Construction Of Eukaryotic Expressing Vectors And Adenovirus Packaging Of HA/HA1 Gene Of Influenza A H1N1 Influenza Virus And Their Primary Expression

The outbreak of Influenza A H1Nl started in Mexico on March18, 2009. Influenza A H1Nl Influenza virus was found to transmit and spread among humans, resulting in outbreaks internationally. According to World Health Organization, the Influenza A H1Nl leaded at least 16,713 people to death worldwide till March 7, 2009. The influenza A H1Nl Influenza virus contains a genome composed of eight segments of single-stranded, which encodes 10 proteins related to the viral structure and function. The hemagglutinin(HA) is the main Protein of surface spikes and a virulence-associated glycoprotein, which can induce neutralizing antibodies in vivo. Therefore, the hemagglutinin plays an important role in diagnose and control of influenza.This research gained 14 nucleotide sequences of H1N1-HA, which were from the different states of the United States in April, 2009. The HA gene consensus sequence was acquired after the sequence analysis. Then the HA gene consensus sequence was optimized in accordance with human codon preference. Synthetic Mod. HA gene and Mod. HA1 gene were inserted into the eukaryotic expression vector pSNAV, which constructed Eukaryotic Expressing Plasmids of Mod.HA and Mod. HA1. Then pSNAV- Mod. H A and pSNAV- Mod. HA1 plasmids were transfected into BHK cells,monoclones were acquired after the selection of G418. Immunofluorescence assay and western blot detected expression in BHK cells, which showed HA and HA1 were both expressed in BHK cells. These cell Monoclones were tested with Immunofluorescence and western blot, and after the 10 generation tests, which showed HA and HA1 were both stably and accurately expressed in BHK cells. The cell lines highly expressing pSNAV-Mo.HA and pSNAV-Mod.HA1 were eventually selected respectively. Meanwhile, the Mod.HA1 gene was inserted into the shuttle plasmid pDC315. The shuttle plasmid pDC315 and backbone plasmid were cotransfected into 293 cells. The recombinant Adenovirus named rAd-Mod.HA1was Packaged, which was tested by PCR,immunofluorescence and Western Blot. The results showed that the packaged rAd-Mod.HA was correct. Consequently, rAd-Mod.HA was packaged successfully.In this study, stable cell lines named pSNAV-Mod.HA-BHK and pSNAV-Mod. HA1-BHK and the recombinant adenovirus named rAd-Mod.HA1 were acquired, which expressed HA and HA1 proteins with 6 His-tag that were easily purified. Therefore, HA and HA1 protein have development prospect in the diagnosis of influenza A H1N1 influenza virus and new subunit vaccine research and provide a foundation for the further study on the development of genetic engineering vaccine and the tested Reagent of H1N1 influenza .