation of the present invention, but the protection scope of the present invention is not limited thereto. Any person familiar with the technology can understand and think of any changes or substitutions within the technical scope disclosed by the present invention, which should be included in the scope of the present invention. Therefore, the protection scope of the present invention should be based on the protection scope of the claims.

Claims

1. An antigenic epitope peptide of NADC30-like PRRSV GP5 protein, characterized in that: The antigen epitope peptide is the peptide segments 155-156 and / or 196-200 of the GP5 protein of porcine reproductive and respiratory syndrome virus, and its amino acid sequence is 155 WR 156 or / and 196 QUR 200 .

2. Use of the antigen epitope peptide as claimed in claim 1 in detecting NADC30-like PRRSV specific neutralizing antibodies.

3. NADC30-like PRRSV GP5 protein monoclonal antibody, characterized in that: The monoclonal antibody is specific for the antigenic epitope peptide described in claim 1.

4. A method for preparing a monoclonal antibody against NADC30-like PRRSV GP5 protein as claimed in claim 3, characterized in that: The steps include: 1) Connect the PRRSV GP5 expression gene to the intermediate expression vector pENTER4-MCS-2HA to obtain the recombinant expression plasmid pENTER4-GP5-2HA; 2) Perform site-specific LR recombination reaction between pENTER4-GP5-2HA and target expression vector pDEST527 to obtain recombinant expression plasmid pDEST527-GP5-2HA; 3) Transform pDEST527-GP5-2HA into DE3 / BL21 competent Escherichia coli, induce expression, and obtain PRRSV GP5 protein; 4) Immunizing mice with PRRSV GP5 protein, taking spleen cells from the immunized mice and fusing them with myeloma cells SP2 / 0 to obtain hybridoma cells; 5) After the hybridoma cells are subcloned, they are injected intraperitoneally to obtain specific monoclonal antibodies against the PRRSV GP5 protein.

5. Use of the NADC30-like PRRSV GP5 protein monoclonal antibody as claimed in claim 3 in immunological detection.

6. The use according to claim 5, characterized in that: Including ELISA test, WB test, and IFA test.

7. Use of the antigenic epitope peptide according to claim 1 and the monoclonal antibody according to claim 3 in diagnosing and detecting PRRSV.

8. The use according to claim 7, characterized in that: The application includes any one or more of the following: 1) The antigen epitope peptide and the monoclonal antibody are used in the preparation of a kit for diagnosing or detecting PRRSV; 2) The antigen epitope peptide and the monoclonal antibody are used in the preparation of a drug for diagnosing or detecting PRRSV infection; 3) Use of the antigen epitope peptide and the monoclonal antibody in the preparation of reagents for diagnosing or detecting PRRSV infection.

9. An indirect ELISA method for detecting NADC30-like PRRSV-specific neutralizing antibodies based on the antigen epitope peptide according to claim 1.

10. The indirect ELISA detection method according to claim 9, characterized in that: The following steps are involved: 1) Using NADC30-like PRRSV GP5 protein epitope peptide 155 WR 156 or / and 196 QUR 200 Mix at a ratio of 1:3 and coat the ELISA plate at a coating concentration of 0.5 ng / μL. Add 100 μL to each ELISA well and incubate overnight at 4°C. Block with 5% BSA for 2 h and then wash. 2) Add the serum to be tested, add 100 μL of PRRSV-positive pig serum diluted 1:10 with PBS buffer to each well, and incubate for 90 minutes; 3) Add enzyme-labeled secondary antibody, add 100 μL of porcine enzyme-labeled antibody diluted 1:10000 with PBS buffer to each well and incubate for 75 minutes; 4) Add color developing solution, color development for 15 min, and add stop solution to terminate the reaction; 5) Measure OD using an ELISA reader 450 When the OD450 value of the sample is greater than 0.240, the sample is judged to be positive.

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