Cloning,Sequence Analysis And Prokaryotic Expression Of Local Isolate Of Avian Influenza HA And NA Gene

Avian influenza (AI) was caused by A type avian influenza virus, a member of Orthomyxoviridae. Avian influenza with low virulence was characterized by respiratory symptom and low productirity. Avian influenza H9N2 subtype has been a main subtype in China since it was reported in 1994, which can infect flow and pig and human being. So it of great interest to study Avian influenza H9N2 subtype from molecular level not only in theory of virology and veterinary science but also in public hygiene.In this study. Virus of the local isolate A/Chicken/Mudanjiang/0823/00(H9N2) (MJ00) was propagated in SPF chicken embryo and nudeic acid was extracted. According to the conservatire sequence of 3' terminus of eight gene fragment from AIV, a piece of primer was designed to reverse transcript RNA. Accordmg to the cDNA sequence of HA gene and NA gene from H9N2 AIV publbhed in GenBank, two pairs of specific primers were designed to amplify HA gene and NA gene by software Oligo4.1 and Primer5.0 and the products of RT-PCR was cloned into pMD18-T vector. The sequencing results show that cDNA of HA gene has a complete open readrng frame of 1707bp which encodes 560 Amino acid residues induding signal peptide of 18 amino acid residues, and HA1 of 320 amino acid residues and HA2 of 222 amino acid residues. By comparing with 24 strains of the same subtype published in GeneBank, the homology of nucleotide of HA gene is between 81.5% and 99%, and the homology of deduced amino acid residues is between 87.5% and 97.9%. The amino acid sequence of cleavage is RSSR↓GLF according with the low virulence AIV strain. cDNA of NA gene is 1448bp in length encoding 469 amino acid residues. By comparing to 24 strains of the same subtype published in GenBank, the homology of nucleotide sequence of NA is between 82.5% and 98.9%, and the homology of deduced amino acid residues is between 85.7% and 98%. Comparing with HongKong strain and domestic strain, there is no deletion of three amino acid residues in stack of MJ00 NA.The HA gene was subcloned into prokaryotic expression vector pGEX-6p-1, and the positrve recombinant plasmid was tramsformed into competemo cell BL21 (DE3) pLyS. The colony containing the positire plasinid pGEX-6P-HA was screened and induced by 1mmol/L IPTG and was analyzed by SDS-PAGE. The result in gel was 88ku fused with GST(26ku), and the target protein is 62ku .In this study, HA gene and NA gene of the local isolate MJ00 were cloned first and HA gene was expressed in prokaryotic expression vector, and lay a theoretical base for preparing gene engineering vaccine and recombinant vaccine against avian influenza. Candidate: Tang Zhifen Major: Preventive Veterinary MedicineSupervisor: Jia YongqingWang Junwei...