Construction Of Report Gene With 3'UTR Sequences Of CSFV And Establishment Of Cell Line Expression MiR-150 ShRNA

microRNAs (miRNAs) are small noncoding RNAs that play important roles in posttranscriptional gene regulation. miRNAs regulate their targets by imperfect complimentary match sequences in target site and result in translational repression. It is reported that miRNAs play important roles in virus infection and it maybe have direct regulatory functions in CSFV 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 3 prime end of GFP mRNA to find the potential miRNAs binding sites on CSFV 3'UTR. And we also constructed a PK-15 cell line that express miR-150 shRNA to research the miRNA function in CSFV infection.1. Identification the replication of CSFV Shimen strain on SUVEC, and according to the 3'UTR sequences of the CSFV Shimen strain published, a pair of primers were designed to get the 3'UTR gene by RT-PCR. After enzymatic assay and recovering the products of PCR, the 3'UTR was cloned to the pEGFP-C1,named pEGFP-3'UTR and the recombination plasmid pEGFP-3'UTR was also identified by sequencing and enzymatic digestion. The the pEGFP -3'UTR was extracted and purified by QIAGENE Plasmid Mini Kit.2. Using the software RNA22 and miRanda, we predicted the target sites of swine and human miRNAs on CSFV genome, and get several miRNAs that may target on the genome RNA. Specially, we focused on the 11 miRNAs that may target to the un-translation region of the genome.3. Two pairs of single DNA chain encoding short hairpin RNA(shRNA)of miR-150 were designed and synthesized, and were ligated to the interference vector pGenesil-1. After sequencing identification and purification, recombination plasmid pGene-miR-150 were transfected into the PK-15 cells. 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 short hairpin RNA persistently.