Expression And Purification Of HA1Gene Of H5N1AIV In E.coli

Avian influenza (AI) is a serious disease of poultry throughout the world. Hemagglutinin(HA) is the major surface glycoprotein of AIV, representing 90% of the envelope protein. It is the most important antigen in developing immunity against the virus. In addition, antigenicity and immunogenicity may very depend on the region of HA expressed, and HA1 is the most suitable expression region.In this study, The HA1 gene of A/Chicken/Guangdong/2/97(H5Nl) AIV were cloned by reverse transcription polymerase chain reaction (RT-PCR) using primers designed and the sequences and amino acids were analyzed with molecular softpakage. The homogeneity of the HA1 of A/chicken/guangdong/2/97(H5N 1 )gene with the published gene of A/Goose/Guangdong/1/9 6(H5N1) and A/Hongkang/156/97(H5Nl) is 98% and 97%, and they all had an insert of basic amino acids at the cleavage site , meaning the H5N1 isolate virus is a highly pathogenic virus.The HA 1 gene was inserted into the bacterial plasmid pGEX-4T-2 and the recombinant plasmids containing HA 1 gene were identified by restriction enzyme analysis and PCR mathod.The recombinant fusion protein was highly expressed in E.coli BL21 induced by Immol/L IPTG in the form of inclusion bodies. The molecular weights of GST-H A1 is 62KD. Western-blot analysis with AIV antibodies against H5 subtype showed the recombinant protein shared good antigencity and specificity.The inclusion bodies of recombinant protein were purified with washing buffer consisting of various urea (2mol/L and 4mol/L) for several times, and then dissolve the fusion protein in the denature buffer using 8mol/L urea as denaturant. After that a good renature result was obteined using gradient with lower protein concentration.The purity of the recombinant protein by affinity-purification of Glutathione Sepharose 4B and electrodialysis method was also attained perfect result.The antigenicity of the GST-HA1 protein were identified using ELISA mathod. The recombinant protein exhibited specific binding to antiviral antibodies of H5 subtype and has no cross-reaction with H7 and H9 subtype. The result show that the purified protein has excellent antigenicity and specificity.The studies provided fundamental data and materials not only for the development of monoclonal antibody (MAb) , but also for the preparation and development of special diagnosis kits of H5 AIV subtypes.