H9n2 subtype aiv multi-copy m2e protein monoclonal antibody, cell line and preparation method and application thereof

Abstract

The application discloses a kind of H9N2 subtype AIV multi-copy M2e protein monoclonal antibody, heavy chain and light chain variable region respectively have the amino acid sequence of sequence table SEQ.ID.NO.1 and SEQ.ID.NO.2.The related cell line and its preparation method are also established according to this.The preservation number of hybridoma cell strain 4E10-A10 of the application is CCTCC NO:C202328, and test shows that the anti-multi-copy M2e protein monoclonal antibody secreted by it has high specificity, sensitivity, can specifically recognize 2M2e, 3M2e and 4M2e recombinant protein and the cell expressing H9N2 subtype avian influenza virus 3M2e protein, and is suitable for indirect immunofluorescence detection and Western-blot identification.In conclusion, the application provides material and technical support for the mechanism research of AIV M2e monoclonal antibody to AIV and the application effect evaluation of broad-spectrum vaccine taking AIV M2e protein as target.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of H9N2 subtype AIV, and particularly relates to an H9N2 subtype AIV multi-copy M2e protein monoclonal antibody, a cell line and a preparation method and application thereof. BACKGROUND

[0002] Influenza virus (IV) belongs to Orthomyxoviridae, which contains four genera, namely Influenza A virus (IAV), Influenza B virus (IBV), Influenza C virus (ICV) and Influenza D virus (IDV). Among them, the susceptible hosts of IAV are the most, including humans, birds, pigs, horses, minks and bats. Avian influenza virus (AIV) belongs to Influenza A, and according to the antigenic differences of Hemagglutinin (HA) protein and Neuraminidase (NA) protein on the AIV particle, AIV can be divided into 16 HA subtypes and 9 NA subtypes. According to the strength of pathogenicity to poultry, AIV can be divided into high pathogenic AIV (HPAIV) and low pathogenic AIV (LPAIV). HPAIV is limited to some subtypes of H5 and H7 subtypes (mainly represented by H5N1 and H7N7 subtype AIV), and LPAIV covers the remaining subtypes of AIV. H7N9, H7N4, H7N7, H5N1, H5N8, H5N6, H6N1, H9N2 and H10N3 subtype AIV have been reported to be able to infect humans, so AIV is considered to have a potential threat to human health.

[0003] AIV is a single-stranded negative-sense RNA virus, and its genome is composed of eight non-contiguous gene segments, namely hemagglutinin (HA), nucleoprotein (NP), neuraminidase (NA), polymerase B2 (PB2), polymerase B1 (PB1), polymerase A (PA), matrix protein (M) and non-structural protein (NS); the ectodomain of M2 protein (M2e) is very conserved in influenza A virus, and is considered as a candidate target gene for developing broad-spectrum influenza A vaccine. Studies have shown that the immunization of experimental animals with the protein coupled with multiple copies of M2e or other proteins (such as NP protein, NA protein and HA protein) or combined with carriers (hepatitis B core particles, human papilloma virus L protein, extracellular membrane complex and flagellin) can improve the immunogenicity of M2e protein, and the antibodies produced by immunization can protect the immunized animals from the attack of different subtypes of influenza A virus. Studies have shown that the 10th-20th amino acids of the M2e protein of human, avian and swine influenza viruses are different, which are P I RN E W G C R C N (people), P T RN G W E C K C S (avians) and P I RN G W E C R C N (swine), and the monoclonal antibody specifically recognizing the M2e protein of human influenza virus can only weakly recognize the M2e protein derived from the avian influenza virus strain. The H9N2 subtype AIV is currently the most widely distributed low pathogenic AIV in poultry, and studies have also shown that the H9N2 subtype AIV has a higher isolation rate in live poultry markets in China. Therefore, it is necessary to carry out research on H9N2 subtype AIV to detect, prevent and treat it. SUMMARY

[0004] The technical problem to be solved by the present application is to provide a H9N2 subtype AIV multiple copy M2e protein monoclonal antibody, a cell line and a preparation method and application thereof, so as to provide material and technical support for the medical application of H9N2 subtype AIV.

[0005] To solve the above technical problems, the present application adopts the following technical solutions:

[0006] The H9N2 subtype AIV multiple copy M2e protein monoclonal antibody has the amino acid sequences of SEQ.ID.NO.1 and SEQ.ID.NO.2 in the variable regions of heavy chain and light chain, respectively.

[0007] The heavy chain subtype is IgG1, and the light chain subtype is Kappa.

[0008] Application of the monoclonal antibody in specific recognition of the multi-copy M2e protein of the H9N2 subtype AIV.

[0009] The multi-copy M2e protein is 2M2e, 3M2e or 4M2e protein expressed in prokaryotes, or 3M2e protein expressed in sf9 cells.

[0010] Application of the monoclonal antibody in biological diagnosis identification.

[0011] The biological diagnosis is indirect immunofluorescence detection or Western-blot identification.

[0012] A cell line secreting the monoclonal antibody.

[0013] The cell line is hybridoma cell line 4E10-A10 with the preservation number CCTCC NO: C202328.

[0014] The preparation method of the hybridoma cell line, in which a H9N2 subtype AIV 3M2e recombinant protein is used as an antigen to immunize a mouse, and spleen cells of the mouse after immunization are collected and fused with myeloma cells to obtain fused cells; the fused cells are cultured in a culture medium containing HAT, and the cell line with stable growth state, high and stable secretion of the monoclonal antibody is screened by indirect ELISA.

[0015] In view of the problems in the current research on the H9N2 subtype AIV, the inventor develops a monoclonal antibody of the multi-copy M2e protein of the H9N2 subtype AIV on the basis of the previous research, and the heavy chain and light chain variable regions have the amino acid sequences of SEQ.ID.NO.1 and SEQ.ID.NO.2 respectively. Accordingly, a related cell line and a preparation method thereof are established. The preservation number of the hybridoma cell line 4E10-A10 is CCTCC NO: C202328, and experiments show that the monoclonal antibody of the multi-copy M2e protein secreted by the hybridoma cell line has high specificity and sensitivity, and can specifically recognize 2M2e, 3M2e and 4M2e recombinant proteins and cells expressing the 3M2e protein of the H9N2 subtype avian influenza virus, and is suitable for indirect immunofluorescence detection and Western-blot identification. In summary, the present application provides material and technical support for the mechanism research of the AIV M2e monoclonal antibody on the AIV and the application effect evaluation of the broad-spectrum vaccine taking the AIV M2e protein as a target. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1Figure 3 is the result of SDS-PAGE of the recombinant protein 3M2e, in which: M. 130 kD protein Marker; 1. pGEX-6p-1 empty vector; 2. uninduced pGEX-3M2e; 3. induced pGEX-3M2e; 4-6. purified 3M2e recombinant protein.

[0017] Figure 2 Figure 4 is the result of SDS-PAGE of mouse ascites, in which: 1: 4E10-A10 ascites, 2: M: 130 kD protein Marker.

[0018] Figure 3 Figure 5 is the result of subtype identification of 4E10-A10 monoclonal antibody.

[0019] Figure 4 Figure 6 is the result of Western-blot identification of monoclonal antibody and recombinant protein, in which: M: 130 kD protein Marker; 1: empty vector control; 2: 2M2e recombinant protein; 3: 3M2e recombinant protein; 4: 4M2e recombinant protein.

[0020] Figure 5 Figure 7 is the result of indirect immunofluorescence identification of 4E10-A10 monoclonal antibody (10x).

[0021] Deposit information:

[0022] Hybridoma cell line 4E10-A10 (Hybridoma cell line 4E10-A10), with preservation number CCTCC NO: C202328, preservation date March 1, 2023, preservation address Wuhan, Wuhan University, postcode 430072, and preservation unit China Center for Type Culture Collection.

[0023] Preservation condition: liquid nitrogen, long-term preservation DETAILED DESCRIPTION

[0024] 1. Materials and methods

[0025] 1.1 Plasmid, cell and test animal

[0026] Recombinant plasmids pGEX-3M2e, pFastBacDual-3M2e (not published) containing 3 copies of M2e protein (M2e amino acid sequence: SEQ. ID. NO. 3) and mouse myeloma cell SP2 / 0 were preserved by the Biotechnology Lab of Guangxi Institute for Animal Health; 8-week-old SPF BALB / c mice were purchased from Changsha Tianqing Biotechnology Co., Ltd. with production license number Scxk (Xiang) 2019-0013.

[0027] 1.2 Main reagents

[0028] 130 kD Protein Marker was purchased from Beijing Solabio Biotechnology Co., Ltd.; BAC Protein Quantitative Kit, BCIP / NBT Alkaline Phosphatase Color Reagent Kit, Alkaline Phosphatase (AP) Labeled Goat Anti-Mouse IgG (H+L), GST Tag Protein Purification Kit and Western Blocking Solution were purchased from Shanghai Biyun Tian Biotechnology Co., Ltd.; HT, HAT culture medium additives, 50% PEG solution and mouse MAb subclass identification kit were purchased from SIGMA company; DMEM / F-12 cell culture medium was purchased from Gibco company; BL21 (DE3) competent cells were purchased from Beijing Quanshi Gold Biotechnology Co., Ltd.

[0029] 1.3 Expression and purification of AIV 3M2e recombinant protein

[0030] The pGEX-3M2e plasmid was transformed into BL21 (DE3) competent cells, and positive colonies were picked and sent to ShangHai Genechem Co., Ltd. for sequencing. 10 μL of recombinant strain pGEX-3M2e was inoculated into 10 mL of ordinary broth medium and cultured at 37℃ until the OD600 was 0.4-0.5. Then 0.5 mmol / L of IPTG was added and induced at 37℃ for 4 h. After that, 10 mL of bacterial solution was centrifuged at 12000 r / min for 5 min, and the bacterial pellet was rinsed with phosphate buffered saline (PBS) for 3 times. Then the recombinant protein was purified using GST tag protein purification kit, and 40 μL of purified recombinant protein was subjected to SDS-PAGE electrophoresis.

[0031] The purified 3M2e recombinant protein concentration was determined using the BAC protein quantification kit, and 8-week-old BALB / c mice were immunized a total of 4 times (the first 3 times were subcutaneous multiple point injection, and the fourth time was intraperitoneal injection), with an interval of 21 days between two immunizations: the first immunization mixed and emulsified complete Freund's adjuvant and the same volume of 3M2e recombinant protein (100 μg per mouse), and the second and third immunizations replaced complete Freund's adjuvant with incomplete Freund's adjuvant with the same protein amount and injection method; the fourth immunization was intraperitoneal injection of 300 μg of unadjuvant recombinant protein per mouse. Cell fusion and culture were performed according to the steps in Document Three (Deng X W, Luo S S, Xie Z X, et al. Development of H4 subtype avian influenza hemagglutinin monoclonal antibody and establishment of sandwich ELISA [J]. Journal of Cell and Molecular Immunology, 2020, 36(01): 69-74.). When the area of the fused cell colony exceeded 1 / 10 of the culture well, 3M2e recombinant protein was used as the detection antigen, and the positive hybridoma cell strain secreting antibody was screened according to the method in Document Four (Wang P, Wang W K, Liang G C, et al. Development of monoclonal antibody against serotype 4 fowl adenovirus fiber protein 2 and its partial property research [J]. Chinese Poultry, 2017, 39(19): 27-31). After obtaining the positive hybridoma cell strain, the preparation of ascites was performed according to the steps in Document Five (Li J X, Sun W Q, Yuan S Q, et al. Preparation and biological property analysis of H9N2 subtype avian influenza virus hemagglutinin monoclonal antibody [J]. Chinese Poultry, 2017, 39(21): 19-23.).

[0032] 1.5 Monoclonal antibody ascites titer determination and subtype identification

[0033] The antibody titer of ascites was determined by the steps of indirect ELISA antibody titer determination in Document Three, and the results were determined by measuring the OD450 value. When the OD value (P value) of the test sample was greater than 2.1, the negative control serum (N value), it was determined to be positive. The ascites obtained in the method of 1.4 was diluted to 1:1000, and the obtained monoclonal antibody was identified according to the instructions of the mouse MAb subclass identification kit.

[0034] 1.6 Monoclonal antibody specificity identification

[0035] Western-blot identification of 3M2e expressed by E. coli prokaryotic expression system in 1.3 and 2M2e (protein size about 31.7kD) and 4M2e protein (protein size about 37.2kD) preserved in the laboratory (Reference 1: Zhang, Xie, Huang, et al. H9N2 subtype avian influenza virus M2e protein in prokaryotic expression system [J]. Animal Husbandry Progress, 2017, 38(4): 7-12.). First, the purified 2M2e, 3M2e and 4M2e recombinant proteins were subjected to SDS-PAGE electrophoresis, and the method in Reference 1 was used for membrane transfer and blocking. The monoclonal antibody prepared in 1.4 was used as the primary antibody (1:1000) for 37°C incubation for 1h, and the AP-labeled goat anti-mouse IgG (1:2000) was used as the secondary antibody for 37°C incubation for 1h. Finally, the BCIP / NBT alkaline phosphatase color developing kit was used for color development.

[0036] Indirect immunofluorescence (IFA) test identified 3M2e protein expressed by Bac-to-Bac baculovirus expression system. First, Sf9 cells were infected with baculovirus expressing 3M2e protein, and after 72h, the supernatant was discarded, 4% paraformaldehyde was used for room temperature fixation for 30min, and after PBS washing, 5% skimmed milk powder was used for 37°C blocking for 1h; PBS was washed for 3 times, the monoclonal antibody prepared in 1.4 was used as the primary antibody (1:1000) for 37°C incubation for 1h; PBS was washed for 3 times, FITC-goat anti-mouse IgG antibody (1:500) was added for 37°C incubation for 1h; after washing with PBST, it was placed under a fluorescence microscope for observation and photography.

[0037] 2Results

[0038] 2.1 Expression and purification of H9N2 subtype AIV 3M2e recombinant protein

[0039] After the recombinant strain was induced by 0.5mmol / L IPTG at 37°C for 4h, 3M2e recombinant protein was purified, and the SDS-PAGE electrophoresis detection result showed that pGEX-3M2e expressed about 34.4kD protein, which was consistent with the expected size Figure 1 ).

[0040] 2.2 Screening of 3M2e hybridoma cell strain and preparation of ascites

[0041] After the spleen cells of immunized mice and SP2 / 0 cells were fused, the hybridoma cells were subjected to 3 times of subcloning by limiting dilution method and screening by indirect ELISA method, and one hybridoma cell strain stably secreting antibodies was screened and named 4E10-A10. The obtained hybridoma cell strain was injected into the abdominal cavity of mice, and after collecting the ascites, SDS-PAGE electrophoresis detection was performed on the obtained ascites, and the result showed that the size of the heavy chain of the antibody was about 55kD, and the size of the light chain was about 25kD Figure 2 ).

[0042] 2.3 Titration and subtype identification of 3M2e monoclonal antibody

[0043] After ascites were obtained, the titers of 4E10-A10 monoclonal antibody were determined by indirect ELISA method, and the results showed that the antibody titers of 4E10-A10 ascites were all 1: 1000000, as shown in Table 1.

[0044] Table 1 Results of monoclonal antibody titer determination

[0045]

[0046] Note: "P / N" means "sample OD 450 value / negative control OD 450 value"

[0047] The obtained monoclonal antibody was identified for subtype, and the results are shown in Figure 3 that the heavy chain subtype of 4E10-A10 monoclonal antibody belongs to IgG1, and the light chain subtype is Kappa.

[0048] 2.4 Specificity identification of monoclonal antibody

[0049] Western-blot identification of 2M2e, 3M2e and 4M2e recombinant proteins was performed using 4E10-A10 monoclonal antibody as primary antibody, and the results showed that the monoclonal antibody 4E10-A10 could specifically react with 2M2e, 3M2e and 4M2e recombinant proteins Figure 4 ). Indirect immunofluorescence was performed on Sf9 cells expressing 3M2e protein using 4E10-A10 monoclonal antibody as primary antibody, and the results showed that the three monoclonal antibodies could specifically bind to Sf9 cells expressing 3M2e protein, showing bright green fluorescence Figure 5 ).

[0050] 3 DISCUSSION

[0051] The development strategy of avian influenza vaccine is to select HA and NA proteins with matching antigenicity according to the current avian influenza virus (H5, H7 and H9 subtype AIV) epidemic strain subtypes, replace the HA and NA genes of the avian influenza virus vaccine strain constructed by reverse genetic technology, and then construct a suitable avian influenza vaccine. However, when the HA gene of avian influenza virus mutates rapidly or a new subtype AIV pandemic occurs, the current vaccine may fail.

[0052] In view of this situation, researchers have carried out a large number of studies on universal vaccines for influenza viruses. Multiple attempts and designs have been made to prepare universal vaccines for influenza A virus based on M2e protein, and a large amount of research data has been obtained. Kim et al. immunized mice with virus-like particles displaying NA protein of human influenza virus H1N1 and H3N2 subtypes and 5M2e protein, and the results showed that the immunized mice could resist the attack of H1N1, rH5N1, H3N2, H9N2 and rH7N9 subtype influenza viruses. Yao et al. chemically coupled HA protein, NP protein and 4M2e protein of human influenza virus H1N1 and H3N2 subtypes to form double-layer protein nanoparticles, and the nanoparticles showed a broad cross-protection effect against the attack of H1N1, H5N1, H3N2 and H7N9 subtype human influenza viruses. Hajam et al. prepared an mRNA vaccine containing H9N2 subtype HA2 protein and 4M2e protein, and the antibodies produced after immunization could effectively protect chickens from the attack of H7N9 and H9N2 subtype AIV. The above data all show that multiple copy M2e protein has good cross-protection ability when used in combination with other proteins of influenza virus. Monoclonal antibodies are a kind of immunoglobulin, which has the advantages of high specificity, high titer and large-scale production. Monoclonal antibodies have important significance in the research of virus prevention, virus infection mechanism and pathogen diagnosis, and are often used as specific detection tools for pathogen proteins and capture antibodies for pathogen sandwich ELISA detection methods.

[0053] Escherichia coli prokaryotic expression system is the most widely used protein expression system at present, which has the advantages of low expression cost, high expression amount and simple operation. Multiple studies have reported that indirect ELISA detection methods are established by using influenza A virus proteins (HA, HA1, NP) expressed by Escherichia coli prokaryotic system to detect the antibody titer of the target protein in the body. The applicant successfully expressed and purified 3M2e protein by using Escherichia coli prokaryotic expression system, which provides biological materials for evaluating the effect of vaccines based on M2e protein. The inventors immunized mice with purified 3M2e protein, and obtained a hybridoma cell strain secreting antibodies against multiple copy M2e protein by monoclonal antibody preparation technology, which was named 4E10-A10. The hybridoma cell strain was injected into mice to obtain corresponding ascites, and the titer of the ascites was 1:1000000. Western-blot identification results showed that 4E10-A10 could specifically react with 2M2e, 3M2e and 4M2e proteins. IFA results showed that 4E10-A10 could specifically react with 3M2e expressed in sf9 cells, and the cells showed specific green fluorescence, indicating that 4E10-A10 monoclonal antibody had good specificity. The subtype identification of the three monoclonal antibodies showed that the heavy chain subtypes of the three monoclonal antibodies were IgG1.

[0054] In summary, the prepared H9N2 subtype AIV multi-copy M2e monoclonal antibody has a higher titer, which can specifically recognize the 2M2e, 3M2e and 4M2e proteins expressed in prokaryotic cells, and can also specifically recognize the 3M2e protein expressed in sf9 cells. Therefore, the purified recombinant protein 3M2e and the screened monoclonal antibody lay a foundation for subsequent evaluation of the effect of the AIV vaccine using the multi-copy M2e protein as an immunogen.

Claims

1. A monoclonal antibody against H9N2 subtype AIV multicopy M2e protein, characterized in that... The variable regions of the heavy and light chains have the amino acid sequences listed in SEQ.ID.NO.1 and SEQ.ID.NO.2, respectively.

2. The monoclonal antibody according to claim 1, characterized in that: The heavy chain subtype belongs to IgG1, and the light chain subtype is Kappa.

3. The non-diagnostic application of the monoclonal antibody of claim 1 in the specific detection of multiple copies of the M2e protein of H9N2 subtype AIV.

4. The application according to claim 3, characterized in that: The multiple copies of M2e protein are 2M2e, 3M2e, and 4M2e proteins expressed in prokaryotes.

5. The application according to claim 4, characterized in that: The multi-copy M2e protein is the 3M2e protein expressed in sf9 cells.

6. The non-diagnostic application of the monoclonal antibody of claim 1 in the bioidentification of H9N2 subtype AIV.

7. The application according to claim 6, characterized in that: The bioassays are indirect immunofluorescence assay and Western blot assay.

8. A cell line that secretes the monoclonal antibody of claim 1, characterized in that... The hybridoma cell line 4E10-A10 is the one with the accession number CCTCC NO: C202328.

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