Constrcted Lentivirus Vector Of SiRNA Targeting Swine FMDV Receptor αv Subunit And Established Anti-FMDV PK-15 Cell Model

Foot-and-mouth diseas (FMD) is an acute, febricity and highly contagious disease caused by foot-and-mouth disease (FMDV), which is classified by Office International des Epizooties (OIE; World Organisation for Animal Health ) as the first one of OIE List diseases.th has been demonstrated that there are at least five receptors for FMDV, including integrandαvβ3,αvβ6,αvβ1,αvβ8 and heparan suifate (HS). Integrandαvβ6 serves as the major receptor that determines the tropism of FMDV for the epithelia normally taergeted by the vieus.To screen the optimum siRNA of porcine integrin alpha V subunit as FMDV receptor. Three target sequences were selected according to porcine integrin alpha V mRNA sequence, the complementary DNA contained both sense and antisense oligonucleotides were designed and synthesized. After PK-15 cells were transected siRNAs by Lipofectamine 2000, real-time RT-PCR and TCID50 were used to evaluate the level ofαv expression and resistance of FMDV O/CHA/99. The result showed that Compared with control and mock group ,the alpha V subunit mRNA expression were obviously suppressed in all three experimental groups(iαv-480,iαv-1719 and iαv-2077), especially the expression rate in iαv-480 group was reduced by 90.1% in 24h. Iαv-480 group had lower virus titers by TCID50 indicated that PK-15 cells increased the resistance of FMDV O/CHA/99. The optimum siRNA screen success of porcine integrinαv subunit gene in this study, which would be helpful work about studying of interference FMDV receptor gene anti-FMD transgene in swine.ShRNA targeting the integrinαv subunit, which is the foot-and-mouth disease virus (FMDV) receptor, plays a key role in virus attachment to susceptible cells. We constructed an RNAi lentiviral vector, iαv-pLenti6/BLOCK-iT which expressed siRNA targeting the FMDV receptor, the porcine integrinαv subunit, on PK-15 cells. We also produced a lentiviral stock, established an iαv-PK-15 cell line, evaluated the gene silencing efficiency of mRNA using real-time RT-PCR, integrandαv expression by IIF and cell ELISA, and investigated the in vivo inhibitory effect of shRNA on FMDV replication in PK-15 cells. Our results indicated successful establishment of the iαv-U6 RNAi entry vector and the iαv-pLenti6/BLOCK-iT expression vector. The functional titer of obtained virus was 1.0×106 TU /mL. To compare with the control and mock group, the iαv-PK-15 groupαv mRNA expression rate in group was reduced by 89.5%, whilst IIF and cell ELISA clearly indicated suppression in the experimental group. Thus, iαv-PK-15 cells could reduce virus growth by more than three-fold, when cells were challenged with 102 TCID50 of FMDV. PK-15 cells were demonstrated as a cell model for anti-FMDV potency testing, and this study suggests that shRNA could be a viable therapeutic approach for controlling the severity of FMD infection and spread.The Anti-FMDV wuzhishan pig transgenic primary cell lines were established by lentivirus of targeting iαv-480 transducted to pig fetal fibroblast cells. Experiments carried out preliminary studies of transgenic pigs by lentiviral transduction of pig cells, nuclear transfer by micromanipulation and access to a large number of 2-4 cells stage embryos were successfully transplanted.