Cloning And Expression Of Partial Gene Of An Avian Influenza Virus

Full-length cDNA of polymerase gene (PB2, PB1 and PA) and non- structural protein gene (NS) of avian influenza virus (CK/GS/2/99) were amplified by RT-PCR method. The cDNA was cloned into pGEM-Teasy vector and sequenced. The HA , NA and NS1 fragment were amplified from pGEM-T-HA , pGEM-T-NA and pGEM-T-NS vector by PCR method, which inserted into plasmid pET-28(a) and obtained recombinant plasmid named pET-HA, pET-NA and pET-NS1, which transformed into E. coli BL 21(DE3) for study the immunologic competence of expressing product respectively.The results show that open reading frame of the PB2, PB1, PA and NS gene cDNA contain 2280 bp, 2274 bp, 2151 bp and 890 bp encoding 759, 757, 716 and 272 amino acids separately. This study suggests that the nucleotide sequence and amino acid identity of polymerase gene (PB2, PB1 and PA) and non- structural protein gene (NS) of avian influenza virus strain Ck/GS/2/99 and Ck/NX/4/99 are 98% beyond and may origin from the same ancestor. Furthermore these genes present more close relation to the strain of Ck/HK/NX/4/99, HK/156/97 Sw/HK/10/98 and Ck/SJZH/2//98 respectively. The pET-HA, pET-NA and pET-NS1 were established successfully and transformed into E. coli BL 21(DE3). And then induced these genes expression with 0.8 mmol/L, 0.9 mmol/L and 0.8 mmol/L IPTG , with 7h, 6h and 6h respectively. The HA and NA protein that the relative molecular mass are approximately 62 000 and 52 000 were observed on the SDS-PAGE gel. The immunoreactivity of expression proteins including HA and NA protein were analyzed by Wesrern blot using positive serum of avian influenza virus H9N2 subtype. The results showed recombinant proteins can bind to positive serum of H9N2 subtype with specificity. This indicates that the recombinant proteins possesse good immunoreactivity. The NS1 protein that relative molecular mass is approximately 25 000 was observed on the SDS-PAGE gel. The purification production of expression protein (NS1) could be used as specificity antigen to detect chickens antibody which induced by live avian influenza virus (H9N2). It could be used to detect NS1 antibody for the differentiation of infected flocks from vaccinate and living ones.