Establishment Of Cell Line Expressing Ssc-let-7c And Regulates CSFV

microRNAs (miRNAs) are small noncoding RNAs that play important roles in posttranscriptional gene regulation. miRNAs regulate their targets by imperfect or perfect base pairing in target site and result in translational repression at posttranscriptional gene regulation level. It is reported that many virus can code miRNAs and they play important roles in virus infection,thus,it is supposed that it maybe have invariably effect in virus pathogenicity and the interaction with the host,then, it maybe have direct regulatory functions in CSFV (Classical Swine Fever Virus) infection by interaction with 3'-noncoding region of CSFV RNA genome. Bioinformatics analysis shows that several miRNAs potentially targeted to CSFV genome. To investigate the function of cellular miRNAs in CSFV infection, we constructed a GFP report vector with a CSFV 3'UTR sequences at the GFP mRNA to find the potential miRNAs binding sites on CSFV 3'UTR. And we also constructed a SUVEC cell line that express ssc-let-7c to research the miRNA function in CSFV infection. The results of this research are as follows:(1)Using the software RNA22 and miRanda, we predicted the target sites of swine and human miRNAs on CSFV 3'UTR, and get 4 miRNAs that may target on the mRNA. According to the ssc-pre-let-7c sequences published, a pair of primers were designed to get the ssc-pre-let-7c by PCR.(2)The ssc-pre-let-7c were designed and synthesized, and were ligated to the vector pGenesil-1.1-dBm2. Named pEGFP-ssc-pre-let-7c and the recombination plasmid pEGFP-ssc-pre-let-7c was also identified by sequencing and enzymatic digestion. The the pEGFP-ssc-pre-let-7c was extracted and purified by QIAGENE Plasmid Mini Kit.(3)The pGenesil-1.1-dBm2 and pGene-pre-let-7c were transfected into SUVEC and expression. The positive cells selected by optimal concentration of G418 were expanded and the monoclonal culture was carried out though the twice limiting dilution assay. Monoclonal strains were subcultured which can transcribe the ssc-let-7c persistently.(4)The two cells lines were dealed with CSFV in the same time, Using the real-time quantitative PCR to detect the expression of CSFV in the two cells lines. Using the MTT to detect the cytoactive. The results of this research are as follows:construction of eukaryotic expression vector pGene-pre-let-7c and pGenesil-1.1-dBm2 can be effectively expressed in cells of SUVEC which infect the CSFV. Under the same conditions, The cell lines which transfected pGene-pre-let-7c can decrease expression of CSFV lower than the cell lines which transfected pGenesil-1.1-dBm2.That indicate let-7c play on the negative regulation of CSFV.