Broad-spectrum monoclonal antibody aiming at avian influenza virus M1 protein and application of broad-spectrum monoclonal antibody

Abstract

The invention belongs to the field of biology, and relates to a broad-spectrum monoclonal antibody aiming at avian influenza virus M1 protein and application of the broad-spectrum monoclonal antibody, the monoclonal antibody has specific reaction with A549 cells infected by avian influenza, has no specific reaction with Newcastle disease virus, duck tembusu virus, goose astrovirus and infectious laryngitis virus, has good specificity, and can be used for preparing the broad-spectrum monoclonal antibody. The 5G9 monoclonal antibody has good reactivity with A549 cells infected by H1-H11 subtype AIV, has good broad spectrum, can be used for detecting M1 protein sub-localization after the AIV infected cells are detected by using the 5G9 monoclonal antibody, and can be used for indicating the infection process of the AIV.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to antibody engineering technology, specifically a broad-spectrum monoclonal antibody that recognizes different subtypes of AIV virus M1 protein and its applications. Background Technology

[0002] Avian influenza virus belongs to influenza A virus of the Orthomyxoviridae family. Its genome consists of eight negative-sense RNA segments encoding 11 viral proteins, including HA, NA, and M1. Among these structural proteins, M1 is one of the most abundant, located between the viral envelope and the viral nucleus, forming the main backbone of the virus's internal structure. The M1 protein plays a crucial role in multiple aspects of the viral life cycle, including viral assembly, budding, viral entry into the nucleus, and intracellular transport. Furthermore, the M1 protein is highly conserved in avian influenza viruses, making it an ideal target for the research of broad-spectrum diagnostic reagents.

[0003] Currently, routine detection methods for avian influenza primarily target HA and NA antigens. While these surface proteins possess good antigenicity, their high mutation rate can easily lead to recognition failures by existing antibodies or vaccines. The M1 protein, however, is highly conserved and functionally crucial in avian influenza viruses, possessing unique advantages in virus detection, infection surveillance, and fundamental mechanism research. Monoclonal antibodies targeting the M1 protein can recognize multiple subtypes, enabling the development of broad-spectrum influenza diagnostic reagents, reducing reliance on subtype-specific antibodies, and facilitating the study of the M1 protein's function in viral replication. Therefore, monoclonal antibodies targeting the M1 protein are of significant value in virus detection, functional studies, and vaccine development. Summary of the Invention

[0004] This study utilized monoclonal antibody 5G9 against avian influenza virus (AIV) to obtain a monoclonal antibody. It exhibits good specificity, showing no cross-reactivity with Newcastle disease virus (NDV), duck Tembusu virus (DTUMV), goose astrovirus (GoAstV), and infectious laryngitis virus (ILTV). It also reacts with all H1-H11 subtypes of the virus, demonstrating broad-spectrum activity. Furthermore, it possesses fluorescent properties, enabling the detection of M1 protein sublocalization after AIV infection of cells. Therefore, this antibody can serve as a basis for establishing methods for AIV detection and tools for functional studies, providing the possibility of developing rapid, sensitive, and broad-spectrum detection methods to meet diverse clinical and practical needs.

[0005] To achieve the above-mentioned objectives, the technical solution adopted by this invention is as follows:

[0006] A broad-spectrum monoclonal antibody against the M1 protein of avian influenza virus, the antibody comprising a heavy chain variable region and a light chain variable region;

[0007] The heavy chain variable region contains V H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes V L CDR1, V L CDR2 and V L CDR3;

[0008] The V mentioned H The amino acid sequence of CDR1 is shown in SEQ ID NO. 4;

[0009] The V mentioned H The amino acid sequence of CDR2 is shown in SEQ ID NO. 8;

[0010] The V mentioned H The amino acid sequence of CDR3 is shown in SEQ ID NO. 12;

[0011] The V mentioned L The amino acid sequence of CDR1 is shown in SEQ ID NO. 20;

[0012] The V mentioned L The amino acid sequence of CDR2 is shown in SEQ ID NO. 24;

[0013] The V mentioned L The amino acid sequence of CDR3 is shown in SEQ ID NO. 28.

[0014] Furthermore, the amino acid sequences of the heavy chain variable regions FR1, FR2, FR3, and FR4 are shown in SEQ ID NO. 2, SEQ ID NO. 6, SEQ ID NO. 10, and SEQ ID NO. 14, respectively; and the amino acid sequences of the light chain variable regions FR1, FR2, FR3, and FR4 are shown in SEQ ID NO. 18, SEQ ID NO. 22, SEQ ID NO. 26, and SEQ ID NO. 30, respectively.

[0015] Furthermore, the heavy chain amino acid sequence is shown in SEQ ID NO: 16; the light chain amino acid sequence is shown in SEQ ID NO: 32.

[0016] The present invention also provides a nucleic acid molecule that encodes the above-mentioned broad-spectrum monoclonal antibody against the M1 protein of avian influenza virus.

[0017] Furthermore, the sequences of the nucleic acid molecules are SEQ ID NO: 15 and SEQ ID NO: 31;

[0018] Sequence SEQ ID NO: 15 encodes the heavy chain variable region of the antibody described;

[0019] The sequence SEQ ID NO: 31 encodes the light chain variable region of the antibody described.

[0020] The present invention also provides an expression vector containing the above-described nucleic acid molecules.

[0021] The present invention also provides a host cell containing the above-described expression vector.

[0022] The present invention also provides a detection reagent comprising the above-described broad-spectrum monoclonal antibody against the M1 protein of avian influenza virus.

[0023] The present invention also provides the application of the above-mentioned broad-spectrum monoclonal antibody against the M1 protein of avian influenza virus in the preparation of avian influenza detection reagents or avian influenza vaccines.

[0024] Furthermore, the avian influenza test kit or avian influenza vaccine is for the H1-H11 subtype of avian influenza.

[0025] This invention amplifies the M1 gene sequence by PCR, constructs the prokaryotic expression vector pGEX-6P-1-M1 using homologous recombination technology, and obtains recombinant M1 protein via IPTG induction. After immunizing 4-6 week old BALB / c mice with the purified recombinant M1 protein, a hybridoma cell line stably secreting anti-M1 monoclonal antibody was successfully obtained by fusing spleen cells with SP2 / 0 myeloma cells and screening by ELISA, IFA, and Western blot, named 5G9. Specificity analysis showed that this monoclonal antibody had no cross-reactivity with Newcastle disease virus, Tembusu virus, goose astrovirus, and infectious laryngotracheitis virus, exhibiting good specificity. Broad-spectrum assays showed that the monoclonal antibody specifically recognized H1-H11 subtypes of AIV, demonstrating good broad-spectrum activity. Furthermore, it exhibits fluorescence properties, allowing detection of M1 protein sublocalization after AIV infection of cells. Therefore, 5G9 is a highly reactive, broad-spectrum monoclonal antibody that can be used to detect various subtypes of avian influenza virus.

[0026] Beneficial effects

[0027] The acquisition of this monoclonal antibody makes it possible to establish a method for simultaneously detecting different subtypes of the virus, and has a wide range of application value. It provides the possibility of establishing rapid, sensitive, and broad-spectrum detection methods to meet different clinical and practical needs. Attached Figure Description

[0028] Figure 1 This is an SDS-PAGE analysis of recombinant AIV M1 protein purified by gradient urea elution; M: molecular weight standard of protein; 1-4: 0M, 2M, 4M and 8M urea-eluted proteins;

[0029] Figure 2 This is the specific result of indirect immunofluorescence detection of 5G9 monoclonal antibody (100×).

[0030] Figure 3 These are the results of Western blot analysis of the specificity of the 5G9 monoclonal antibody; M: 180 kDa protein marker, 1: Vero cells; 2: Vero cells infected with AIV; 3: Vero cells infected with TMUV; 4: A549 cells; 5: A549 cells infected with AIV; 6: A549 cells infected with NDV; 7: LMH cells; 8: LMH cells infected with AIV; 9: LMH cells infected with GoAstV; 10: LMH cells infected with ILTV;

[0031] Figure 4 The result is a broad-spectrum result (630×) from the indirect immunofluorescence assay of the 5G9 monoclonal antibody.

[0032] Figure 5 This is a broad-spectrum result from Western blot analysis of the 5G9 monoclonal antibody; M: 180 kDa protein marker, 1: A549 cells; 2-12: A549 cells infected with H1-H11 subtype AIV;

[0033] Figure 6 This is the result of the IFA assay (630×) used to locate the distribution of AIV in A549 cells using the 5G9 M1 monoclonal antibody. Detailed Implementation

[0034] Example 1

[0035] Expression and purification of M1 recombinant protein

[0036] 1.1 Construction of recombinant plasmids

[0037] Based on the gene sequence of AIV A / Mallard / Huadong / S / 2005 (SY) strain, the M1 gene was amplified, the target band was recovered and purified. Then, the pGEX-6P-1 vector was double-digested and ligated with the purified M1 gene fragment for homologous recombination to obtain a recombinant plasmid, which was finally named pGEX-6P-1-M1.

[0038] 1.2 Induced expression of M1 protein

[0039] After the recombinant plasmid pGEX-6P-1, which had been verified by sequencing, was transformed into competent *E. coli* Rosetta (DE3) cells, single colonies with regular morphology were aseptically picked and inoculated into LB medium containing 100 μg / mL ampicillin. The bacterial culture was incubated at 37°C with shaking for 12 h at a speed of 220 r / min. On day 2, the overnight culture was transferred to fresh resistant LB medium at a volume ratio of 1:100 and cultured at 37°C with shaking for 220 r / min. When the OD600 value of the bacterial culture reached 0.6 as measured by spectrophotometer, isopropyl-β-D-thiogalactoside (IPTG) was added to a final concentration of 1 mM, and the culture was transferred to a 25°C low-temperature shaking incubator with the shaking speed adjusted to 200 r / min to induce expression for 12 h.

[0040] After induction, the bacterial culture was collected and centrifuged at 4°C. The precipitate was resuspended in pre-cooled PBS buffer. The resuspended cells were then sonicated in an ice bath at 200 W for 15 min (2 s sonication followed by a 2 s pause). After lysis, the cells were centrifuged at 12000 r / min for 10 min at 4°C to separate the components. The supernatant containing soluble proteins and the precipitate containing inclusion bodies were collected separately. Electrophoresis samples were prepared according to standard procedures, and the expression of the target protein was analyzed by SDS-PAGE electrophoresis.

[0041] 1.3 Purification and refolding of recombinant M1 protein

[0042] After protein expression was induced, the bacterial culture was centrifuged to obtain a bacterial pellet. The pellet was then fully resuspended in PBS and subjected to ultrasonic lysis in an ice bath.

[0043] After centrifuging the lysis buffer at 12000 r / min for 10 min, the supernatant was discarded. The collected precipitate was dissolved and purified by elution with urea. All samples were prepared using standard methods, and the purification effect of the target protein was detected by SDS-PAGE. Successfully purified proteins were then tested for their corresponding concentrations using a BCA kit and stored at -70 ℃ for later use.

[0044] Example 2

[0045] Preparation of monoclonal antibodies

[0046] 2.1 Animal Immunization

[0047] For the initial immunization, 100 μg of recombinant AIV M1 protein antigen and 40 μL of MONTANIDE GEL 01 PR adjuvant were vortexed to form a stable emulsion, which was then administered to 6-week-old female BALB / c mice via multiple subcutaneous injections at the nape of the neck. A booster immunization was performed 14 days later using the same method. Serum antibody titers were then measured by collecting blood from the orbital sinus veins. When the antibody titer reached the desired level, a booster immunization was administered via intraperitoneal injection of an adjuvant-free antigen solution. Cell fusion was prepared 72 hours later.

[0048] 2.2 Integration

[0049] Under aseptic conditions, a suspension of immunized mouse spleen cells and SP2 / 0 myeloma cells in logarithmic growth phase were mixed at a ratio of 6:1 in a 50 mL centrifuge tube. After centrifugation at 1000 rpm for 10 min, the supernatant was discarded. The tube was gently tapped to loosen the cell pellet, and then placed in a 37°C water bath. Over 1 min, 1 mL of pre-warmed PEG1500 fusion agent was slowly added dropwise while gently agitating the tube to promote complete cell fusion. At the fusion termination stage, 30 mL of pre-warmed DMEM complete medium was injected uniformly over 1 min, and the mixture was allowed to stand for 10 min to complete the fusion termination. After centrifugation at 1000 rpm for 10 min, the cell pellet was collected and gently resuspended in HAT selective medium. Finally, the cell suspension was seeded into 96-well plates containing a feeder layer. Strict aseptic conditions must be maintained throughout the entire process, and transfer should be performed gently to avoid cell damage. The culture plates were incubated at 37°C in a 5% CO2 incubator, avoiding unnecessary movement during the initial fusion phase. During subsequent culture, half of the HAT medium was replaced on day 4 after fusion, and the entire medium was replaced with HT medium on day 7. Cell growth was monitored daily. Screening was performed when the culture medium turned pale yellow or typical hybridoma cell clusters were observed under a microscope. Specific methods included ELISA, IFA, and Western blot.

[0050] 2.3 Screening of hybridoma cells

[0051] 2.3.1 Indirect ELISA screening

[0052] After the supernatant in the cell wells of the fused cell plate turned yellow, the supernatant was used for indirect ELISA detection. Immunized mouse fusion serum was used as the positive sample, normal BALB / c mouse serum as the negative sample, and PBS was set up as the blank control. In the experimental design, the antigen was serially diluted longitudinally with coating buffer. The first row of wells was coated with 100 μL of M1 protein solution with an initial concentration of 10 μg / mL. After overnight coating at 4°C, the wells were washed three times with PBST, and then blocked with 5% skim milk at 37°C for 2 h. Positive serum was serially diluted horizontally at an initial dilution of 1:100, and negative control wells were treated simultaneously. PBS was set up as the blank control. All reaction wells were incubated at 37°C for 90 min, following the same washing steps as before. Then, 100 μL of HRP goat anti-mouse IgG + IgM secondary antibody diluted 1:8000 was added to each well, and the wells were incubated at 37°C for 45 min. In the colorimetric stage, TMB substrate chromogenic solution was added first, and the reaction was carried out at room temperature in the dark for 15 min before the stop solution was added. Finally, the OD450 value was measured. By comparing the detection parameters of positive sample group with OD450 values ​​close to 1.0 and P / N values ​​≥ 2.1, the optimal antigen coating concentration and corresponding serum dilution factor were determined.

[0053] 2.3.2 Western Blot Screening

[0054] In a 6-well plate, with 1.2 × 10⁻⁶ ppm per well... 6 A549 cells were seeded at a density of [number] cells and cultured. After cell growth stabilized, the culture medium was removed, and the cells were gently washed three times with PBS buffer. The AIV virus suspension was diluted to an MOI of 0.1 using serum-free MEM medium, and uninfected A549 cells were used as a blank control. After virus inoculation, the 6-well plates were incubated at 37°C for 1 h, then replaced with MEM containing 1% TPCK trypsin for maintenance culture and continued incubation for 24 h in an incubator. The culture supernatant was then removed, and protein samples were prepared according to standard procedures.

[0055] The prepared sample was added to the wells of the SDS-PAGE protein gel at a rate of 10 μL / well for electrophoresis. After electrophoresis, the gel was removed from the apparatus, and the gel was placed between filter paper and a pre-activated NC membrane in transfer buffer. The layers were stacked sequentially from bottom to top: thick sponge, filter paper, gel, NC membrane, filter paper, and thick sponge. The electrophoresis program was set to a constant current of 250 mA for 1.5 h. After transfer, the NC membrane was removed from the apparatus and blocked with 5% skim milk at room temperature for 1 h. After blocking, the NC membrane was washed three times with TBST buffer for 10 min each time, and then incubated overnight at 4°C on a shaker with primary antibody. After incubation, the washing step was repeated with TBST buffer. Next, HRP-labeled goat anti-mouse IgG + IgM secondary antibody diluted 1:8000 with TBST buffer was added, and the mixture was incubated at room temperature for 1 h. After incubation, the membrane was washed again with TBST buffer. Finally, the protein bands on the membrane were imaged and analyzed using a chemiluminescence imaging system under light-protected conditions. The results of the protein blot were observed and recorded. The presence of the target band indicated a positive result.

[0056] 2.3.3 Indirect immunofluorescence screening

[0057] Positive wells selected by Western blotting were further screened using indirect immunofluorescence. First, A549 cells were plated and the culture medium removed. The cells were then washed three times with preheated PBS (37°C). Finally, the AIV virus solution was serially diluted to 10⁻¹⁰ using MEM culture medium. -3 100 μL of cells were seeded into each well of a cell culture plate, with uninfected A549 cells serving as a blank control. After 12 h of infection, the culture medium was removed, and the cells were gently washed three times with PBS at 37°C. Then, pre-chilled paraformaldehyde fixative was added for 15 min, followed by repeated washing. The cells were then permeabilized with 0.5% Triton X-100 solution for 15 min and blocked with 3% BSA solution at 37°C for 1 h, with repeated washing between each step. Hybridoma supernatant was then added to the wells of both the virus-infected and control groups, and incubated at 37°C for 1 h. After three washes with PBST solution, 100 μL of Alexa Fluor diluted 1:1000 was added to each well under light-protected conditions. TM 488 sheep anti-mouse antibodies were reacted at 37°C for 1 hour, followed by repeated washing. Wells showing specific reactions to the H5 subtype AIVNP monoclonal antibody served as positive controls. If wells infected with AIV showed specific fluorescence, while uninfected wells did not, it indicated that the antibody secreted by the hybridoma cells specifically targeted the avian influenza virus, meeting the screening requirements.

[0058] 2.4 Subcloning of hybridoma cells

[0059] For hybridoma cell lines that tested positive by ELISA, IFA, and Western Blot, a limiting dilution method was used for three-stage subcloning. First, feeder cells were prepared. After detaching the hybridoma cells to be cloned by pipetting in HT medium, 100 μL of cell suspension was added to the first well of the first column of a 96-well plate, followed by serial dilutions along the columns. After standing for 5 minutes to allow cell sedimentation, the cell density in each well of the first column was observed under a microscope. Wells with approximately 100 cells were selected, resuspended by pipetting, and transferred to centrifuge tubes containing 5 mL of HT medium. After thorough mixing, 100 μL of cell suspension was seeded into each well of the second to fourth columns sequentially. Medium was added to a total volume of 5 mL, mixed again, and evenly distributed to the fifth to eighth columns. Finally, 3.5 mL was added to complete the seeding of columns 9 to 12. A sterile environment was maintained throughout the procedure, and the culture plate was incubated at 37°C in a 5% CO2 incubator. When the culture medium turned pale yellow or typical white cell clumps appeared at the bottom of the wells, immediate testing was performed. This subcloning process needs to be repeated at least three times until the supernatant from all cloning wells shows a positive reaction for the target antibody before hybridoma establishment can proceed.

[0060] 2.5 Preparation of Ascites

[0061] Multiparous BALB / c female rats were selected and sensitized by intraperitoneal injection of 0.5 mL of sterile paraffin oil. Seven days later, hybridoma cells in the logarithmic growth phase were injected with 1.2 × 10⁻⁶ cells... 6 Cells were inoculated intraperitoneally only. The degree of abdominal swelling was observed daily, and ascites was collected by aseptic puncture when the abdominal cavity was significantly distended. After preliminary clarification by centrifugation at 1000 rpm for 10 min, the collected fluid was filtered through a 0.45 μm microporous membrane to remove lipid components, aliquoted, and stored at -70°C.

[0062] Example 3

[0063] Identification of monoclonal antibody characteristics

[0064] 3.1 Monoclonal antibody specificity identification

[0065] 3.1.1 Indirect immunofluorescence assay

[0066] Vero cells were infected with AIV and TUMV, A549 cells with AIV and NDV, and LMH cells with AIV, GoAstV, and ILTV, all at an MOI of 0.1. After 36 hours, the specificity of the monoclonal antibody in ascites was detected by indirect immunofluorescence assay. The results showed that the monoclonal antibody did not exhibit specific fluorescence in cells infected with other avian viruses, but did exhibit specific fluorescence in cells infected with AIV, indicating that the 5G9 monoclonal antibody has good specificity. Figure 2 ).

[0067] 3.1.2 Western Blot Detection

[0068] Vero cells were infected with AIV and TUMV, A549 cells with AIV and NDV, and LMH cells with AIV, GoAstV, and ILTV, all at an MOI of 0.01. After incubation at 37°C for 48 h, protein samples were prepared according to standard procedures. Western blot analysis was then performed to determine the specificity of the monoclonal antibodies in ascites, and the results showed that all five antibodies exhibited specific reactive bands with AIV but no reaction with NDV, TMUV, ILTV, and GoAstV, indicating that all five monoclonal antibodies possessed good specificity. Figure 3 )

[0069] 3.2 Identification of the broad spectrum of monoclonal antibodies

[0070] 3.2.1 Indirect immunofluorescence assay

[0071] A549 cells were infected with AIV of subtypes H1-H11 at MOI=1 and incubated at 37°C for 1 h. For low-pathogenic avian influenza viruses, the culture medium was replaced with maintenance medium containing 1% TPCK trypsin. Cells were then incubated at 37°C for 12 h before indirect immunofluorescence assay. Results showed that the monoclonal antibody 5G9 exhibited specific fluorescence for both H1-H11 subtype AIV, indicating its good broad-spectrum activity. Figure 4 ).

[0072] 3.2.2 Western Blot Detection

[0073] A549 cells were infected with H1-H11 AIV at MOI=0.1. After viral adsorption at 37℃ for 1 h, if low pathogenic avian influenza virus was present, the culture medium was replaced with maintenance medium containing 1% TPCK trypsin, and the cells were cultured in an incubator for another 36 h. Cell lysis products were collected for Western blotting. The results showed that the monoclonal antibody specifically responded to A549 cells infected with different AIV subtypes, indicating that this monoclonal antibody has good broad-spectrum activity against H1-H11 AIV subtypes. Figure 5 ).

[0074] 3.3 Subclass Identification

[0075] Following the operating instructions of the Biodragon commercial antibody subclass identification kit, the subclass of the monoclonal antibody in ascites was identified. The results showed that 5G9 was classified as the IgG1 subclass, and the light chain subclass as the κ subclass. The nucleotide and amino acid sequences of the variable region are described below.

[0076] 3.4 Monoclonal antibody localization of AIV M1 protein distribution on infected cells

[0077] A549 cells were divided into groups of 1.2 × 10⁻⁶. 6 Cells were seeded at a density of 1 / well in 6-well plates. After the cells were in good growth condition, the culture medium was discarded, and the cells were washed three times with PBS. The AIV virus suspension was then diluted with serum-free DMEM-F12K to MOI=10 to infect the cells, and an uninfected cell control group was set up. The virus was adsorbed at 37℃ for 1 h, and the culture medium was replaced with maintenance medium containing 1% TPCK trypsin for continued culture. Cell samples were fixed at three time points: 1 h, 2.5 h, and 4.5 h post-infection. The localization of M1 protein in cells was detected by indirect immunofluorescence, using a self-made M1 monoclonal antibody 5G9 as the primary antibody to determine the role of this monoclonal antibody in localizing AIV in infected cells. The results showed that the 5G9 monoclonal antibody had different fluorescence responses at different time points after AIV infection of A549 cells. After 1 h of AIV infection, the fluorescence showed punctate aggregation; after 2.5 h of infection, the fluorescence decreased significantly; and after 4.5 h of infection, the fluorescence showed intranuclear distribution, indicating that the 5G9 monoclonal antibody could accurately locate the position of AIV infection in A549 cells. Figure 6 ).

[0078] The nucleotide or amino acid sequences involved in this invention are specifically as follows:

[0079] SEQ ID NO. 1 (nucleotide sequence of 5G9-mAb heavy chain FR1)

[0080] GAAGTGATGCTGGTGGAGTCTGGGGGAGGCTTAGTGAAGCCTGGAGGGTCCCTGAAACTCTCCTGTGCAGCCTCT;

[0081] SEQ ID NO. 2 (Amino acid sequence of FR1 heavy chain of 5G9-mAb)

[0082] EVMLVESGGGLVKPGGSLKLSCAAS;

[0083] SEQ ID NO. 3 (nucleotide sequence of CDR1 in the 5G9-mAb heavy chain)

[0084] GGATTCGCTTTCAGTAGCTATGAC;

[0085] SEQ ID NO. 4 (Amino acid sequence of CDR1 in the 5G9-mAb heavy chain)

[0086] GFAFSSYD;

[0087] SEQ ID NO. 5 (nucleotide sequence of 5G9-mAb heavy chain FR2)

[0088] ATGTCTTGGGTTCGCCAGACTCCGGAGAAGAGGCTGGAGTGGGTCGCATAT;

[0089] SEQ ID NO. 6 (amino acid sequence of FR2 heavy chain of 5G9-mAb)

[0090] MSWVRQTPEKRLEWVAY;

[0091] SEQ ID NO. 7 (nucleotide sequence of 5G9-mAb heavy chain CDR2)

[0092] ATTAGTAGTGGTGGTGGTAACACC;

[0093] SEQ ID NO. 8 (Amino acid sequence of CDR2 in the heavy chain of 5G9-mAb)

[0094] ISSGGGNT;

[0095] SEQ ID NO. 9 (nucleotide sequence of 5G9-mAb heavy chain FR3)

[0096] TACTATCCAGACACTGTGAAGGGCCGATTCACCATCTCCAGAGACAATGCCAAGAGCACCCTGAACCTGCAAATGAGCAGTCTGAGGTCTGAGGACACAGCCATGTATTACTGT;

[0097] SEQ ID NO. 10 (Amino acid sequence of FR3 heavy chain of 5G9-mAb)

[0098] YYPDTVKGRFTISRDNAKSTLNLQMSSLRSEDTAMYYC;

[0099] SEQ ID NO. 11 (nucleotide sequence of CDR3 of 5G9-mAb heavy chain)

[0100] GCAAGACCCCATTACTACGGTAGTAGCTTCGAGTACTTCGATGTC;

[0101] SEQ ID NO. 12 (Amino acid sequence of CDR3 in the 5G9-mAb heavy chain)

[0102] ARPHYYGSSFEYFDV;

[0103] SEQ ID NO. 13 (nucleotide sequence of 5G9-mAb heavy chain FR4)

[0104] TGGGGCGCAGGGACCTCAGTCACCGTCTCGAGC;

[0105] SEQ ID NO. 14 (amino acid sequence of FR4 heavy chain of 5G9-mAb)

[0106] WGAGTSVTVSS;

[0107] SEQ ID NO. 15 (nucleotide sequence of 5G9-mAb heavy chain V(D)J-IMGT)

[0108] GAAGTGATGCTGGTGGAGTCTGGGGGAGGCTTAGTGAAGCCTGGAGGGTCCCTGAAACTCTCCTGTGCAGCCTCTGGATTCGCTTTCAGTAGCTATGACATGTCTTGGGTTCGCCAGACTCCGGAGAAGAGGCTGGAGTGGGTCGCATATATTAGTAGTGGTGGTGGTAACACCTACTATCCA GACACTGTGAAGGGCCGATTCACCATCTCCAGAGACAATGCCAAGAGCACCCTGAACCTGCAAATGAGCAGTCTGAGGTCTGAGGACACAGCCATGTATTACTGTGCAAGACCCCATTACTACGGTAGTAGCTTCGAGTACTTCGATGTCTGGGGCGCAGGGACCTCAGTCACCGTCTCGAGC;

[0109] SEQ ID NO. 16 (Amino acid sequence of 5G9-mAb heavy chain V(D)J-IMGT)

[0110] EVMLVESGGGLVKPGGSLKLSCAASGFAFSSYDMSWVRQTPEKRLEWVAYISSGGGNTYYPDTVKGRFTISRDNAKSTLNLQMSSLRSEDTAMYYCARPHYYGSSFEYFDVWGAGTSVTVSS;

[0111] SEQ ID NO. 17 (nucleotide sequence of 5G9-mAb light chain FR1)

[0112] GATATTGTGATCACCCAGACTCCATCATCTCTGGCTGTGTCTGCAGGAGAAAAGGTCACTATGAGCTGTAAGTCCAGT;

[0113] SEQ ID NO. 18 (Amino acid sequence of FR1 light chain of 5G9-mAb)

[0114] DIVITQTPSSLAVSAGEKVTMSCKSS;

[0115] SEQ ID NO. 19 (nucleotide sequence of 5G9-mAb light chain CDR1)

[0116] CACAGTGTTTTCGACAGTTCAAATCAGAAGAACTAC;

[0117] SEQ ID NO. 20 (Amino acid sequence of the light chain CDR1 of 5G9-mAb)

[0118] HSVFDSSNQKNY;

[0119] SEQ ID NO. 21 (nucleotide sequence of 5G9-mAb light chain FR2)

[0120] TTGGCCTGGTACCAGCAGAAACCAGGGCAGTCTCCTAAACTGCTGATCTAC;

[0121] SEQ ID NO. 22 (amino acid sequence of FR2 light chain of 5G9-mAb)

[0122] LAWYQQKPGQSPKLLIY;

[0123] SEQ ID NO. 23 (nucleotide sequence of 5G9-mAb light chain CDR2)

[0124] TGGGCATCC;

[0125] SEQ ID NO. 24 (Amino acid sequence of the light chain CDR2 of 5G9-mAb)

[0126] WAS;

[0127] SEQ ID NO. 25 (nucleotide sequence of FR3 light chain of 5G9-mAb)

[0128] ACTAGGGAATCTGGTGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTTTACTCTTACCATCAGCAGTGTACAACCTGAAGACCTGGCATTTTATTACTGT;

[0129] SEQ ID NO. 26 (Amino acid sequence of FR3 light chain of 5G9-mAb)

[0130] TRESGVPDRFTGSGSGTDFTLTISSVQPEDLAFYYC;

[0131] SEQ ID NO. 27 (nucleotide sequence of 5G9-mAb light chain CDR3)

[0132] CATCAATACCTCTCCTCGTACACG;

[0133] SEQ ID NO. 28 (Amino acid sequence of the light chain CDR3 of 5G9-mAb)

[0134] HQYLSSYT;

[0135] SEQ ID NO. 29 (nucleotide sequence of FR4 light chain of 5G9-mAb)

[0136] TTCGGAGGGGGGACCAAGCTTGGAAAATAAAA;

[0137] SEQ ID NO. 30 (Amino acid sequence of FR4 light chain of 5G9-mAb)

[0138] FGGGTKLEIK;

[0139] SEQ ID NO. 31 (nucleotide sequence of 5G9-mAb light chain V(D)J-IMGT)

[0140] GATATTGTGATCACCCAGACTCCATCATCTCTGGCTGTGTCTGCAGGAGAAAAGGTCACTATGAGCTGTAAGTCCAGTCACAGTGTTTTCGACAGTTCAAATCAGAAGAACTACTTGGCCTGGTACCAGCAGAAACCAGGGCAGTCTCCTAAACTGCTGATCTACTGGGCATCCACTAGGGAATCTGGTGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTTTACTCTTACCATCAGCAGTGTACAACCTGAAGACCTGGCATTTTATTACTGTCATCAATACCTCTCCTCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAA;

[0141] SEQ ID NO. 32 (Amino acid sequence of 5G9-mAb light chain V(D)J-IMGT)

[0142] DIVITQTPSSLAVSAGEKVTMSCKSSHSVFDSSNQKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQPEDLAFYYCHQYLSSYTFGGGTKLEIK。

Claims

1. A broad-spectrum monoclonal antibody against the M1 protein of avian influenza virus, characterized in that, The antibody comprises a heavy chain variable region and a light chain variable region; The heavy chain variable region contains V H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes V L CDR1, V L CDR2 and V L CDR3; The V mentioned H The amino acid sequence of CDR1 is shown in SEQ ID NO. 4; The V mentioned H The amino acid sequence of CDR2 is shown in SEQ ID NO. 8; The V mentioned H The amino acid sequence of CDR3 is shown in SEQ ID NO. 12; The V mentioned L The amino acid sequence of CDR1 is shown in SEQ ID NO. 20; The V mentioned L The amino acid sequence of CDR2 is shown in SEQ ID NO. 24; The V mentioned L The amino acid sequence of CDR3 is shown in SEQ ID NO.

28.

2. The broad-spectrum monoclonal antibody against the M1 protein of avian influenza virus according to claim 1, characterized in that, The amino acid sequences of the heavy chain variable regions FR1, FR2, FR3, and FR4 are shown in SEQ ID NO. 2, SEQ ID NO. 6, SEQ ID NO. 10, and SEQ ID NO. 14, respectively; the amino acid sequences of the light chain variable regions FR1, FR2, FR3, and FR4 are shown in SEQ ID NO. 18, SEQ ID NO. 22, SEQ ID NO. 26, and SEQ ID NO. 30, respectively.

3. The broad-spectrum monoclonal antibody against the M1 protein of avian influenza virus according to claim 1, characterized in that, The heavy chain amino acid sequence is shown in SEQ ID NO: 16; the light chain amino acid sequence is shown in SEQ ID NO:

32.

4. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes a broad-spectrum monoclonal antibody against the M1 protein of avian influenza virus as described in any one of claims 1 to 3.

5. The nucleic acid molecule according to claim 4, characterized in that, The sequences of the nucleic acid molecules are SEQ ID NO: 15 and SEQ ID NO: 31; Sequence SEQ ID NO: 15 encodes the heavy chain variable region of the antibody described; The sequence SEQ ID NO: 31 encodes the light chain variable region of the antibody described.

6. An expression carrier, characterized in that, The expression vector contains the nucleic acid molecule as described in claim 4 or 5.

7. A host cell, characterized in that, The host cell contains the expression vector as described in claim 6.

8. A detection reagent comprising a broad-spectrum monoclonal antibody against the M1 protein of avian influenza virus as described in any one of claims 1 to 3.

9. The use of the broad-spectrum monoclonal antibody against the M1 protein of avian influenza virus as described in any one of claims 1-3 in the preparation of avian influenza detection reagents or avian influenza vaccines.

10. The application according to claim 9, characterized in that, The avian influenza test kit or avian influenza vaccine is for the H1-H11 subtype of avian influenza.

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