Expression Of Optimized H7 Subtype Avain Influenze HA Gene

Avian influenza is an infection caused by avian influenza (flu) viruses. Highly pathogenic avian influenza was a type A acute infectious disease by Office International Desépizooties (OIE). Avian influenza virus(AIV)are type A members of the Orthomyxoviridae family. These subtypes differ because of changes in certain proteins on the surface of the influenza A virus (hemagglutinin [HA] and neuraminidase [NA] proteins). There are 16 known HA subtypes and 9 known NA subtypes of influenza A viruses. Many different combinations of HA and NA proteins are possible. Therefore, development of the susceptible, subtype specific diagnosis methods and safe, effective vaccines is urgently needed for control the avian influenza.The hemagglutinin (HA) is a virulence-associated glycoprotein. The hemagglutinin can induce neutralizing antibodies in vivo. Therefore, the hemagglutinin play a important role in diagnose and control avian influenza. In this experiment, Based on the sequence of optiHA gene, A set of primers were designed and synthesized to amplify the optiH7HA gene of H7 subtype AIV by PCR from the optiH7HA-puc19 plasmid which had been constructed. The restriction site were introduces into the 5'and 3'ends of the primer. The amplified fragments were cloned into the plasmid vector pMD18-T. After identifying, positive clone was digested with BamHI and HindIII, then subcloned into the plasmid vector pFastBacHTB cleaved with BamHI and HindIII. The recombinant optiH7HA-pFastBacHTB plasmids were identified by sequenced, PCR and endonucleases digestion. The optiH7HA-pFastBacHTB plasmids were transported into DH10Bac competent cells, then the recombinant Bacmid DNA were extracted and identified by PCR using PUC/M13 primers. The recombinant baculovirus were obtained by transfecting s.frugiperda (Sf9) cells with recombinant bacmid. For protein expression analysis, Sf9 cells were harvested and lysised when cell CPE(cytopathogenic effect) were observed. Total cells lysate was submitted to HA test, SDS-PAGE analysis and the expression proteins were confirmed by Western-blotting and IFA. The results showed that the optiH7HA gene was specifically expressed in Sf9 cells with the expected sizes and the expression proteins had immunoreactivity in immunoblot assay. .