| The influenza virus genome contains eight single-stranded RNA segments of negative polarity, coding for nine structural proteins and two nonstructural polypeptides designated NS1 and NS2. NS1 is expressed soon after virus entry into the cell and acts at an early stage of infection. NS1 is a conservative gene. NS1A protein plays a predominant role in virus reproduction and pathogenicity though it isn't exist in the virus particle. Influenza virus induces apoptosis in infected cells in vivo and vitro. There was evidence to make clear Apoptosis begain after influenza virus entry into the cell. The influenza virus factors dsRNA, NS1, NA and PB1-F2 all can induce the cell apoptosis directly. It is not clear that the pathway and mechanism of NS1 induce the cell apoptosis. The thesis is study on Cloning and expression of NS1A gene of H9N2 avian influenza and Induce apoptosis of Hela cells. The result is following. Cloning of NS1A gene and its sequence analysis the NS1A gene was amplified by RT-PCR, the product of PCR were cloned into a clone vector pMD18-T. By double enzyme digest with Salâ… and XhoI, the NS1A gene was cloned into an expression vector pET-20b, and then the gene was sequenced and analyzed by computer software. It was confirmed that the gene sequence was consistent with the sequence in GENEBANK. The positive recombinant plasmids were then transfered into host strain BL21 (DE3). Expression of NS1A gene in E.coli and preparation of its antibody Recombinant expression plasmid pET-20b-NS1A was constructed according to recombinant plasmid pMD18-T-NS1A. Then the recombinants were transfered into the host strain BL21 (DE3) to induce NS1A protein expression by IPTG when OD600 of the culture was about 0.6. The specific protein expressed (about 30KDa) was detected by SDS-PAGE. NS1A protein was expressed at high level, amounting to 35% of the total bacterial protein as confirmed by thin-layer scanning. After purified by electroelution in dialysis bags, NS1A protein was used to induce the production of polyclonal antibodies against rabbits and ELISA detection showed the antigenicity of NS1A protein was satisfactory. Western blot showed that the antiserum raised against the recombinant protein in rabbits could react to the protein expressed specifically. The construction of eukaryotic expression vector of NS1A gene and induces apoptosis in tranfected Hela cells The NS1A gene was cloned and the cloning vector pMD18-T-NS1A was constructed. And then the NS1A gene was subcloned into the multiclone sites of vector pVAX1 between the site of BamHâ… and Xhol I to construct the eukaryotic expression vector of NS1A gene (pVAX1-NS1A). We transfected the gene of pVAX1-NS1A into Hela cells by lipidosome. The results showed that NS1A gene had been successfully expressed in Hela cells as confirmed with ELISA and Western blot. Apoptosis of Hela cells was determinated by light microscopy, fluoromicroscopy and fluorescence activated cell sorter (FACS) analysis, direct cell number counting and MTT assay 48 hours after transient transfection. The results showed that remarkable apoptotic characteristics such as nuclear shrinkage and strong fluorescent staining signals appeared in tumor cells, the apoptosis persent were higher than control, the cell cycle were exchanged also detected by FACS, but few apoptotic characteristics were observed in control groups which transfected with blank plasmids. It was suggested... |
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