Cloning And Sequencing Of HA Gene Of H5 Subtype Avian Influenza Virus Isolate

Content : Avian influenza(AI) is one of infectious diseases which cause serious loss to poultry industry. Hemagglutinin(HA) is proved to be the main immune pathogen of chicken accounting for inducing of antibody against AI and main target antigen causing humoral immunity in chicken. It plays an important role in anti-infection immunity. The purpose of this research is to clone the HA gene which will provide the basis for the producing of nucleic acid vaccine in the future. 12-day-old SPF chicken embryo was inoculated with various concentration of AIV (H5 subtype). Then allantoidal fluid was collected and virus titer was measured. After the collected allantoidal fluid was concentrated, RNA of AIV was extracted using One-step-method. One pair of specific primers PAI1 and PAI2 was designed according to the sequence of HA gene of AIV H5 subtype reference strain. The fragment of HA gene was amplified by RT-PCR using its genome RNA as template. The obtained target fragment was cloned into the vector pMD18-T and then E.coli was transformed. .Following the restrictive enzyme of BamH, HindHI digestion and PCR identification, the positive recombinant plasmid was named for pHABZ. The sequencing was performed by dideoxy method, the results shows that the cloned fragment is about 900 bases and the homology of nucleotide sequence among this isolate and other typical strains (H5 subtype) such as HN/2002, HK/2000, SH/2000, HK/97, Michigan/80 reported in the world is 80.0%-98.6% and that of the deduced amino acid is 89.7%-98.3% by NCBI blastn analysis. The homogeneity between the isolate and the strain A/chicken/Hong Kong/481/97(H5N1) is up to 97.8%. There is minor variation in some nucleotide sequences. And the results indicate by analysis that the deduced amino acid sequence contains characteristic sequence of splitting site of high pathogenic AI strain. The product cloned will provide reliable evidence for the establishment of quick and accurate molecular biological diagnostic method and lay a profound foundation for the study of gene engineering vaccine of AI.