Identifing "Different Genes" Of CSFV In SVEC And Extending CDNA Sequences And Inhibiting CSFV Propagation In PK-15 Cells By ShRNA

Classical swine fever virus(CSFV) effected specificly pathopoiesis for swine,CSFV could propagate in more swine cells in vitro culture without cytopathic effect(CPE),but had CPE in swine vascellum endothelial cells(SVEC).It's that one or some protease which one or some"sensitive genes"(they were named with CSFV"sensitive genes") expressed splited p125 protein to p80 protein after CSFV infected in SVEC possibly.On basis of previous study, we designed small hairpin RNA(shRNA) for four genes of eleven CSFV"sensitive genes"by biology software and synthetized them.We Screened and identified"different genes"by vector transcribing shRNAs in SVEC. The"different genes"sequences by Rapid Amplification of cDNA Ends(RACE) extending provided a basis for researching functions of different genes in PK-15 cells.It was a basis for gene functions and anti-virus by transfecting shRNAs recombinant vectors targeting CSFV NS3 into PK-15 cells.This study obtained following results:1. We designed shRNAs for four"different genes"by biology software and synthetized them.we annealed double-strands and ligated them into RNAi vector and transformed competent cell of E.coli DH5α.We screened and indentified recombinant plasmids, the results indicated that recombinant vectors were constructed successfully.These recombinant plasmids were transfected into SVEC and inoculated CSFV and observed cells pathological changes during different times.The results indicated that SVEC's appearance and survival rate was better than control cells and other cells by transfecting recombinant vectors targeting for Y8"different gene".The Y8"different gene"is CSFV"sensitive gene"with SVEC CPE possibly.2. Did CSFV infect PK-15 cells with CPE by"different genes"?cDNA sequcnce of"different gene"was transcribed reversely by RT primers. 5'-uptream unknows sequenc of Y8"different gene"was amplificated by two inverse PCR primers.3'-downstream unknows sequence of Y8"different gene"was amplificated by specific upstream primer and 3'-adaptor primer.Objective genes was ligated into pMD18-T and pMD19-T simple vector and transformed competent cell of E.coli DH5α.We identified recombinant plasmids by restriction analysis and sequence analysis.The results indicated that 5'upstream unknows sequence and 3'downstream unknows sequence of Y8"different gene"were extended by 5'RACE and 3'RACE.3. We analyzed and designed shRNAs for CSFV NS3 gene by biology software and synthetized them.We annealed double-strands and ligated them into pGenesil-1 vector, the constructing recmbinant vectors for pGene-NS3-1,pGene-NS3-2,pGene-NS3-3 and pGene-NS3-Neg negative control were transformed competent cell of E.coli DH5α. Recombinant vectors were transfected into PK-15 cells by liposome and screened positive cells.Cells were collected for Real-Time PCR and ELISA after CSFV were inoculated into cells in 72h.Real-Time PCR results indicated that shRNAs that were transcribed by pGene-NS3-1 and pGene-NS3-2 and pGene-NS3-3 silenced CSFV gene partly;ELISA results indicated that shRNAs inhibitted CSFV propagation partly.