Lentviral-mediated RNAi to inhibit target gene expression of the porcine integrin αv subunit, the FMDV receptor, and against FMDV infection in PK-15 cells

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• 作者：Jihuai Luo (1)
  Junzheng Du (1)
  Shandian Gao (1)
  Guofeng Zhang (1)
  Jingjing Sun (1)
  Guozheng Cong (1)
  Junjun Shao (1)
  Tong Lin (1)
  Huiyun Chang (1)
  
• 刊名：Virology Journal
• 出版年：2011
• 出版时间：December 2011
• 年：2011
• 卷：8
• 期：1
• 全文大小：393KB
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• 作者单位：Jihuai Luo (1)
  Junzheng Du (1)
  Shandian Gao (1)
  Guofeng Zhang (1)
  Jingjing Sun (1)
  Guozheng Cong (1)
  Junjun Shao (1)
  Tong Lin (1)
  Huiyun Chang (1)
  
  1. State Key Laboratory of Veterinary Etiological Biology, National Foot and Mouth Disease Reference Laboratory, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou, 730046, China
  
• ISSN：1743-422X

文摘

Background shRNA targeting the integrin αv subunit, which is the foot-and-mouth disease virus (FMDV) receptor, plays a key role in virus attachment to susceptible cells. We constructed a RNAi lentiviral vector, iαv pLenti6/BLOCK -iT? which expressed siRNA targeting the FMDV receptor, the porcine integrin αv 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 qRT-PCR, integrand αv expression by indirect immunofluorescence assay (IIF) and cell enzyme linked immunosorbent assays (cell ELISA), and investigated the in vivo inhibitory effect of shRNA on FMDV replication in PK-15 cells. Results 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 group αv 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 and there was a > 99% reduction in virus titer when cells were challenged with 102 TCID50 of FMDV. Conclusions Iαv-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.