Application of H5-Re11 hemagglutinin protein monoclonal antibody and its antigenic epitope

Abstract

The present invention discloses the application of H5-Re11 hemagglutinin protein monoclonal antibody and its antigenic epitope, and belongs to the field of biotechnology. The present invention solves the problem that the broad-spectrum vaccine for 2.3.4.4 branch H5 subtype avian influenza virus is relatively scarce. The monoclonal antibodies 2D7 and 3C8 are secreted and produced by hybridoma cell lines with deposit numbers CGMCC No.45926 and CGMCC No.45927, respectively. Sites 131, 136 and 172 of the hemagglutinin protein are key antigenic epitopes recognized by the two monoclonal antibodies. The artificial recombinant vaccine H5-Re11+3 constructed based on the epitope has good immunogenicity, can provide broad-spectrum immune protection for 2.3.4.4 branch H5 subtype avian influenza virus, and has good development and application prospects. The monoclonal antibodies and the epitopes recognized by the present invention have high application value in the field of avian influenza vaccine preparation.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and specifically relates to a monoclonal antibody of hemagglutinin protein of H5-Re11 vaccine strain and application of its recognition epitope. Background Art

[0002] Avian influenza is an infectious disease of poultry caused by influenza A virus, with many subtypes, wide hosts and serious harm. According to the antigenic and genetic characteristics of the glycoproteins hemagglutinin (HA) and neuraminidase (NA) on the surface of influenza virus particles, influenza A virus can be divided into 18 HA subtypes and 11 NA subtypes. Among the many subtypes, the H5 subtype highly pathogenic avian influenza virus is the most harmful. According to statistics, since 2004, more than 36 million poultry in my country have been culled for the prevention and control of H5 subtype avian influenza, causing huge economic losses to the poultry farming industry. The H5 subtype avian influenza virus can not only cause a large number of deaths in poultry, but also break through the interspecies barrier to infect humans. According to statistics from the World Health Organization (WHO), as of February 26, 2024, 887 people have been infected with the H5N1 subtype avian influenza virus, 462 people have died, and the mortality rate is as high as 52%.

[0003] At present, vaccine immunization is the most effective means of preventing and controlling avian influenza. In the process of epidemic, influenza virus is very easy to mutate to form new antigen groups. In order to prevent and control H5 subtype avian influenza, the National Avian Influenza Reference Laboratory needs to update the vaccine strain every few years to match the latest epidemic strains so that the vaccine can achieve the best protection effect. In view of the antigenic characteristics of the currently popular 2.3.4.4 branch H5 subtype avian influenza virus, the National Avian Influenza Reference Laboratory has updated the H5-Re8, H5-Re11, H5-Re13 and H5-Re14 series of vaccines. However, the update of vaccines always lags behind the evolution of the virus. If a broad-spectrum vaccine against the 2.3.4.4 branch H5 subtype avian influenza virus can be developed, it will greatly improve the level of vaccine immune protection and reduce the frequency of epidemics caused by H5 subtype avian influenza mutant viruses. Summary of the invention

[0004] The invention discloses a monoclonal antibody of hemagglutinin protein of H5-Re11 vaccine strain and application of its recognition epitope, solving the problem of scarcity of broad-spectrum vaccines against 2.3.4.4 branch H5 subtype avian influenza virus.

[0005] The technical solution of the present invention is as follows:

[0006] A hybridoma cell line that secretes monoclonal antibodies to the hemagglutinin protein of the H5-Re11 vaccine strain, wherein the hybridoma cell line is hybridoma cell line 2D7, and the hybridoma cell line 2D7 is deposited in the General Microbiology Center of the China Culture Collection of Microorganisms, with the deposit address being No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, with the deposit number being CGMCC No.45926, and the deposit time being April 24, 2024; or, the hybridoma cell line is hybridoma cell line 3C8, and the hybridoma cell line 3C8 is deposited in the General Microbiology Center of the China Culture Collection of Microorganisms, with the deposit address being No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, with the deposit number being CGMCC No.45927, and the deposit time being April 24, 2024.

[0007] The monoclonal antibody is 2D7, which is secreted by hybridoma cell line 2D7, and the deposit number of hybridoma cell line 2D7 is CGMCC No.45926. It is deposited in the General Microbiology Center of China National Microbiological Culture Collection Committee, and the deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit time is April 24, 2024. The nucleotide sequence of the heavy chain variable region of the monoclonal antibody 2D7 is shown in SEQ ID NO.1, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.2, the nucleotide sequence of the light chain variable region is shown in SEQ ID NO.3, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.4; or the monoclonal antibody is monoclonal antibody 3C8, which is secreted by hybridoma cell line 3C8, and the deposit number of the hybridoma cell line 3C8 is CGMCC No.45927, deposited in the General Microbiology Center of China National Culture Collection Administration, the deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit time is April 24, 2024. The nucleotide sequence of the heavy chain variable region of the monoclonal antibody 3C8 is shown in SEQ ID NO.5, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.6, the nucleotide sequence of the light chain variable region is shown in SEQ ID NO.7, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.8.

[0008] Furthermore, the key antigenic epitopes recognized by the monoclonal antibody 2D7 are glutamine at position 131 and arginine at position 136 of the H5-Re11 hemagglutinin protein, and the key antigenic epitope recognized by the monoclonal antibody 3C8 is alanine at position 172 of the H5-Re11 hemagglutinin protein.

[0009] The monoclonal antibody of the hemagglutinin protein of the H5-Re11 vaccine strain is used in the preparation of H5 subtype avian influenza vaccine.

[0010] A recombinant avian influenza virus, the recombinant avian influenza virus is named avian influenza virus H5-Re11+3, and is deposited in the General Microbiology Center of China Culture Collection Administration, the deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, the deposit number is CGMCC No.45965, and the deposit time is April 24, 2024.

[0011] A vaccine for preventing and treating H5 subtype avian influenza virus, wherein the vaccine contains the artificial recombinant avian influenza virus H5-Re11+3.

[0012] Beneficial effects: The present invention discloses two hybridoma cell lines that can secrete monoclonal antibodies of the hemagglutinin protein of the H5-Re11 vaccine strain, and the hybridoma cell lines are obtained by fusing spleen cells of immunized mice and mouse myeloma cells. It has been identified that sites 131, 136 and 172 of the hemagglutinin protein are the key antigenic epitopes recognized by the two monoclonal antibodies. The present invention constructs an artificial recombinant virus H5-Re11+3 based on the key antigenic epitopes recognized by the above monoclonal antibodies. Compared with the H5-Re11 vaccine strain, the artificial recombinant virus H5-Re11+3 has good cross-reactivity and reacts well with the serum of the 2.3.4.4 branch H5-Re8, H5-Re11, H5-Re13 and H5-Re14 series vaccine strains. The artificial recombinant virus H5-Re11+3 is a broad-spectrum vaccine. When it is used as an immunogen to prepare a vaccine for immunizing chickens, it will produce good immune protection against attacks by wild-type viruses corresponding to the 2.3.4.4 branches H5-Re8, H5-Re11, H5-Re13 and H5-Re14, and has good development and application prospects.

[0013]

Biological Deposit Information

[0014] The hybridoma cell line 2D7 is classified and named as a hybridoma cell line, and is deposited in the General Microbiology Center of the China Culture Collection Administration. The deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing. The deposit number is CGMCC No. 45926, and the deposit date is April 24, 2024.

[0015] The hybridoma cell line 3C8 is classified and named as a hybridoma cell line, and is deposited in the General Microbiology Center of the China Culture Collection Administration. The deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing. The deposit number is CGMCCNo.45927, and the deposit date is April 24, 2024.

[0016] The avian influenza virus H5-Re11+3, classified and named avian influenza virus, is deposited in the General Microbiology Center of the China Culture Collection Administration. The storage address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing. The storage number is CGMCC No. 45965 and the storage time is April 24, 2024. DETAILED DESCRIPTION

[0017] In order to explain the present invention more clearly, the present invention will be described in detail below in conjunction with specific embodiments, but the present invention is not limited to the following embodiments.

[0018] Example 1

[0019] Preparation and identification of monoclonal antibodies against the hemagglutinin protein of the H5-Re11 vaccine strain.

[0020] 1.1 Preparation of immunogens and animal immunization

[0021] The H5-Re11 vaccine strain was diluted 10-fold 4 After that, inoculate 9-day-old SPF chicken embryos, collect hemagglutination-positive chicken embryo allantoic fluid, add β-propiolactone to inactivate, use ultrafiltration tube to concentrate the inactivated virus allantoic fluid to make its hemagglutination titer reach above 8log2, and store it at -70℃ for future use after sterility test.

[0022] The frozen pCAGGS-Re11 HA positive bacterial liquid was inoculated into liquid LB medium at a ratio of 1:1000. After overnight culture at 37°C, the bacteria were collected by centrifugation. The plasmid was extracted using the plasmid extraction kit of Novozymes. The concentration was adjusted to 500 ng / μl and stored in a -70°C refrigerator for future use.

[0023] The prepared inactivated virus and plasmid were used to immunize mice according to the immunization program in Table 1. The first immunization was performed by intramuscular injection of eukaryotic expression plasmids, with an immunization dose of 50 μg / mouse; 14 days after the first immunization, the mice were immunized for the second time by intraperitoneal injection of inactivated virus, with an immunization dose of 500 μL / mouse; 14 days later, the mice were immunized for the third time by intraperitoneal injection of inactivated virus, with an immunization dose of 500 μL / mouse. Three days before fusion, mice were given a booster immunization with intraperitoneal injection of H5-Re11 live virus, with an immunization dose of 1 mL / mouse.

[0024] Table 1 Immunization procedure for preparing monoclonal antibodies against hemagglutinin protein of H5-Re11 vaccine strain

[0025]

[0026] 1.2 Hybridoma cell preparation

[0027] The spleen of the mouse was collected aseptically, and the spleen was ground with the piston of a 2.5mL syringe to evenly disperse the spleen cells, and then transferred to a sterile 50mL centrifuge tube. SP2 / 0 cells were blown down with serum-free DMEM and placed in the centrifuge tube containing spleen cells, gently mixed, centrifuged at 800rpm for 10min, and the supernatant was discarded. Then the centrifuge tube was placed in a 37℃ water bath, and 1mL of preheated PEG was slowly added within 45s, while shaking the centrifuge tube to mix it evenly. After standing for 1min, serum-free DMEM was slowly added to 40mL, centrifuged at 800rpm for 10min, and the supernatant was discarded. The fused cells were resuspended in DMEM containing 1% HAT and 20% FBS preheated at 37℃ and distributed into 96-well plates, 100μL per well, and then placed in a 37℃ cell culture incubator containing 5% CO2 for culture.

[0028] 1.3 Screening and subcloning of positive hybridoma cells

[0029] When the fused cells grow to more than 1 / 2 of the size of the bottom of the 96-plate, the positive hybridoma cells are screened by the hemagglutination inhibition method. 25 μL of the hybridoma cell supernatant is aspirated into the hemagglutination plate, 25 μL of PBS is added, and then 4 units of H5-Re11 antigen are added to the hybridoma cell supernatant for 15 minutes, and then 25 μL of 1% chicken red blood cells are added, and the results are recorded after waiting for about 25 minutes. The cells in the wells with hemagglutination inhibition activity are resuspended, and subcloned by cell counting and limiting dilution. After three subclones, it is ensured that each hybridoma cell is a single cell clone. Finally, the positive cell lines are frozen and the cell supernatant is collected for standby use. Finally, two hybridoma cells that can secrete monoclonal antibodies to the hemagglutinin protein of the H5-Re11 vaccine strain were screened, named hybridoma cells 2D7 and hybridoma cells 3C8, respectively. The cell line is deposited in the General Microbiology Center of China Culture Collection Administration, located at the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The deposit numbers are CGMCC No.45926 and CGMCC No.45927, and the deposit date is April 24, 2024.

[0030] 1.4 Preparation of monoclonal antibodies against the hemagglutinin protein of the H5-Re11 vaccine strain

[0031] The mice were intraperitoneally injected with Freund's incomplete adjuvant, 0.5 mL per mouse. Three days after the injection, the hybridoma cells in good cell condition were blown off, centrifuged at 800 rpm for 10 min, the supernatant was discarded, the cells were resuspended with sterile PBS, centrifuged again, and the supernatant was discarded. Sterile PBS was added to resuspend the cells, and the cells were counted and diluted to contain 5×10 per 500 μL. 5cells. Evenly mix the cell dilution and then inject it into the mouse peritoneal cavity, 500 μL per mouse. Within 7-10 days after cell injection, observe the mouse status every day. When the mouse abdomen is swollen, use a syringe to draw the mouse ascites, after overnight at 4°C, centrifuge at 4500rpm for 10 minutes, collect the ascites supernatant, and place it in a -70°C refrigerator for later use.

[0032] 1.5 Identification of subclasses of monoclonal antibodies against the hemagglutinin protein of the H5-Re11 vaccine strain

[0033] Hybridoma cells 2D7 and 3C8 were cultured in serum-free medium, and the cell supernatants were collected. The subclasses of monoclonal antibodies in the cell supernatants were identified according to the instructions of the antibody subclass identification kit. It was determined that the heavy chain subclasses of monoclonal antibodies 2D7 and 3C8 were IgG2b and IgG2a, respectively, and the light chain subclasses were both κ chains.

[0034] Specificity detection of monoclonal antibodies against the hemagglutinin protein of 1.6H5-Re11 vaccine strain

[0035] The cross-hemagglutination inhibition experiment was conducted with the monoclonal antibody 2D7 and monoclonal antibody 3C8 against the hemagglutinin protein of the H5-Re11 vaccine strain and the 2.3.4.4 branch series antigen vaccine strains H5-Re8, H5-Re11, H5-Re13 and H5-Re14, respectively. The results are shown in Table 2: Monoclonal antibody 2D7 only reacts with H5-Re11, but has no cross-reaction with H5-Re8, H5-Re13 and H5-Re14, indicating that monoclonal antibody 2D7 has good specificity for H5-Re11. In addition to having a good reaction with H5-Re11, monoclonal antibody 3C8 also has a low level of cross-reaction with H5-Re14, but does not react with H5-Re8 and H5-Re13.

[0036] Table 2 Specificity detection of monoclonal antibodies against hemagglutinin protein of H5-Re11 vaccine strain

[0037]

[0038] 1.7 Determination of the variable region sequence of the monoclonal antibody against the hemagglutinin protein of the H5-Re11 vaccine strain

[0039] Total RNA from hybridoma cells 2D7 and 3C8 was extracted using an animal tissue / cell total RNA extraction kit (Tian Gen, Catalog No. DP451), and then cDNA was prepared using SMARTer RACE 5' / 3'kit (Takara, Catalog No. 634858). PCR amplification was performed using the conserved sequence of mouse antibody genes as primers, and the PCR products were sequenced using the Big Dye Terminator kit to obtain the variable region sequences of monoclonal antibodies 2D7 and 3C8.

[0040] The nucleotide sequence of the variable region heavy chain of monoclonal antibody 2D7 is:

[0041] CAGATCCAGTTGGTGCAGTCTGGACCTGAGCTGAAGAAGCCTGGAGAGACAGTCAAGATCTCCTGCAAGGCTTCTGGATATTCCTTCACAAACTATGGAATGAACTGGGTGAAGCAGGCTCCAGGAAAGGGTTTAAAGTGGATGGGCTGGATAGACACCTACACTGGAGAGCCGACATATGCTGATGACTTCAAG GGACGGTTTGCCTTCTTTGGAAACCTCTGCCAGCACTGCCTATTTGCAGATCAACAACCTCAAAAATGAGGACATGGCTACATATTTCTGTGCAAGACGAGATTACTACGGTGGTGCCAACTGGTATATCGATGTCTGGGCCGCAGGGACCACGGTCACCGTCTCCTCAGCCAAAACAACACCCCCATCA(SEQ ID NO.1).

[0042] According to the codon rules, the amino acid sequence of the heavy chain of the variable region of monoclonal antibody 2D7 can be obtained as follows:

[0043] QIQLVQSGPELKKPGETVKISCKASGYSFTNYGMNWVKQAPGKGLKWMGWIDTYT GEPTYADDFKGRFAFSLETSASTAYLQINNLKNEDMATYFCARRDYYGGANWYIDVWAA GTTVTVSSAKTTPPS (SEQ ID NO. 2).

[0044] The nucleotide sequence of the variable region light chain of monoclonal antibody 2D7 is:

[0045] GACATTTGTGCTCACCCAATCTCCAGCTTCTTTGGCTGTGTCTCTAGGGCAGAGAGCCACCATCTCCTGCAGAGCCAGTGAAAGTGTTGAATATTATGGCACAAGTTTAGTGCAGTGGTACCAACATAAACCAGGACAGCCACCCAAACTCCTCATCTATGCTGCATCCAA CGTAGAATTCTGGGGTCCCTGCCAGGTTTAGTGGCAGTGGGTCTGGGACAGACTTCAGCCTCAACATCCATCCTGTGGAGGAGGATGATATTGCAATGTATTTCTGTCACCAAAGTAGGAAGGTTCCTTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAACGG(SEQ ID NO.3).

[0046] According to the codon rules, the amino acid sequence of the light chain of the variable region of monoclonal antibody 2D7 can be obtained as follows:

[0047] DIVLTQSPASLAVSLGQRATISCRASESVEYYGTSLVQWYQHKPGQPPKLLIYAASNVESGVPARFSGSGSGTDFSLNIHPVEEDDIAMYFCHQSRKVPWTFGGGTKLEIKR (SEQ ID NO. 4).

[0048] The nucleotide sequence of the variable region heavy chain of monoclonal antibody 3C8 is:

[0049] GAGATCCAGTTGCAGCAGTCTGGACGTGAGCTGGTGAAGCCTGGGGCTTCAGTGAAGGTTTCCTGCAAGGCTTCTGGTTATCCATTCACTAACTACcACAtgTACTGGgTGAAGCAGAGCCATGGAGAGAGCCTTGAGTGGATTGGATATATTGATCCTTACAATGGTGTTATTAACTTCAACC AGAAGTTCAAGGGCAAGGCCACATTGACTGTTGACAAGTCCTCCAGCACAGCCTACATACATCTCAACAGCCTGACATCTGAGGACTCTGCAGTCTATTACTGtGCAAGATCAGATGGTAACGACCCCCATTTCTATGCTTTGGCTATTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA(SEQ IDNO.5).

[0050] According to the codon rules, the amino acid sequence of the heavy chain of the variable region of monoclonal antibody 3C8 can be obtained as follows:

[0051] EIQLQQSGRELVKPGASVKVSCKASGYPFTNYHMYWVKQSHGESLEWIGYIDPYNG VINFNQKFKGKATLTVDKSSSTAYIHLNSLTSEDSAVYYCARSDGNDPHFYALAIGVKEPQS PSP (SEQ ID NO. 6).

[0052] The nucleotide sequence of the variable region light chain of monoclonal antibody 3C8 is:

[0053] GACATTTGTGCTGACACAGTCTCCTGCTTCCTTAGCTGTATCTCTGGGGCAGAGGGCCACCATCTCATACAGGGCCAGCAAAAGTGTCAGTACATCTGGCTATAGTTATATGCACTGGAACCAACAGAAACCAGGACAGCCACCCAGACTCCTCATCTATCTTGTA TCCAACCTAGAATCTGGGGTCCCTGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACCCTCAACATCCATCCTGTGGAGGAGGAGGATGCTGCAACCTATTACTGTCAGCACATTAGGGAGCTTACACGTTCGGAGGGGGGACCAAGCTGGAAATAA(SEQ ID NO.7).

[0054] According to the codon rules, the amino acid sequence of the light chain of the variable region of monoclonal antibody 3C8 can be obtained as follows:

[0055] DIVLTQSPASLAVSLGQRATISRASKSVSTSGYSYMHWNQQKPGQPPRLLIYLVSNLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHIRELTRSEGGPSWK (SEQ ID NO. 8).

[0056] Example 2

[0057] Identification of key antigenic sites recognized by monoclonal antibodies against the hemagglutinin protein of the H5-Re11 vaccine strain.

[0058] 2.1 Rescue of H5-Re11 HA single-point mutation virus

[0059] The results of specificity experiments showed that monoclonal antibodies 2D7 and 3C8 could react with H5-Re11 virus, but could not react with H5-Re8 virus (Table 2). Comparison of the HA1 protein sequences of H5-Re8 and H5-Re11 revealed that the HA1 proteins of H5-Re8 and H5-Re11 had 21 different amino acid sites (Table 3), suggesting that these 21 different amino acids may contain key antigenic epitopes recognized by monoclonal antibodies 2D7 and 3C8.

[0060] Table 3 Different amino acid sites of HA1 protein between H5-Re8 and H5-Re11

[0061]

[0062] In order to identify the key antigen epitopes recognized by monoclonal antibodies 2D7 and 3C8, the recombinant plasmid pBD-H5-Re11 HA was used as the backbone, and the 21 different amino acid sites between the HA1 protein sequences of H5-Re8 and H5-Re11 were mutated one by one. The 21 recombinant mutant plasmids constructed were mixed with 7 pBD bidirectional transcription vector plasmids of PB2, PB1, PA, NP, NA, M and NS genes of A / PR / 8 / 34 (H1N1), and transfected into 293T cells under the action of transfection reagent LTX. The cells and supernatant harvested after culture were inoculated into chicken embryos to obtain 21 artificial recombinant mutant viruses with hemagglutination activity (Table 4).

[0063] Table 4 Cross-hemagglutination inhibition titer of H5-Re11 hemagglutinin protein monoclonal antibody and mutant virus

[0064]

[0065] / : indicates that no cross-hemagglutination inhibition titer was detected.

[0066] 2.2 Screening of key antigen epitopes recognized by monoclonal antibodies against H5-Re11 hemagglutinin protein

[0067] The cross-hemagglutination inhibition experiment was performed with the rescued 21 recombinant mutant viruses and monoclonal antibodies 2D7 and 3C8 to screen the key antigenic epitopes recognized by monoclonal antibodies 2D7 and 3C8. The results are shown in Table 4: When the H5-Re11 hemagglutinin protein undergoes Q131L or R136S mutation, the monoclonal antibody 2D7 and its hemagglutination inhibition titer can be completely eliminated, indicating that the glutamine (Q) at position 131 and the arginine (R) at position 136 are the key antigenic epitopes recognized by the monoclonal antibody 2D7; when the H5-Re11 hemagglutinin protein undergoes A172T mutation, the monoclonal antibody 3C8 and its hemagglutination inhibition titer are also completely eliminated, indicating that the alanine (A) at position 172 is the key antigenic epitope recognized by the monoclonal antibody 3C8.

[0068] Example 3

[0069] Application of monoclonal antibodies to recognize epitopes of the hemagglutinin protein of the H5-Re11 vaccine strain.

[0070] 3.1 Preparation and identification of recombinant virus H5-Re11+3

[0071] (1) HA gene mutation primers were designed, and pBD-Re11 HA was used as a template. The H5-Re11 HA gene inserted into the pBD vector was mutated to Q131L, R136S, and A172T by PCR amplification, and the mutant vector pBD-Re11 HA+3 was constructed.

[0072] (2) The mutant vector pBD-Re11 HA+3 constructed in (1) was mixed with 7 pBD bidirectional transcription vector plasmids containing the PB2, PB1, PA, NP, NA, M and NS genes of the influenza virus chicken embryo high titer adapted strain A / PR / 8 / 34 (H1N1), and transfected into 293T cells under the action of the transfection reagent LTX. The cells and supernatant harvested after culture were inoculated into chicken embryos to obtain an artificial recombinant virus with hemagglutination activity. The artificial recombinant virus was named avian influenza virus H5-Re11+3 and deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration with a deposit number of CGMCC No.45965 and a deposit date of April 24, 2024.

[0073] 3.2 Antigenicity analysis of recombinant virus H5-Re11+3

[0074] The recombinant virus H5-Re11+3 was subjected to a cross-hemagglutination inhibition test with vaccine sera from different subbranches of 2.3.4.4 (H5-Re8, H5-Re11, H5-Re13 and H5-Re14). The results are shown in Table 5. The antigenicity difference between H5-Re11+3 and vaccine sera from different branches was within 8 times, and it had good cross-reactivity.

[0075] Table 5 Antigenicity analysis of recombinant virus H5-Re11+3

[0076]

[0077] 3.3 Analysis of immunogenicity of recombinant virus H5-Re11+3

[0078] The recombinant virus H5-Re11+3 was inactivated with 0.2% formaldehyde, and the hemagglutination titer was adjusted to 8log2. Lytol white oil from Sornneborn was used as an adjuvant to prepare an oil emulsion inactivated vaccine. 3-week-old SPF chickens were immunized with 0.3 ml / chicken by intramuscular injection. A blank control group of PBS was set up at the same time. The hemagglutination inhibition antibody titer of all chicken sera was tested 21 days after immunization, and 10 5 EID 50 The doses were used to attack the strong wild-type strains corresponding to the H5-Re8, H5-Re11, H5-Re13 and H5-Re14 vaccines. The chickens were observed for 14 days after the challenge, and the incidence and mortality of the chickens were recorded. On the 3rd and 5th days after the challenge, the chickens' laryngeal and cloacal swabs were collected and inoculated into chicken embryos to detect the detoxification.

[0079] The results are shown in Table 6: 21 days after immunization, the average HI antibody titer against the recombinant virus H5-Re11+3 in the sera of the immunized chickens in different groups was between 7.2 and 7.7 log2, while the average HI antibody titer against the recombinant virus H5-Re11+3 in the sera of the chickens in the control group was less than 2 log2 (HI<2log2 was judged as HI antibody negative). 21 days after immunization, the wild-type strong strains H5-Re8 WT, H5-Re11 WT, H5-Re13 WT and H5-Re14 WT corresponding to the H5-Re8, H5-Re11, H5-Re13 and H5-Re14 vaccines were challenged by nasal infection. During the 14-day observation period, all immunized chickens survived without obvious clinical symptoms, and the virus detection of the laryngeal and cloacal swab samples on the 3rd and 5th days after the challenge was negative; while the chickens in the control group all became ill and died within 4 days after the challenge, and the virus isolation results of the laryngeal and cloacal swab samples of the dead chickens were all positive. The above results show that the vaccine prepared with the recombinant virus H5-Re11+3 has good immunogenicity and can provide complete immune protection against H5 subtype avian influenza viruses of different subbranches 2.3.4.4.

[0080] Table 6 HI antibody, virus shedding and survival of chickens immunized with the vaccine prepared from the recombinant virus H5-Re11+3

[0081]

[0082] * All chickens in the control group died within 4 days after infection. Virus excretion was detected in the larynx and cloaca of all dead chickens. The excretion results were combined and counted into the results on the 3rd day after infection.

[0083] / No chickens survived on day 5 after challenge.

Claims

1. A hybridoma cell line secreting monoclonal antibodies against the hemagglutinin protein of the H5-Re11 vaccine strain, characterized in that: The hybridoma cell line is hybridoma cell line 2D7, which is deposited in the General Microbiology Center of China National Microorganism Culture Collection Administration, with the deposit address being No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, with the deposit number being CGMCC No. 45926, and the deposit time being April 24, 2024; or, the hybridoma cell line is hybridoma cell line 3C8, which is deposited in the General Microbiology Center of China National Microorganism Culture Collection Administration, with the deposit address being No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, with the deposit number being CGMCC No. 45927, and the deposit time being April 24, 2024.

2. A monoclonal antibody against the hemagglutinin protein of the H5-Re11 vaccine strain, characterized in that: The monoclonal antibody is 2D7, which is secreted by hybridoma cell line 2D7, and the deposit number of the hybridoma cell line 2D7 is CGMCC No.45926. It is deposited in the General Microbiology Center of China National Microbiological Culture Collection Committee, and the deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing. The deposit time is April 24, 2024. The heavy chain variable region nucleotide sequence of the monoclonal antibody 2D7 is shown in SEQ ID NO.1, the heavy chain variable region amino acid sequence is shown in SEQ ID NO.2, the light chain variable region nucleotide sequence is shown in SEQ ID NO.3, and the light chain variable region amino acid sequence is shown in SEQ ID NO.4; or the monoclonal antibody is monoclonal antibody 3C8, which is secreted by hybridoma cell line 3C8, and the deposit number of the hybridoma cell line 3C8 is CGMCC No.45927, deposited in the General Microbiology Center of China National Culture Collection Administration, the deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit time is April 24, 2024. The nucleotide sequence of the heavy chain variable region of the monoclonal antibody 3C8 is shown in SEQ ID NO.5, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.6, the nucleotide sequence of the light chain variable region is shown in SEQ ID NO.7, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.

8.

3. An artificial recombinant avian influenza virus, characterized in that: The artificial recombinant avian influenza virus is named avian influenza virus H5-Re11+3, and is deposited in the General Microbiology Center of the China Culture Collection Administration. The deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing. The deposit number is CGMCC No.45965, and the deposit time is April 24, 2024.

4. A vaccine for preventing and treating H5 subtype avian influenza virus, characterized in that: The vaccine comprises the artificial recombinant avian influenza virus H5-Re11+3 described in claim 3.

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