The Matrix Protein Of VSV Induces Apoptosis In Human Lung Carcinoma Cells In Vitro

The Vesicular Stomatitis virus genome contains five single-stranded RNAsegments of negative polarity, coding for five proteins . M is expressed soonafter virus entry into the cell and acts at an early stage of infection. M proteinplays a predominant role in virus reproduction and pathogenicity. VesicularStomatitis virus induces apoptosis in infected cells in vivo and vitro. There wasevidence to make clear Apoptosis begain after Vesicular Stomatitis virus entryinto the cell. The cell-rounding activity by M protein is caused by apoptosis.The thesis is study on Cloning and expression of M gene of VesicularStomatitis virus and Induce apoptosis of Lung cancinoma cells. The result isfollowing:The pMD18-T-M was digested with Handâ…¢ and XhoI, the M gene wassubcloned into an expression vector pET-22b, And then the gene was sequencedand analyzed by computer software. It was confirmed that the gene sequencewas consistent with the sequence in GENBANK. The positive recombinantplasmids were then transfered into host strain BL21 (DE3).Expression of M gene in E.coli and preparation of its antibodyRecombinant expression plasmid pET-22b-M was constructed according torecombinant plasmid pMD18-T-M. Then the recombinants were transfered intothe host strain BL21 (DE3) to induce M protein expression by IPTG whenOD600 of the culture was about 0.6. The specific protein expressed (about30KDa) was detected by SDS-PAGE. M protein was expressed at high level,amounting to 25% of the total bacterial protein as confirmed by thin-layerscanning. After purified, M protein was used to induce the production ofpolyclonal antibodies against rabbits and ELISA detection showed theantigenicity of M protein was satisfactory.The construction of eukaryotic expression vector of M gene and inducesapoptosis in tranfected Lung cancinoma cellsThe M gene was cloned and the cloning vector pMD18-T-M1 wasconstructed. And then the M gene was subcloned into the multiclone sites ofvector pVAX1 between the site of Nheâ… and XhoI to construct the eukaryoticexpression vector of M gene (pVAX1-M). We transfected the pVAX1-M intoLung cancinoma cells by lipidosome. The results showed that M gene had beensuccessfully expressed in Lung cancinoma cells as confirmed with Western blot.Apoptosis of Lung cancinoma cells was determinated by light microscopy andfluorescence activated cell sorter (FACS) analysis. The results showed thatremarkable apoptotic characteristics such as cell-rounding appeared in tumorcells, the apoptosis persents were higher than control, but few apoptoticcharacteristics were observed in control groups which transfected with blankplasmids. It was suggested that the M protein could induce apoptosis in Lungcancinoma cells effectively.