Cloning And Sequence Analysis Of HA Gene Of H9 (N2) Avian Influenza Virus

Haemagglutinin is one of two kinds of surface glycoprotein,which primarily induce humoral immunity. In this study,avian influenza isolate (H9)was propagated in SPF chicken embryo. Virus RNA was extracted from allantoic fluid by means of guandidinium isothiocyanate. The full-length HA cDNA was amplified using RT-PCR.Then I purified and extracted the target fragment by using UNIQ-10 pillar purification kit and cloned the product added A-tail into pGEM-T easy vector by T-A ligation.The JM109 competent cells were transformed with the recombinant plasimid. Consequently,the recombinant plasimid was amplified and extracted from the transformants and tested the inserting of DNA fragment. Sequencing the HA fragment of the positive clone showed that we have cloned the three strains whole length of HA gene(about). Compared with the published HA sequence,the highest homology is 98%,showing that these viruses were closely related ;Phylogenetic analysis of the three viruses revealed that Zhuhai isolates cluster with Eurasian origin;none of the Zhuhai AIVs had multiple basic amino acids at the cleavage site that confer high pathogeicity to some H5 and H7 AIVs. The product provided a good foundation for the study of gene chips of AIV.