Preparation and application of antigenic epitope and antibody of porcine reproductive and respiratory syndrome virus helicase

Abstract

The present invention relates to an antigen epitope targeting PRRSV helicase, a method for preparing a monoclonal antibody and its application. The prokaryotic expression vector pCold-I-Nsp10 is constructed, recombinant Nsp10 is expressed in Escherichia coli, Balb / c mice are immunized after purification, and after four immunizations, the mouse spleen cells are fused with myeloma SP2 / 0 cells to prepare hybridoma cells, and then subcloned by an indirect ELISA method to obtain a monoclonal antibody against PRRSV Nsp10, and the antigen epitope corresponding to the antibody is identified as 425 TDKRVVDSLR 434 .

Description

Technical Field

[0001] The present invention relates to the field of biology, and specifically provides an antigenic epitope of a PRRSV helicase, a method for preparing a monoclonal antibody and applications thereof. Background Art

[0002] Porcine reproductive and respiratory syndrome (PRRS), commonly known as "blue ear disease", is a contact infectious disease caused by porcine reproductive and respiratory syndrome virus (PRRS virus, PRRSV). The main symptoms are reproductive disorders in pregnant sows and respiratory diseases in pigs of all ages. In 1996, PRRSV was isolated for the first time in my country. Currently, strains from multiple sources are mixed and prevalent in clinical practice, and recombination between strains may occur, as well as mixed secondary infections with other viral and bacterial pathogens. In view of the complex and severe epidemic situation of the virus, the preparation of monoclonal antibodies against important proteins encoded by the virus and related to replication and transcription will provide an important experimental tool for conducting basic and applied research on PRRSV.

[0003] Helicase (Nsp10) is relatively conservative in evolution and is an important component of the PRRSV replication-transcription complex (RTC). It plays an important role in all cellular processes involving nucleic acids and is crucial for viral RNA synthesis, making Nsp10 an important target for drug development. PRRSV helicase belongs to the UPF1-like family helicase in the SF1 superfamily. It can unwind double-stranded DNA and double-stranded RNA in the direction of 5′→3′ and has ATPase activity. Upf1-like family helicases all have two very conserved domains: the N-terminal cysteine-anthocyanin-rich domain (zinc finger domain, ZBD), 1B domain, and C-terminal domain (CTD). CTD contains two RecA-like folds, namely 1A and 2A subdomains, which constitute the helicase core. PRRSV helicase has conserved domains, and its key domains also have unique spatial conformations.

[0004] Helicase plays a vital role in PRRSV infection and pathogenicity: First, it can exert its own helicase activity, open specific RNA structures during viral transcription and replication, and directly promote the synthesis of viral genomic RNA; second, it can guide the variation of viral replication efficiency and virulence through mutations in its own key amino acid domains and key amino acid sites, regulate important biological phenotypes such as viral pathogenicity, transmission ability, and immune escape, and facilitate its own prevalence and spread; third, it can participate in regulating the host immune response, induce the activation of important immune molecules, participate in the formation of immunosuppression and the polarization phenotype of macrophages, and promote self-infection. In addition, as one of the most conservative viral non-structural proteins, PRRSV helicase can be used as a good candidate molecule for diagnostic markers.

[0005] The present invention successfully obtained a monoclonal antibody against PRRSV helicase Nsp10 by cell fusion and subclone screening, and identified its antigenic epitope, and found that it can recognize the 425-434 peptide segment of Nsp10, and its amino acid sequence is ( 425 TDKRVVDSLR 434 ). Software analysis revealed that the antigen epitope is highly conserved in PRRSV-2. The monoclonal antibody has strong specificity and can differentiate and diagnose PRRSV of different genotypes, providing a valuable tool for future basic and applied research on PRRSV. Summary of the invention

[0006] In view of the gaps in the prior art, the present invention provides a monoclonal antibody of PRRSV helicase and a preparation method and application thereof. The technical scheme of this method is: constructing a prokaryotic expression vector by cloning the PRRSV Nsp10 gene into the pCold-I expression vector, purifying the protein by eluting with different concentrations of imidazole, immunizing Balb / c mice with the purified Nsp10 as an antigen, fusing the mouse spleen cells with myeloma cells SP2 / 0 cells to prepare hybridoma cells, coating plates with purified Nsp10, and screening positive hybridoma cells that can specifically recognize Nsp10 by indirect ELISA.

[0007] The specific invention is: a monoclonal antibody that can specifically recognize PRRSV Nsp10 was obtained. It can be used for IFA and Western blotting detection, and can well recognize Nsp10. Subtype identification shows that the heavy chain of the monoclonal antibody is IgG1 and the light chain is a Kappa chain. By constructing truncated Nsp10 and synthesizing a series of Nsp10 short peptides, it was finally determined that the monoclonal antibody can specifically recognize the 425-434 peptide segment of PRRSV Nsp10, and its amino acid sequence is 425 TDKRVVDSLR 434, as shown in SEQ ID NO.1. BRIEF DESCRIPTION OF THE DRAWINGS

[0008] Figure 1 This is the electrophoresis diagram of the PCR amplification of the gene sequence fragment encoding PRRSV Nsp10 in Example 1 of the present invention. Among them, the first lane is the DNA standard DL2000, and the second lane is the PRRSV Nsp10 gene fragment, which is about 1323 bp in size, which is in line with expectations, indicating that the fragment of the correct size has been successfully amplified.

[0009] Figure 2 It is the Coomassie brilliant blue staining and purified protein Western blotting identification diagram of Nsp10 induced expression in Example 2. Among them, lane M is the protein molecular mass standard, lane 1 is the pCold-I empty vector induction control, lane 2 is the whole bacteria after pCold-I induction, lane 3 is the whole bacteria before pCold-I-Nsp10 induction, lane 4 is the whole bacteria after pCold-I-Nsp10 induction, lane 5 is the supernatant after pCold-I-Nsp10 induction, and lane 6 is the precipitate after pCold-I-Nsp10 induction. It shows that the expression level of recombinant Nsp10 in the supernatant is high and can be used to purify antigen protein. After elution with different gradient imidazoles, lane 7 is the recombinant protein eluted with 200mM imidazole, lane 8 is the recombinant protein eluted with 400mM imidazole, lane 9 is the recombinant protein eluted with 200mM imidazole detected by His tag antibody, and lane 10 is the recombinant protein eluted with 400mM imidazole detected by His tag antibody. This shows that the purification effect of the recombinant Nsp10 antigen protein is good, the band is single, and the size is about 48.5kDa, which is in line with expectations and can react specifically with the His tag, indicating that the purified protein is correct and can be used for later immunization.

[0010] Figure 3 This is an IFA image of the MARC-145 cells infected with PRRSV vHuN4 strain using the 10F41 monoclonal antibody in Example 5, indicating that the antibody can specifically recognize PRRSV Nsp10.

[0011] Figure 4 This is a Western blotting identification diagram of the 10F41 monoclonal antibody used to detect PRRSV vHuN4 strain infected MARC-145 cells at different times in Example 5, indicating that the antibody can specifically recognize PRRSV Nsp10, and the expression level of Nsp10 gradually increases at 12h, 24h, and 36h after PRRSV infection.

[0012] Figure 5 This is the subtype detection result of the 10F41 monoclonal antibody in Example 5, indicating that the heavy chain of the antibody is IgG1 and the light chain is Kappa. Figure 6 This is a Western blotting identification diagram of the antigen epitope recognized by the 10F41 monoclonal antibody in the preliminary segmented screening in Example 6, indicating that the truncated Nsp10 fragments recognized by the antibody all contain 425 TDKRVVDSLR 434 Peptide fragment.

[0013] Figure 7 This is the ELISA identification diagram of the minimum antigen epitope in Example 6, indicating that the minimum epitope recognized by the 10F41 monoclonal antibody is 425 TDKRVVDSLR 434 Peptide fragment.

[0014] Figure 8 The IFA, Western blotting identification diagram and amino acid sequence alignment results of using the 10F41 monoclonal antibody to detect multiple strains of PRRSV infecting MARC-145 cells respectively in Example 7 indicate that the antibody specifically recognizes PRRSV-2. DETAILED DESCRIPTION

[0015] In order to make the purpose, technical scheme and advantages of the present invention more clearly understood, the present invention is further described below in conjunction with specific embodiments. It should be understood that the following embodiments are only used to illustrate the present invention and are not used to limit the scope of the present invention.

[0016] Example 1 Construction of the recombinant prokaryotic expression vector pCold-I-Nsp10

[0017] The inventors first constructed the pCold-I-Nsp10 prokaryotic expression plasmid and designed the primers as follows:

[0018] Nsp10-F: 5′-GGGGTACCGGGAAGAAGTCCAGAATGTG-3′ (SEQ ID NO. 2)

[0019] Nsp10-R: 5′-CGGAATTCTTCCAGGTCTGCGCAAATAG-3′ (SEQ ID NO.3)

[0020] The above primers were all pCold-I-Nsp10 specific sequences after BLAST analysis, and the primers were synthesized by Shanghai Qingke Biotechnology Co., Ltd. The template was the gene sequence encoding Nsp10 of the PRRSV vHuN4 strain (GenBank: EF635006.1) in NCBI, and was connected to the pCold-I vector. After sequencing, it was completely consistent with the PRRSV Nsp10 gene sequence and named pCold-I-Nsp10. The specific process is as follows:

[0021] The PCR reaction conditions were as follows: pre-denaturation at 95°C for 30s; denaturation at 95°C for 10s; annealing at 65°C for 30s; extension at 72°C for 2min, with 30 cycles from denaturation to extension; final extension at 72°C for 10min. After electrophoresis, the samples were observed under UV light and photographed. The synthesized plasmids were identified by PCR using the designed primers. After electrophoresis, the specific bands of about 1323bp were amplified ( Figure 1 ), the results are consistent with expectations.

[0022] Example 2 Induced expression and purification of the recombinant prokaryotic expression vector pCold-I-Nsp10

[0023] Transform BL21 competent cells with pCold-I-Nsp10 and pCold-I empty vectors, evenly spread on LB solid agar medium with ampicillin resistance, and invert incubator at 37°C. Randomly pick a single colony and inoculate it in LB liquid medium with ampicillin resistance, and shake the bacteria in a 37°C constant temperature shaker overnight. The next day, take 1 mL of bacterial solution and add it to two new tubes of LB liquid medium with ampicillin resistance, and shake and culture in a 37°C constant temperature shaker. When the bacterial solution OD 600nm When it is about 0.6-0.8, add IPTG to one of the tubes as the induction group. The other group is not treated and is the control group. Except for not adding the inducer IPTG, the other conditions of the control group are the same as those of the induction group. Low-temperature induction at 16°C for 16 hours. Take 1mL of bacterial solution from the induction group and the control group, centrifuge and collect and process the bacteria, and perform SDS-PAGE electrophoresis identification. According to the results, analyze the induced expression of plasmid pCold-I-Nsp10. After successful identification, centrifuge the remaining bacterial solution of the induction group at high speed, collect the bacteria, and then perform ultrasound. Then perform SDS-PAGE electrophoresis to analyze whether the recombinant Nsp10 is expressed in the supernatant or the inclusion body. Figure 2 As shown, recombinant Nsp10 is mainly expressed in the supernatant. The recombinant protein was purified using QIAGEN's Ni-NTA Agarose. The general process is: collect a large amount of bacterial liquid expressing Nsp10 with a His tag, wash it twice with sterile PBS, then use an ultrasonic disruptor to break it, centrifuge it at high speed, and collect the supernatant. Combine the supernatant with the pre-treated Ni-NTA Agarose at 4°C overnight. Then wash the Ni-NTA Agarose with a washing solution containing 30mM, 50mM, 100mM, 200mM and 400mM imidazole, collect the eluate of 200mM and 400mM imidazole, perform SDS-PAGE gel electrophoresis, and observe the purification effect of the recombinant protein. After SDS-PAGE electrophoresis, the purified protein was transferred to the NC membrane for Western blotting experiments to identify the reactivity of the purified protein with the His tag ( Figure 2 ).

[0024] Example 3 Animal Immunization

[0025] Five 4-week-old female Balb / C mice were immunized with the recombinant Nsp10 purified antigen protein. Another group of 5 mice was used as a negative control. Before immunization, the concentration of the purified protein was determined. After 100 μg of purified protein was emulsified with an equal volume of Freund's complete adjuvant, the mice were immunized by subcutaneous injection on the back. Two weeks later, the protein was emulsified with Freund's incomplete adjuvant and injected into the mice in the same way. Four weeks later, the third immunization was performed. The method is similar to the second immunization. Three days after the third immunization, the mice were tail-cut and blood was collected, and the serum antibody titer was determined by ELISA. After both test results met the standard, a booster immunization was performed.

[0026] Example 4 Preparation of PRRSV Nsp10 Monoclonal Antibody

[0027] (1) Recovery and culture of SP2 / 0 cells

[0028] About 5 days before fusion, resuscitate SP2 / 0 cells and adjust the cell density. Specific method: Take out the SP2 / 0 frozen cells from liquid nitrogen, put them in a sealed bag, quickly put them in a 37°C water bath, and shake them to thaw. After complete thawing, centrifuge horizontally at 1000rpm for 5 minutes, aspirate and discard the supernatant in the biosafety cabinet, blow and mix the cell clusters with 1mL of culture medium, spread them flat in the cell culture dish, add culture medium to 10mL, culture at 37°C until the cells adhere to the wall, and replace with fresh culture medium.

[0029] (2) Preparation of feeder cells

[0030] One day before fusion, the control group mice were blooded by eye sampling to obtain serum as a negative control, and the mice were killed and disinfected by soaking in 75% alcohol. In the biosafety cabinet, the mouse abdomen was fixed on a sterilized foam board with the abdomen facing up. After the abdominal skin was clamped with tweezers, a small hole was cut in the skin at the fat layer below the mouse abdomen. The skin was torn open from the small hole with tweezers and scissors to expose the abdominal cavity. 5mL DMEM culture medium was injected into the abdominal cavity. Use tweezers to carefully tap both sides of the peritoneum to suspend the mouse peritoneal macrophages in the culture medium. After several taps, the culture medium was aspirated. Repeat this step and divide the aspirated culture medium into 96-well plates, 100μL per well. Place in a 37℃, 5% CO2 incubator for culture. The next day, observe the growth status of the feeder layer cells. If the status is good, fusion can be performed.

[0031] (3) Preparation of spleen cells

[0032] Blood was collected from the eyeballs of the mice in the immunized group as a positive control, and the mice were killed. The killed mice were soaked in 75% alcohol for 5 minutes, the mice were fixed, and the abdominal skin of the mice was gently cut open with sterilized scissors. The spleen of the immunized mice was removed under sterile conditions and placed in a culture dish. A 40μm cell sieve was placed on a 50ml centrifuge tube, and the spleen was ground with a disposable grinding rod. DMEM was added to rinse the spleen, and the grinding liquid was collected and centrifuged at 1000rpm for 5 minutes.

[0033] (4) Fusion of hybridoma cells

[0034] The SP2 / 0 myeloma cell suspension and immune spleen cell suspension were mixed in a 50ml centrifuge tube at a ratio of 1:3, and DMEM was added to 30ml. After sufficient mixing, the cells were centrifuged at 1000rpm for 10min, the supernatant was discarded, and the bottom of the tube was tapped to disperse the cell precipitate into a paste. The centrifuge tube containing spleen cells and SP2 / 0 cells was placed in a foam box filled with 37℃ warm water. While rotating the centrifuge tube, 1ml of PEG2000 preheated in a 37℃ incubator was slowly dripped in. After adding within 1min, it was allowed to stand for 2min. Then DMEM basal culture medium was slowly dripped to dilute PEG to terminate fusion, and centrifuged at 1000rpm for 10min. The cells were resuspended in 37℃ preheated HAT culture medium containing 20% ​​serum, and added to a 96-well cell culture plate with feeder cells at a volume of 100μL / well, and cultured in a 37℃, 5% CO2 incubator. After fusion for 5 days, the cell fusion was observed and the HAT culture medium was appropriately supplemented. Ten days after fusion, 50 μL of cell culture supernatant was carefully aspirated with a pipette, and the positive hybridoma cell wells that could secrete antibodies were screened by the indirect ELISA method.

[0035] (5) Screening of positive hybridoma cells

[0036] The supernatant of fused cells was used as the primary antibody, the supernatant of SP2 / 0 cells was used as the negative control, the mouse serum before fusion was used as the positive control, and the HRP-labeled goat anti-mouse IgG was used as the secondary antibody. The specific implementation method is as follows: the ELISA plate was coated with purified nsp10 protein, the coating amount per well was 200 ng / well, 100 μL per well, and the plate was coated overnight at 4°C. 200 μL of 5% skim milk was added to each well for blocking at 37°C for 2 hours, and after washing 3 times with PBST containing 5‰ Tween-20, 100 μL of cell supernatant was added to the protein-coated plate and mixed, and after incubation at 37°C for 1 hour, the plate was washed 3 times with PBST containing 5‰ Tween-20, each time for 5 minutes. Then 100 μL of secondary antibody diluted at a ratio of 1:6000 was added, and after incubation at 37°C for 30 minutes, the plate was washed 3 times with PBST containing 5‰ Tween-20. Add 100 μL TMB colorimetric solution to each well and incubate at room temperature for 15 min in the dark. Finally, add 50 μL 2M H2SO4 to each well to terminate the reaction and measure the OD with an enzyme-labeled instrument. 450 The positive cell well with the highest OD value was selected for expansion culture, and then subcloned three times, and each subclone was screened by the above-mentioned indirect ELISA method. The hybridoma cell line 10F41 that stably secreted nsp10 antibodies was selected for expansion culture and ascites was prepared.

[0037] (6) Preparation of ascites

[0038] 7 days in advance, 8-week-old Balb / c female mice were intraperitoneally injected with incomplete Freund's adjuvant, 0.2 mL per mouse. One week later, the hybridoma cells that were growing well were blown up and transferred into a 15 mL centrifuge tube, at 1000 rpm for 5 min. The cell pellet was resuspended in PBS and intraperitoneally injected into mice, 10 6 After 7 days, the abdominal cavity of the mice was inflated, and the ascites was collected, centrifuged, and the supernatant was aspirated and frozen at -80°C.

[0039] Example 5: Specificity detection and subtype identification of monoclonal antibodies

[0040] To verify the specificity of the monoclonal antibody, the PRRSV vHuN4 strain was infected with MARC-145 cells, and IFA and Western blotting analysis were performed. The IFA results showed that the 10F41 monoclonal antibody could specifically recognize PRRSV-infected cells and showed green fluorescence, while the control group had no fluorescence ( Figure 3 ). Western blotting results showed that 10F41 monoclonal antibody could recognize a specific protein band with a molecular weight of about 48.5 kDa, and the band was clear ( Figure 4The antibody subtype was further identified using the monoclonal antibody subtype identification kit from Proteintech. The results showed that the heavy chain of the antibody was IgG1 and the light chain was Kappa( Figure 5 ). Subsequently, the collected ascites was purified using Protein G, and the titer of the purified 10F41 monoclonal antibody was detected by ELISA to be 1:256000.

[0041] Example 6 Identification of epitopes recognized by Nsp10 monoclonal antibodies

[0042] First, PRRSV Nsp10 (1-441aa) was divided into three segments (S1: 1-133aa; S2: 113-275aa; S3: 255-441), and expression plasmids were constructed according to the primers in Table 1 (SEQ NO: 4-9). The segmentation schematic diagram is shown in Figure 6 A. The PCR reaction conditions were: pre-denaturation at 95°C for 30s; denaturation at 95°C for 10s; annealing at 65°C for 30s; extension at 72°C for 40s, with 35 cycles from denaturation to extension; final extension at 72°C for 10min. The fragments were connected to the pCold-TF vector respectively, and positive clones were selected after transformation with BL21. Single colonies were selected for verification. After verification, expression was induced. The bacteria were cultured in a 37°C shaker until the OD value reached 0.6-0.8, and 1mM IPTG was added to induce expression. After induction at 16°C for 16h, the bacterial solution was collected for ultrasonication. The results showed that the epitope recognized by the monoclonal antibody 10F41 was located in the S3 region (255-441aa) ( Figure 6 B). The S3 fragment was further truncated into three segments (S3-1: 255-330aa; S3-2: 311-385aa; S3-3: 366-441aa), and amplified and induced expression was identified as described above according to the primers in Table 1 (SEQ NO: 10-15). The results showed that the epitope of the monoclonal antibody 10B14 was located in the S3-3 region (366-441aa) ( Figure 6 C).

[0043] In order to further accurately identify the B cell epitope recognized by the 10F41 monoclonal antibody, 9 different truncated peptides (SEQ ID NO.1 and SEQ ID NO.16-23) were synthesized by Gill Biochemical (Shanghai) Co., Ltd., and the 9 peptides were used to coat the ELISA plate for indirect ELISA identification. The results showed that the minimum epitope of Nsp10 recognized by the 10F41 monoclonal antibody was a peptide segment of amino acids 425-434, and its sequence was 425 TDKRVVDSLR 434 ( Figure 7 ).

[0044] Example 7 Application of Nsp10 monoclonal antibody

[0045] To verify the application range of 10F41 monoclonal antibody, representative strains of PRRSV-2, including highly pathogenic strong strain vHuN4, highly pathogenic weak strain vHuN4-F112, classic strain vAPRRS, NADC30-like strain vZJqz21 and representative strain of PRRSV-1 vSHE, were used to infect MARC-145 cells, and IFA and Western blotting analysis were performed. IFA results showed that 10F41 monoclonal antibody can specifically recognize cells infected with PRRSV-2 (vHuN4, vHuN4-F112, vAPRRS, ZJqz21), showing green fluorescence, but cannot recognize cells infected with PRRSV-1 (vSHE). At the same time, the blank cell control group had no fluorescence ( Figure 8 A). Western blotting results showed that 10F41 monoclonal antibody could recognize PRRSV-2 infected cells and produce a specific protein band of about 48.5 kDa. The band was clear, but it could not recognize PRRSV-1 infected cells ( Figure 8 B) Comparison of the Nsp10 amino acid sequences of the above five representative strains with the Nsp10 amino acid sequences of the NADC34-like and QYYZ-like strains currently prevalent in my country revealed that 425 TDKRVVDSLR 434 The peptides are highly conserved in all PRRSV-2. The above results indicate that the 10F41 monoclonal antibody can specifically recognize PRRSV-2 and can also be used for differential diagnosis of different genotypes of PRRSV.

[0046] Table 1 Primer sequences used in the present invention

[0047]

[0048] In summary, the present invention provides an antigenic epitope targeting PRRSV helicase, a method for preparing a monoclonal antibody and its application. A prokaryotic expression vector capable of expressing PRRSV Nsp10 was constructed by cloning the PRRSV Nsp10 coding sequence into the pCold-I vector, and PRRSV Nsp10 was obtained by purification using a His tag. Balb / c mice were immunized with the purified protein as an immunogen, and monoclonal antibodies against PRRSV Nsp10 were obtained through cell fusion and subclone screening. The identification results showed that the heavy chain of the prepared monoclonal antibody was IgG1 and the light chain was Kappa. The results of IFA and Western blotting analysis showed that the obtained monoclonal antibody could specifically recognize PRRSV Nsp10 and could be used to detect PRRSV Nsp10 antigen under reducing conditions. The epitope sequence recognized by it is 425 TDKRVVDSLR 434 , further demonstrating the specificity of the antibody.

[0049] It should be understood that although the present invention has been described in detail above with general descriptions and specific embodiments, some modifications or improvements can be made on the basis of the present invention, which is obvious to those skilled in the art. Therefore, these modifications or improvements made on the basis of not departing from the spirit of the present invention all belong to the scope of protection claimed by the present invention.

Claims

1. An antigenic epitope peptide of porcine reproductive and respiratory syndrome virus (PRRSV) helicase Nsp10, wherein the antigenic epitope peptide is a 425-434 peptide segment of PRRSV Nsp10, and its amino acid sequence is 425 TDKRVVDSLR 434 .

2. Use of the antigenic epitope peptide according to claim 1 in differential diagnosis or detection of genotype 2 PRRSV, wherein the use is for non-disease diagnosis and treatment purposes.

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