Preparation Of Monoclonal Antibodies Against HA Protein Of AIV H5N1 Subtype And High-efficiency Expression And Purification Of Hemagglutinin Protein From Avian Influenza Virus H5N1

In this study, monitoring of avian influenza in recent years, the separation of avian influenza virus subtype H5N1 strain A/Chicken/Guangdong/S2261/2009 (H5N1) and A/Chicken/Jiangsu/ S1147/2010 (H5N1) is by immunizing 4 to 6 weeks BALB/c mice immunized three times and strengthen the immune spleen cells and myeloma cells SP2/0 fusion, two strains of the virus to the whole virus as coating antigen for ELISA, and the hemagglutination inhibition test (HI) test screening, the positive cells by limited dilution and selection for indirect ELISA test, by 4 times to obtain the six hybridoma subclone, can steadily secrete anti-HA antibody, followed by the name GD2611A4, GD2612F5, GD2613D3, JS1471B5, JS1474D1, JS1475C5. HA-specific monoclonal antibody of subtype H5 sub-type identification that, GD2612F5 and JS1474D1 for the IgM class, the rest of the IgG2a subclass, light chain areκchain. The hemagglutination inhibition test, HA-specific and most of the six strains of hybridoma cells can be combined with the avian flu virus strain has good broad-spectrum and stability. Chromosome number found myeloma cells with spleen cells from the number of chromosomes and. Six hybridoma HI titers were as follows: 2¹², 2¹²,2¹¹,2¹⁰,2¹²,2¹⁰. Hemagglutination test results show that the prepared hybridoma cells and other subtype avian influenza virus and no cross reaction of Newcastle disease virus. After cryopreservation, the still high levels of antibodies, hybridoma cells were obtained and infected with H5 subtype AIV in 293T corresponding specific binding occurs, capture fluorescently labeled secondary antibodies, the fluorescent bright green under a microscope fluorescence. H5 subtype of avian influenza-specific anti-HA monoclonal antibody was successfully prepared for AIV surveillance, diagnosis and differential diagnosis of the material to provide a good foundation for the study of structure and function of viral HA antigen to provide the material means.According to AIV HA strain genome sequence, specific PCR primers designed and synthesized, amplified by conventional PCR H5 subtype avian influenza HA gene, cloned HA gene inserted into the transfer vector PFastbac1 point in The recombinant transfer vector was constructed good recombinant transfer vector transposition DH10 competent, then the recombinant of transposons, transfected insect cells by recombinant baculovirus, a large proliferation of suspension culture was expressed protein electropHoresis for protein transfer and conventional Western- blotting identified, while doing immunofluorescence, positive identification of the purified protein with His tag protein purification methods, and good protein purified through gel filtration chromatograpHy oligomer and trimer, obtained higher concentrations and has a blood clotting protein trimer activity for H5 subtype of influenza virus may antigenic structure and location of sites provide the basis for the forecast.