Monoclonal antibody combined with multi-branch H5Nx subtype avian influenza virus HA protein in broad spectrum and application

Abstract

The invention discloses a monoclonal antibody in broad-spectrum combination with multi-branch H5Nx subtype avian influenza virus HA protein and application of the monoclonal antibody. The monoclonal antibody is named as 1G10, belongs to an IgG1 subclass and comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprises VHCDR1, VHCDR2 and VHCDR3 of which the amino acid sequences are respectively shown as SEQ ID NO.5-7, and the light chain variable region comprises VLCDR1, VLCDR2 and VLCDR3 of which the amino acid sequences are respectively shown as SEQ ID NO.8-10. The monoclonal antibody can specifically recognize the HA protein in an indirect immunofluorescence test, a protein immunoblotting test and a neutralization test. Besides, the monoclonal antibody can be subjected to a specific hemagglutination inhibition reaction with H5Nx subtype avian influenza viruses such as H5N1, H5N6, H5N8 and the like of different HA gene evolution branches such as 2.3. 2.1 d, 2.3. 4.4 b, 2.3. 4.4 d and the like, which indicates that the epitope recognized by the 1G10 monoclonal antibody is a broad-spectrum neutralizing epitope of the H5 subtype avian influenza viruses. Through a chick embryo immune escape test and sequence alignment, the neutralizing antigen epitope identified by the 1G10 monoclonal antibody is screened and contains 170th and 231th amino acid sites of HA protein, and the mutation of the neutralizing antigen epitope significantly influences antigenicity.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to a monoclonal antibody capable of broadly binding to multi-branched H5Nx subtype avian influenza virus HA protein, and neutralization epitope identification and application. Background Art

[0002] The H5 subtype highly pathogenic avian influenza virus (AIV) is distributed worldwide, seriously endangering the healthy development of the poultry industry, and can cause infection and disease in a variety of mammals including humans, so it also has important public health significance. Statistics show that from January 2005 to November 2022, 8534 H5 subtype highly pathogenic avian influenza outbreaks have been reported worldwide, with up to 389 million poultry deaths and culling (Shi J, Zeng X, Cui P, et al. Alarming situation of emerging H5 and H7 avian influenza and effective control strategies [J]. Emerg Microbes Infect, 2023, 12(1):2155072). As of July 19, 2024, the World Health Organization reported that the H5N1 subtype AIV has caused a total of 896 confirmed human infections since 2003, of which 463 have died, with a mortality rate of more than 50% (https: / / www.who.int / ). In addition, H5N2, H5N6 and H5N8 subtype AIVs can also cause infection and disease in humans.

[0003] Hemagglutinin (HA) is the most important antigenic glycoprotein on the surface of AIV. It has the ability to agglutinate red blood cells and is the core target of vaccines, neutralizing antibodies and diagnostic preparations. However, the HA gene is also the gene with the highest degree of variation in the AIV genome, which directly affects the antigenicity of the virus. It is known that the H5 subtype highly pathogenic AIV has derived many HA gene evolutionary branches (Clades), including the primary branches of Clade 0~9 and their multiple subbranches. Among them, the H5 subtype highly pathogenic AIV of the 2.3.2 and 2.3.4 branches have long been dominant in my country, and there are obvious antigenic variations between the two branches. Therefore, different vaccine strains have been developed to cope with their epidemics. In addition, the virus strains within the 2.3.2 and 2.3.4 branches are also constantly evolving and mutating, resulting in antigenic differences. In response to the further derivation of 2.3.2.1d, 2.3.2.1e and other epidemic branches from the 2.3.2 branch, the recombinant inactivated vaccine of H5 subtype avian influenza virus approved for use in China was updated from Re-6 to Re-12; and in response to the continuous evolution of 2.3.4 branch into mainstream sub-branches such as 2.3.4.4g, 2.3.4.4h, and 2.3.4.4b, the vaccine strains have at least gone through Re-5, Re-8, Re-11 to the currently used Re-13 and Re-14 (Zeng Xianying, Tian Guobin, Chen Hualan. Progress in the development and application of H5 / H7 subtype avian influenza vaccines in China [J]. Science China: Life Sciences, 2023, 53(12): 1700-1712).

[0004] Since the antigenic matching degree between epidemic strains and vaccine strains is the key to determine the immune protection efficacy of vaccines, it is crucial to monitor and screen epidemic strains with consistent antigenicity as vaccine candidates. Moreover, commercial vaccines for new variants of H5 subtype AIV usually lag behind the emergence of epidemic strains, which is likely to exacerbate the spread of the epidemic caused by the variant strains escaping vaccines with poor antigenic matching. Therefore, the development of a universal vaccine with broad protective effects is of great practical significance for the effective prevention and control of H5 subtype highly pathogenic avian influenza. However, the premise of universal vaccine design is to discover conservative broad-spectrum antigenic epitopes. One of the main ways to discover broad-spectrum antigenic epitopes is to analyze the antigenic epitopes targeted by monoclonal antibodies with broad-spectrum recognition ability. Since antibodies targeting the globular head domain of HA protein can specifically neutralize the virus and can be detected by hemagglutination inhibition test, many influenza universal vaccine designs also focus on this region of HA protein.

[0005] In recent years, monoclonal antibodies against HA proteins have also been widely used to study the antigenicity of influenza viruses. For example, Zheng Qingbing et al. used multiple HA protein monoclonal antibodies against the 2009 influenza A (H1N1) vaccine strain A / Califomia / 07 / 2009 to analyze the antigenic differences of virus isolates and identified that mutations in key amino acids such as positions 128 and 158 in the Sa antigen region, position 225 in the Ca2 antigen region, and position 188 in the Sb antigen region can significantly affect the antigenicity of the virus (Zheng Qingbing, Chen Yixin, Zeng Fen, et al. Monitoring the antigenic variation of influenza A (H1N1) virus using hemagglutinin monoclonal antibodies [C] / / Chinese Medical Association, Chinese Society of Microbiology and Immunology. Proceedings of the 2012 National Symposium on Clinical Microbiology and Infection Immunity. National Infectious Disease Diagnostic Reagents and Vaccine Engineering Technology Research Center, School of Public Health, Xiamen University, 2012: 1). Zhang et al. identified positions 120, 126, 141, 156, 185, and 189 as important antigenic sites by combining a group of monoclonal antibodies against the HA protein of the H5 subtype AIV vaccine strain Re-8, which are of great significance for the drawing of the antigenic map of the 2.3.4.4 branch H5 influenza virus (Zhang Y, Cui P, Shi J, et al. Key amino acid residues thatdetermine the antigenic properties ofhighly pathogenic H5 influenza virusesbearing the clade 2.3.4.4 hemagglutinin gene [J]. Viruses. 2023, 15(11):2249).

[0006] However, since monoclonal antibodies target a single antigenic epitope, their reaction spectrum is usually limited and they can often only recognize viruses belonging to the same HA branch. Therefore, the identification of broadly neutralizing monoclonal antibodies that can simultaneously recognize the HA proteins of multiple-branched H5 viruses will provide important biological materials for H5 subtype AIV antigen monitoring and vaccine development. Summary of the invention

[0007] The present invention discloses a monoclonal antibody 1G10 that can broadly bind to the HA protein of multi-branched H5Nx subtype avian influenza viruses. The monoclonal antibody targets the HA protein, can specifically immunologically bind to the HA protein in both indirect immunofluorescence and Western blot tests, and can specifically inhibit hemagglutination with H5Nx subtype avian influenza viruses such as H5N1, H5N6 and H5N8 of multiple HA evolutionary branches such as 2.3.2.1d, 2.3.4.4b, 2.3.4.4d, and has neutralizing activity.

[0008] The present invention discloses a monoclonal antibody that broadly binds to the HA protein of multi-branched H5Nx subtype avian influenza virus, wherein the monoclonal antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises V H CDR1, V H CDR2 and V H CDR3, light chain variable region contains V L CDR1, V L CDR2 and V L CDR3; The V H The amino acid sequence of CDR1 is shown in SEQ ID NO. 5; The V H The amino acid sequence of CDR2 is shown in SEQ ID NO. 6; The V H The amino acid sequence of CDR3 is shown in SEQ ID NO. 7; The V L The amino acid sequence of CDR1 is shown in SEQ ID NO. 8; The V L The amino acid sequence of CDR2 is shown in SEQ ID NO. 9; The V L The amino acid sequence of CDR3 is shown in SEQ ID NO. 10.

[0009] Furthermore, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO. 2, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO. 4.

[0010] In the present invention, the nucleotide sequence of the heavy chain variable region of the monoclonal antibody that broadly binds to the multi-branched H5Nx subtype avian influenza virus HA protein is shown in SEQ ID NO. 1, and the nucleotide sequence of the light chain variable region is shown in SEQ ID NO. 3. The antibody can be expressed by conventional genetic engineering or protein engineering methods, avoiding the loss of antibody genes during long-term cryopreservation of hybridoma cells, and is conducive to optimizing antibodies at the gene and protein levels, thereby improving the specificity and affinity of the antibody.

[0011] The present invention also discloses an expression vector, which contains the nucleotide sequence of the heavy chain variable region and the nucleotide sequence of the light chain variable region mentioned above.

[0012] The present invention also discloses a host cell, wherein the host cell contains the above-mentioned expression vector.

[0013] The invention also discloses the application of the monoclonal antibody in the analysis of antigenic variation of influenza virus.

[0014] Furthermore, the invention discloses the use of the monoclonal antibody in preparing a detection reagent for H5 subtype avian influenza virus.

[0015] The present invention also provides the identification of the neutralizing antigenic epitope recognized by the monoclonal antibody, by incubating a wild-type H5 subtype avian influenza virus liquid with the HA protein monoclonal antibody with neutralizing properties of the present invention, inoculating SPF chicken embryos to prepare an escape mutant strain with significantly reduced hemagglutination inhibition titer, and comparing the HA gene sequence of the escape mutant strain with the wild type, determining that the 170th and 231st differential amino acid sites are the antigenic epitopes recognized by the monoclonal antibody, and big data analysis indicates that 170N and 231T are well conserved in H5 subtype influenza viruses of different evolutionary branches.

[0016] The present invention also provides the use of the monoclonal antibody in studying antigenic variation of H5 subtype avian influenza virus, and by screening escape mutants, it is identified that HA protein N170D and T231N mutations can significantly affect antigenicity.

[0017] The characteristics and advantages of the present invention are as follows: the present invention discloses a neutralizing monoclonal antibody 1G10 which broadly binds to the HA protein of multi-branched H5Nx subtype avian influenza virus, can undergo hemagglutination inhibition reaction with multiple strains of 2.3.2.1 and 2.3.4.4 epidemic branches H5N1, H5N6 and H5N8 subtype avian influenza viruses, and can specifically identify the HA protein of the H5 subtype avian influenza virus in both indirect immunofluorescence and Western blot tests, and can be further applied to the preparation of H5 subtype avian influenza virus detection reagents, the design of universal vaccines and the study of antigenic variation. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The results of indirect immunofluorescence assay (IFA) of monoclonal antibody 1G10 and 293T cells transfected with pCAGGS-171033-HA are shown in Figure 1. A shows IFA of 293T cells transfected with pCAGGS-171033-HA using mouse ascites 1G10 as primary antibody, and bright green fluorescence can be seen; B shows IFA of 293T cells transfected with pCAGGS-171033-HA using mouse positive serum as primary antibody, and bright green fluorescence can be seen, which is the positive control; C shows IFA of 293T cells transfected with empty pCAGGS vector using mouse ascites 1G10 as primary antibody, and no green fluorescence can be seen, which is the negative control; D shows IFA of normal 293T cells not transfected with plasmid using mouse ascites 1G10 as primary antibody, and no green fluorescence can be seen, which is also the negative control.

[0019] Figure 2 This is a graph showing the results of a Western blot assay of monoclonal antibody 1G10 and 293T cells transfected with pCAGGS-171033-HA.

[0020] Figure 3 The HA gene evolution tree of different branches of H5Nx subtype avian influenza virus used to determine the broad-spectrum reactivity of monoclonal antibody 1G10. Among them, the red triangle marks the virus strain used for detection, and the others are reference strains.

[0021] Figure 4 Spatial structural localization of the key antigenic sites of the H5 subtype avian influenza virus escape mutants screened for monoclonal antibody 1G10 in the HA protein.

[0022] Figure 5 The amino acid conservation of the antigenic epitope of the monoclonal antibody 1G10 targeting the HA protein. Among them, (A) shows the conservation of amino acids at positions 170 and 231 of the HA gene of the H5 subtype influenza virus of the 2.3.2.1 branch; (B) shows the conservation of amino acids at positions 170 and 231 of the HA gene of the H5 subtype influenza virus of the 2.3.4.4 branch. Different color legends represent different amino acids, namely: A, Ala (alanine); D, Asp (aspartic acid); E, Glu (glutamic acid); G, Gly (glycine); H, His (histidine); L, Leu (leucine); K, Lys (lysine); I, Ile (isoleucine); N, Asn (aspartic acid); P, Pro (proline); T, Thr (threonine); S, Ser (serine). DETAILED DESCRIPTION

[0023] The present invention is further described below in conjunction with the accompanying drawings and specific examples so that those skilled in the art can better understand the present invention and implement it, but the examples are not intended to limit the present invention. The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials and reagents used can be obtained from commercial sources unless otherwise specified. The materials and reagents used are as follows: Dimethyl sulfoxide (DMSO), Freund's complete adjuvant, Freund's incomplete adjuvant, polyethylene glycol 1500 (PEG1500), HAT, and HT were products of Sigma; DMEM culture medium and fetal bovine serum (FBS) were products of Gibco; goat anti-mouse IgG FITC and monoclonal antibody subclass identification kit (Mouse Monoclonal Antibody Isotyping Kit) were purchased from ThermoFisher Scientific.

[0024] Example 1: Obtaining hybridoma cell line 1G10 Preparation of immunogen The Clade 2.3.2.1d H5N1 subtype avian influenza virus strain A / duck / China / 1033 / 2017 (Ge Z, Gu M, Cai T, et al. Phylogenetic tracing and biological characterization of a novel clade 2.3.2.1reassortant of H5N6 subtype avianinfluenza virus in China [J]. TransboundEmerg Dis, 2021, 68(2): 730-741) stored in the laboratory was inactivated with β-propiolactone, and the allantoic fluid of the inactivated virus (abbreviated as 171033) was centrifuged at 10000r / min and 4°C for 30min, and all the supernatant was aspirated. 20% sucrose solution was placed at the bottom, and all the supernatant was slowly added along the wall of the tube with a syringe, and the supernatant was discarded after ultracentrifugation at 30000r / min and 4°C for 2h. The ultracentrifuged precipitate was resuspended in sterile PBS buffer stored at 4°C to fill the ultracentrifuge tube, and then ultracentrifuged again at 30,000 r / min and 4°C for 2 hours, and the supernatant was discarded to remove the sucrose in the virus solution. The precipitate was resuspended in sterile PBS buffer stored at 4°C, and the amount of PBS buffer was about 1 / 50 of the amount of allantoic fluid. After the precipitate was resuspended, the HA titer was determined, filtered through a 0.45 μm filter, aliquoted, and stored at -70°C.

[0025] Animal Immunization Take an appropriate amount of the purified virus and Freund's complete adjuvant and mix them in equal proportions. Homogenize at 5500r / min, 15s each time, and homogenize 6 times to fully emulsify it; immunize 6-week-old BALB / c mice with a dose of 0.2mL / mouse and multiple subcutaneous injections. After the first immunization, perform another immunization 2-3 weeks apart. Note that Freund's incomplete adjuvant is used for antigen emulsification in the second and third immunizations. 10 days after the third immunization, obtain the three-immunity serum from the tail vein blood of the immunized mice, and determine the antibody titer in the serum by the hemagglutination inhibition (HI) method.

[0026] Cell fusion Preparation of myeloma cells One week before cell fusion, myeloma cells Sp2 / 0 were revived and cultured for about 3 times to stabilize the cell state and then expanded to a suitable amount for use. Before fusion, the cells were gently blown off with 10 mL of incomplete DMEM medium and collected in a 50 mL fusion tube for use.

[0027] Preparation of spleen lymphocytes 3 days before fusion, select mice with high titer of triple-immune serum antibodies for shock immunization, i.e., intraperitoneal injection of 0.2 mL of purified virus; on the day of fusion, blood was collected from the eyeballs to collect positive serum, and the mice to be fused were placed in 75% alcohol for 10 minutes after being killed, and then fixed in a supine position on a dissecting board in a clean bench; the spleen of the mouse was aseptically removed and placed in incomplete DMEM culture medium for washing to remove excess connective tissue and fat; the spleen cells were then transferred to a cell sieve and ground in another dish containing incomplete DMEM culture medium, and the ground spleen cell suspension was collected in a centrifuge tube and placed in a 5%-10% CO2, 37°C cell culture incubator for 10-15 minutes.

[0028] Preparation of feeder cells One day before fusion, 8-week-old negative ICR mice were taken, and the mice were killed by eye bleeding. The serum was collected as a negative control and mixed with 15% FBS DMEM HAT medium. The cells were plated in a 96-well plate at a volume of 100 μL per well, placed in 75% alcohol for 10 minutes, and then fixed on the dissection board in the clean bench in a supine position; the skin was cut to expose the peritoneum; 10mL HAT medium was drawn with a 10mL syringe and carefully injected into the abdominal cavity, and the abdomen was gently massaged with an alcohol cotton ball to draw back the fluid in the abdominal cavity. After dilution, it was dispensed into a 96-well cell culture plate and placed in a saturated humidity incubator containing 5%-10% CO2 and 37°C for culture.

[0029] Fusion: Pipette the spleen cells prepared above, leaving the orange-red impurities at the bottom of the centrifuge tube, transfer to the same fusion tube with Sp2 / 0 cells, mix, centrifuge at 100r / min for 10min to remove the supernatant, gently wipe the bottom of the fusion tube to disperse the precipitated cells, slowly add 1mL preheated PEG1500 in a 37℃ water bath environment to complete the fusion of the two, and terminate the fusion reaction with antibody-free and serum-free DMEM medium. After standing at 37℃ for 10min, centrifuge, suspend the cells with 15% FBS DMEM HAT medium, and evenly drop them into the 96-well cell plate with feeder cells on the bottom, 100 µL / well, and culture in a 5%-10% CO2, 37℃ incubator; half-change the medium with fresh HAT medium after 5d; fully change the medium with HT medium after 7-10d; observe the growth of hybridoma cells, and when the cell culture supernatant turns yellow or the cells are distributed to more than 1 / 10 of the bottom area of ​​the well, hybridoma cells can be screened, and the cell supernatant can be aspirated to detect antibodies.

[0030] Screening of hybridoma cells: The hybridoma cell supernatant in the 96-well cell plate was taken as the detection antibody, and the immunogenic virus strain 171033 was prepared into a four-unit antigen solution for HI test. The cell wells with HI results showing that the red blood cells did not agglutinate were positive cell wells with HI activity, and vice versa. The cell wells that were positive in three tests were selected for subcloning, and the hybridoma cells were cloned by the limiting dilution method; after cloning 3-5 times, the cell lines that could still stably secrete antibodies were selected for expansion and culture. Finally, a positive hybridoma cell line 1G10 was obtained.

[0031] Embodiment 2: Large-scale production of monoclonal antibodies BALB / c female mice were selected for intraperitoneal injection of sterilized liquid paraffin (0.3-0.5 mL / mouse). After 7-10 days, the hybridoma cells cultured above were cultured at a rate of about 1×10 6 / mice were injected intraperitoneally. After 5 days, the abdomen of the mother mice was observed every day. When the abdomen was obviously swollen, the ascites was collected with a 16# needle. After centrifugation at 5000r / min for 5 minutes, the supernatant was aspirated and stored in aliquots at -70℃.

[0032] Subclass identification and HI titer determination According to the instructions of the mouse monoclonal antibody (mAb) subclass identification kit, the subclass of the obtained monoclonal antibody was identified; the titer of hybridoma cell supernatant and mouse ascites was tested by HI test, and the maximum dilution multiple of the positive well was the HI titer. The results showed that 1G10 belonged to the IgG1 class antibody, the HI titer of hybridoma cell supernatant was 4 log2, and the HI titer of mouse ascites was 13 log2.

[0033] Example 3: Monoclonal Antibody Variable Region Gene Sequencing Take 1×10 6 Hybridoma cells were collected and total cellular RNA was extracted using Trizol reagent (purchased from Life Technologies, catalog number 15596026), and RNA bands were detected by 1% agarose gel electrophoresis. Subsequently, the extracted RNA was reverse transcribed into cDNA using cDNA Synthesis Kit (purchased from Nanjing Novogene Biotech Co., Ltd., catalog number R312-02). Next, the heavy chain variable region (V H ) and the light chain variable region (V L) and detected the PCR amplification product by 1.5% agarose gel electrophoresis. Then, the PCR product was connected to the pMD19-T vector and transformed into DH5α competent cells, and then spread on ampicillin-resistant LB plates; single colonies formed by overnight culture were picked and inoculated into LB liquid medium containing ampicillin, and after culturing at 37°C for 12 h, the bacterial solution was PCR- and electrophoresed using M13+ and M13- primers. Finally, the positive bacterial solution was subjected to plasmid extraction, and DNA sequencing was performed using M13+ and M13- primers to deduce the corresponding amino acid sequence.

[0034] After sequencing, the V H IgG1 subtype, V L It is a κ subtype. H The nucleotide sequence is shown in SEQ ID NO. 1, V H The amino acid sequence is shown in SEQ ID NO. 2, V L The nucleotide sequence is shown in SEQ ID NO. 3. L The amino acid sequence is shown in SEQ ID NO. 4. H and V L The complementarity-determining region (CDR) of the mitochondria includes three subregions: CDR1, CDR2 and CDR3. H The amino acid sequence of CDR1 is shown in SEQ ID NO. 5, V H The amino acid sequence of CDR2 is shown in SEQ ID NO. 6, V H The amino acid sequence of CDR3 is shown in SEQ ID NO. 7; V L The amino acid sequence of CDR1 is shown in SEQ ID NO. 8, V L The amino acid sequence of CDR2 is shown in SEQ ID NO. 9, V L The amino acid sequence of CDR3 is shown in SEQ ID NO. 10.

[0035] Example 4: Identification by indirect immunofluorescence assay (IFA) First, the HA gene of the 171033 virus strain (GenBank accession number MK554842.1) was connected to the eukaryotic expression vector pCAGGS (purchased from Miaoling Biotechnology, product number P0165) to construct the eukaryotic expression plasmid pCAGGS-171033-HA expressing the HA protein. The construction steps are as follows: Use a conventional commercial RNA extraction kit to extract the 171033 virus RNA according to the instructions, and then add the AIV universal reverse transcription primer Uni12 (Hoffmann E, Stech J, Guan Y, et al. Universal primer set for the full-length amplification of all influenza A viruses [J]. Arch Virol, 2001, 146(12): 2275-2289) for reverse transcription to obtain cDNA; design a plasmid carrying Sac I restriction site upstream primer 5'-GGCAAAGAATTCGAGCTCATGGAGAAAATAGTTCTTTTCTTTG-3' (SEQ ID NO. 11) and Nhe The full length of the HA gene was amplified by PCR using the downstream primer 5'-GGGAAAAAGATCTGCTAGCTTAAATGCAAATTCTGCACTG-3' (SEQ ID NO. 12) of the restriction site I, and the PCR product was recovered by 1% agarose gel electrophoresis using a conventional commercial DNA gel recovery kit; Nhe I and Sac I. When the empty vector is used, AGGS is double-digested and linearized by electrophoresis and gel recovery; then, the full-length HA obtained by gel recovery is connected with the linearized empty vector pCAGGS according to the instructions using a homologous recombination kit (purchased from Beijing Quanshijin Biotechnology Co., Ltd., product number CU201-02), and the connection product is transformed into DH5α competent cells, coated with LB plates containing ampicillin and cultured at 37°C, and then a single colony is picked and inoculated in ampicillin-resistant LB liquid culture medium and shaken for 12 hours before bacterial liquid PCR identification; the positive bacterial liquid is taken to use a conventional commercial plasmid extraction kit for plasmid extraction, and the plasmid that is finally verified by sequencing is the successfully constructed eukaryotic expression plasmid pCAGGS-171033-HA.

[0036] The pCAGGS-171033-HA eukaryotic expression plasmid was transfected into 293T cells, and the pCAGGS empty vector was transfected as a negative control. After 36 hours, the cell supernatant was discarded, and the cells were washed three times with PBS precooled at 4°C, and then fixed with precooled 4% paraformaldehyde at 4°C for 20 minutes. The fixative was discarded, and after it evaporated naturally, 5% skim milk was prepared with PBST and allowed to stand at room temperature for 1 hour for blocking. After blocking, the cells were washed three times with PBST, each for 5 minutes. The mouse ascites to be tested was added to the cell wells as the primary antibody, incubated overnight at 4°C, and washed three times as described above. FITC-labeled goat anti-mouse IgG fluorescent secondary antibody diluted 1:500 with PBST was added, and the cells were incubated at room temperature for 1 hour in a light-proof shaking incubator. After washing three times as above, the cells were observed for green fluorescence under a microscope. The results are shown in the attached figure. Figure 1 As shown, green fluorescence reaction can be observed in mouse ascites 1G10, mouse positive serum and 293T cells transfected with pCAGGS-171033-HA plasmid ( Figure 1 A and B); 293T cells transfected with mouse ascites 1G10 and pCAGGS empty vector and normal 293T cells showed no fluorescence reaction ( Figure 1 (C, D).

[0037] Example 5: Western blot identification The pCAGGS-171033-HA eukaryotic expression plasmid was transfected into 293T cells. After 36 hours, the cell culture medium was discarded, and the cells were washed with PBS. The excess water was sucked off with a pipette tip and placed on ice to lyse the cells. The lysate was centrifuged at 10000 r / min for 10 min, and the supernatant was collected as the protein sample. The protein sample prepared above was mixed with a certain amount of loading buffer and boiled for 5 min. After loading at 10 μL / well, SDS-PAGE was performed and the membrane was transferred to the PVDF membrane by conventional wet transfer method. After the membrane was blocked with 5% skim milk at room temperature for 2 h, 5% skim milk was used as a primary antibody with a dilution of 1: 2000 for mouse ascites, and incubated overnight at 4°C on a shaker. The membrane was washed 3 times with TBST, 5 min each time, and 1: 5000 dilution of goat anti-mouse IgG-HRP was used as a secondary antibody with TBST, and incubated at room temperature on a shaker for 1 h. After washing the membrane 3 times as above, it was developed by ultrasensitive ECL and the results were observed by imaging. The results are attached Figure 2 As shown, 1G10 can react specifically with the HA protein produced by pCAGGS-171033-HA plasmid transfection, with an obvious band at around 70 kDa; the cell samples transfected with the empty vector pCAGGS had no reaction band.

[0038] Example 6: Neutralization test identification Primary CEF cells were cultured at 5 × 10 5The density of cells / mL was plated on a 96-well cell plate, and the 171033 allantoic fluid was diluted 10 times with maintenance solution (V solution), and 5 wells of cells were inoculated at each dilution. After adsorption for 1 hour, the cells were rehydrated and placed in a 5%-10% CO2, 37°C cell culture incubator for culture. After 72 hours, the TCID was calculated based on the hemagglutination (HA) titer of the cell supernatant and the Reed-Muench method. 50 . Measure the TCID 50 The 171033 strain was diluted with V solution to 200 times TCID 50 Reserve, and dilute to 100 times TCID 50 , 10 times TCID 50 , 1 times TCID 50 , 0.1 times TCID 50 The virus solution was used for TCID 50 Regression test. Take the sterilized 1G10 mouse ascites, dilute it 10 times with V solution in advance, and then dilute it 2 times in a new 96-well plate with 50μL system, and make 4 replicates for each dilution. Next, add 200 times TCID 50 Add 50 μL / well of the diluted monoclonal antibody to a 96-well plate, and gently pipette to mix the virus solution and the monoclonal antibody evenly. At this time, the mixed virus solution contains 100 times the TCID per 100 μL. 50 At the same time, set up the regression test wells for the virus, with 4 wells for each dilution, and incubate them in a 37°C incubator for 1 hour. Discard the CEF cell culture medium cultured in the 96-well plate, transfer the mixture of virus and monoclonal antibody that has been neutralized for 1 hour in the previous step to a 96-well cell plate covered with a monolayer of CEF cells, and then add 150 μL of V solution to each well, and culture in a 5%~10% CO2, 37°C incubator. Take out the cell plate at 72 hours, detect the HA in the supernatant of each well, and calculate the neutralization titer of the monoclonal antibody by the Reed-Muench method. The results are as follows: TCID of 171033 virus on CEF cells 50 For 10 7.5 10 times the TCID in the virus regression assay for cell neutralization assay 50 All the supernatants of the inoculated cell wells can agglutinate red blood cells, while 0.1 times TCID 50 The supernatant of the inoculated cell wells was all non-agglutinated red blood cells, indicating that the virus solution dilution multiple was accurate and the neutralization test results were reliable. After determination, monoclonal antibody 1G10 had neutralization properties, and the neutralization titer was 1: 8129.

[0039] Example 7: Broad spectrum analysis The HA gene genetic tree of H5Nx subtype AIV viruses from different HA evolutionary branches is shown in the attached figure. Figure 3As shown, the red-marked strains are the strains to be tested, which are distributed in the 2.3.2.1d, 2.3.2.1e, 2.3.4.4b and 2.3.4.4d branches, including H5N1, H5N2, H5N6 and H5N8 subtypes. Through the HI test, the HI titer of 1G10 monoclonal antibody ascites was measured with each virus strain to evaluate the broad-spectrum reactivity of 1G10. The results are shown in Table 1 (HI titer was only measured to 10 log2, so the reading value of 10 is indicated by "≥10", and non-reactive is indicated by "-"). Monoclonal antibody 1G10 has good reactivity with the virus strain 171033 corresponding to the immunogen and some H5N1 strains in the 2.3.2.1d subbranch; although it does not react with the two tested H5 virus strains in the 2.3.2.1e subbranch, 1G10 can show good cross-reaction with some H5N6 and H5N8 strains in the 2.3.4.4b and 2.3.4.4d branches. This shows that monoclonal antibody 1G10 has a relatively broad spectrum of reactivity and can react specifically with H5Nx viruses of different HA branches. The recognized antigenic epitope may be a conserved broad-spectrum neutralizing epitope shared by multiple branches.

[0040] Table 1 HI reactivity of mAb 1G10 with clade 2.3.2.1 and 2.3.4.4 H5Nx viruses Note: - indicates no specific reactivity in the HI test.

[0041] Example 8: Identification of antigenic epitopes Take 200 μL of the 1G10 monoclonal antibody ascites that has been sterilized by filtration and mix it with 200 μL of 10 5 TCID 50 The allantoic fluid of 171033 or 181123 strains was mixed evenly and incubated in a 37°C incubator for 1 h. Subsequently, the mixture of the above monoclonal antibody and virus was diluted to 1 mL with sterile PBS and inoculated into 10-day-old SPF chicken embryos via the allantoic cavity route, with 0.2 mL inoculated per embryo. 72 hours after inoculation, the chicken embryo allantoic fluid was collected for HA test, and the allantoic fluid with HA titer was further subjected to HI test with 1G10. The allantoic fluid with significantly reduced HI titer was considered positive, and the virus was further purified by limiting dilution method. In brief, 200 μL of positive allantoic fluid was mixed with an equal volume of 1G10 monoclonal antibody, and after 1 h at 37°C, the mixture was diluted 10 times continuously, and 10 -5 , 10 -6 and 10 -7Dilution, each dilution was inoculated with 0.2mL / embryo for 2 10-day-old SPF chicken embryos; after 72h, the chicken embryo allantoic fluid was collected, and the chicken embryo allantoic fluid with HA titer and significantly reduced HI titer and the highest dilution multiple was used as the monoclonal antibody escape strain obtained by screening, and the HA gene sequence of the escape strain was further determined, and the mutation site was compared with the original sequence to analyze the key amino acids of the antigen epitope recognized by monoclonal antibody 1G10. The results are shown in Table 2. For strains 171033 and 181123, monoclonal antibody 1G10 screened out 1 escape mutant strain, both of which lost HI reactivity with monoclonal antibody 1G10, and the HI titer was 0. Through HA gene sequencing, the key amino acid site mutations of the escape mutant virus were identified, and it was found that the escape mutant for strain 171033 had a double site mutation of N170D and T231N, while the escape mutant for strain 181123 had only a single site mutation of N170K. The results showed that the antigen epitope recognized by monoclonal antibody 1G10 included amino acid sites 170 and 231 of HA protein, among which 170N might be more critical.

[0042] Table 2 Amino acid site mutations in the HA gene of the escape mutants screened by mAb 1G10 Note: - indicates no specific reactivity in the HI test; the mutation site number is based on the H5 subtype HA gene coding region.

[0043] On the one hand, the spatial structure location analysis of the key antigenic sites identified above was performed. Using the Swiss-Model homology modeling website (https: / / swissmodel.expasy.org / ), the H5N1 subtype avian influenza virus HA protein monomer structure numbered 6PCX included in the PDB protein structure database (https: / / www.rcsb.org / ) was used as a template to perform homology modeling on the HA protein of the 171033 virus strain, and the three-dimensional molecular modeling software PyMOL (https: / / pymol.org / ) was used to display the protein spatial structure and key amino acid sites. As shown in the attached figure. Figure 4 The side view of the spatial structure simulation diagram of the HA protein shown in the figure shows that the two sites 170N (red mark) and 231T (green mark) are both located in the globular head region of the HA protein, which is the main structural domain of the HA protein for antigen recognition function.

[0044] On the other hand, the conservation of the identified key antigenic sites was analyzed. The amino acid sequences of the HA genes of H5 subtype influenza viruses of the 2.3.2.1 branch (involving 2.3.2.1a, 2.3.2.1b, 2.3.2.1c and 2.3.2.1 without further subdivision, n=3082) and 2.3.4.4 branch (involving 2.3.4.4b, 2.3.4.4c, 2.3.4.4e, 2.3.4.4g, 2.3.4.4h and 2.3.4.4 without further subdivision, n=21775) that can be directly retrieved were downloaded from the GISAID database (https: / / gisaid.org / ). The amino acids at positions 170 and 231 of the HA gene were compared and analyzed, and the results are shown in the attached figure. Figure 5 As shown, although position 170 is mainly D in the 2.3.2.1 branch, 170N still accounts for a relatively high proportion (1026 / 3082, 33.29%), indicating that 170N is somewhat conservative in the 2.3.2.1 branch ( Figure 5 In the 2.3.4.4 branch, 170N is highly conserved (21130 / 21775, 97.04%) ( Figure 5 For 231T, both in the 2.3.2.1 branch (3050 / 3082, 98.96%) ( Figure 5 The 2.3.4.4 branch (21723 / 21775, 99.76%) is highly conserved ( Figure 5 Middle (B). This indicates that the identified antigenic epitopes are well conserved in the HA genes of viruses from different epidemic branches and can be further applied to the development of universal vaccines against influenza viruses.

Claims

1. A monoclonal antibody that broadly binds to the HA protein of multi-branched H5Nx subtype avian influenza virus, characterized in that: The monoclonal antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises V H CDR1, V H CDR2 and V H CDR3, light chain variable region contains V L CDR1, V L CDR2 and V L CDR3; The V H The amino acid sequence of CDR1 is shown in SEQ ID NO. 5; The V H The amino acid sequence of CDR2 is shown in SEQ ID NO. 6; The V H The amino acid sequence of CDR3 is shown in SEQ ID NO. 7; The V L The amino acid sequence of CDR1 is shown in SEQ ID NO. 8; The V L The amino acid sequence of CDR2 is shown in SEQ ID NO. 9; The V L The amino acid sequence of CDR3 is shown in SEQ ID NO.

10.

2. The monoclonal antibody that broadly binds to the multi-branched H5Nx subtype avian influenza virus HA protein according to claim 1, characterized in that: The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO. 2, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.

4.

3. The monoclonal antibody that broadly binds to the multi-branched H5Nx subtype avian influenza virus HA protein according to claim 1, characterized in that: The nucleotide sequence of the heavy chain variable region is shown in SEQ ID NO. 1, and the nucleotide sequence of the light chain variable region is shown in SEQ ID NO.

3.

4. An expression vector, characterized in that: The expression vector contains the heavy chain variable region nucleotide sequence and the light chain variable region nucleotide sequence according to claim 3.

5. A host cell, characterized in that The host cell contains the expression vector according to claim 4.

6. Use of the monoclonal antibody according to any one of claims 1 to 3 in the analysis of antigenic variation of influenza virus.

7. Use of the monoclonal antibody according to any one of claims 1 to 3 in the preparation of a detection reagent for H5 subtype avian influenza virus.

Images