Expression And Identification Of Truncated Hemagglutinin (HA) Of Influenza A/H1N1/2009 Virus

Influenza virus is a member of the Orthomyxoviridae family,and is negative strand RNA viruses. Its genome consists of eight segments, and encode eleven proteins at least, Hemagglutinin(HA) is one of the most important surface antigen. The mainly function of HA is that HA can bind to sialic acid on the surface of the host cells, then virus bind to the host cells, moreover HA enhance membrane fusion and endocytosis. The HA of human influenza viruses and avian influenza viruses have different sialic acid receptor, which is one of the mechanism decide virus interspecies infection. HA is one of mainly Virulence factor.HA is also a neutralize antigen of virus. Pandemic A(H1N1) 2009 influenza emerged in april 2009, in Mexico and America, caused by aquadruple reassortant virus fron swine, avian and human influence virus. Since Pandemic Spanish influence broken out, there are many influence caused by HINlsubtype strain nearly a century, so the research about the A(H1N1) 2009 influenza virus play a important role in public heath.To express prokaryotically a truncated HA of influenza A/H1N1/2009 virus using the pET expression vector and to purify and identify the expressed HA protein.The gene encoding HA of A/California/04/2009 H1N1 influenza virus was synthesized, which was used as a template for amplification of a segment of HA gene encoding a truncated HA protein (consists of HA1 and HA2, but with the signal peptide, transmembrane domain and intracellular region of HA removed) by PCR. The amplified PCR product was cloned into pET 28(a), and was expressed with IPTG induction in E. coli BL21 (DE3). The expressed protein was detected by SDS-PAGE and was purified using Ni-NTA His Bind purification kit. The antigenic reactivity of the truncated HA protein was analyzed by western blot.The truncated HA protein was expressed in prokaryotic express system and a higher-purified protein was obtained. The truncated protein showed reactivity with antibody specific to HA of 2009 H1N1 influenza virus. These provide useful material for detection of influenza A/H1N1/2009 virus.In order to express recombinant HA protein, our study made use of the Bac-to-Bac Baculovirus Expression System, then identify the expression of the recombinant HA protein by western blot and IFA. Method:A/California/04/2009(H1N1)HA gene was amplified by PCR, and cloned to the pFastBacHT A vector, the recombinant plasmid pFastBacHT-HA was identified and made sure by restriction enzyme digestion and gene sequencing. Then the recombinant plasmid pFastBacHT-HA was transformed into E.coli DH10Bac component cell, the recombinant rbacmid-HA was identified by Bluo-gal blue/white selection and PCR. The rbacmid-HA DNA was Isolated, then transfected sf9, we obtained Pl viral stock and amplified it. Baculovirus infected sf9 cell,then harvested the cell to analysis expression of recombinant protein by western blot and IFA. Conclusion:Our study successful constructed expression vector of A/H1N1 influence virus HA gene, which was transfected into insect cell, then obtained higher viral titer recombinant baculovirus. recombinant protein was expressed by recombinant baculovirus infecting insect cell and identified by western blot and IFA, the result demonstrate that the truncated protein showed reactivity with antibody specific to HA of 2009 H1N1 influenza virus.