Cytopathic Effect Of The CSFV NS3 Protein To PK-15 Cells And Establisment Of The Cell Strains Transcribing Shrna Target To The CSFV 3'-UTR

It had previously been recognized that the NS3 protein has important role in the (cytopathic effect)CPE induced by the CSFV and there is a relationship between the expression of the NS3 and the CPE occured in the porcine cell line. It's not only useful for us to further researsh the relationship between the CSFV and host cell by expressing NS3 protein in porcine cell, and also to reveal the pathopoiesis mechanism about CSFV. The proliferation of CSFV in cell is the base to know the pathopoiesis mechanism, 5'-UTR and 3'-UTR can regulate the duplicating of the virus genom and expression of the virus polyprotein. So the researsh on inhibition the CSFV to proliferate by RNAi targeted to the 3'-UTR is the other way to reveal the virus's pathopoiesis mechanism.1. According to the NS3 sequences of the CSFV Shimen strain published, a pair of primers P1/P2 which 5'terminal were added the XhoI and KpnI enzyme site respectively were designed to got the NS3 gene by RT-PCR. After sequencing and accrediting the products of PCR, the NS3 gene was subcloned to the pEGPF-C1,named pEGPF-NS3 and the recombination plasmid pEGPF-NS3 was also identified by XhoI and KpnI. The concentration of the pEGPF-NS3 was 0.6μg/μL when purified by QIAGENE Plasmid Mini Kit.2. The plasmid pEGFP-NS3 and pEGFP-C1 were transfected into the PK-15 cells respectively by Lipofectamine 2000 Regent and we got the positive pEGFP-NS3 and pEGFP-C1 cells by optimal concentration of G418 after 12 d. The NS3 protein expressed in PK-15 was identified by the SDS-PAGE and Western-blot and was about 68 ku. The CPE was appeared in positive pEGFP-NS3 cells at 72 h after passaged, the cells became round gradually and the number of the detached cells were increased.3. The apoptosis detection was performed on the positive pEGFP-NS3 and pEGFP-C1 cells at 72 h after passaged by Tunel assay respectively. The results suggested that the positive pEGFP-NS3 cells generated apoptosis and presented CPE. There is a relationship between the expression of NS3 and the apoptosis. The rate of the apoptosis in the positive pEGFP-NS3 and pEGFP-C1 cells were 43.4 % and 13.1 % per 1 000 cells respectively(P<0.05).4. Three pairs of single DNA chain encoding short hairpin RNA(shRNA)which target to the CSFV Shimen strain 3'-UTR 114～132 sites, 136～154 sites and 209～227 sites were designed and synthesized and were ligated to the interference vector pGenesil-1. After sequencing identification and purification, recombination interference plasmid pGene-1, pGene-2 and pGene-3 were gotten and 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. Three monoclonal strains were subcultured which can transcribe the short hairpin RNA persistly.