靶向VP1和VP2双拷贝shRNA重组表达质粒抑制传染性法氏囊病病毒复制的研究

Inhibition of infectious bursal disease virus replication by duplex shRNAs targeting the VP1 and VP2 genes driven by chicken U6 promoter

为抑制传染性法氏囊病病毒(IBDV)的复制,本研究构建了靶向IBDV VP1和VP2基因的鸡双向U6启动子(chU6)双拷贝shRNA重组表达质粒pchU6-shRNA12,将其转染鸡胚成纤维细胞(CEF),再接种IBDV,72 h后,未出现细胞病变(CPE),病毒滴度小于101 TCID50/0.1mL;而转染空质粒和未转染两个对照组均出现明显的CPE,病毒滴度均达到108.75 TCID50/0.1mL。此外,将pchU6-shRNA12与IBDV混合,接种于10日龄SPF鸡胚尿囊腔,96 h后,鸡胚发育正常,病毒滴度小于101 ELD50/0.1mL,实时荧光定量RT-PCR检测VP1和VP2基因,比单纯接种IBDV组分别降低92%和95%;而含有空质粒和不含质粒两个对照组鸡胚则全部死亡,病毒滴度均达到107.00 ELD50/0.1mL。本研究结果表明,chU6启动子在双拷贝shRNA表达质粒pchU6-shRNA12中能高效驱动双拷贝shRNA的转录,生成的siRNA在CEF(in vitro)和鸡胚体内(in vivo)均能有效抑制IBDV的复制。

To inhibit the replication of infectious bursal disease virus （IBDV）, two short hairpin RNAs （shRNAs） targeting VP1 and VP2 genes of IBDV were designed and synthesized. The recombinant plasmid pchU6-shRNA12 containing chicken U6 promoters on each side of the shRNAs based on pSilencer2, l-U6 vector was constructed. In the IBDV inhibition test （in vitro） of the chicken embryo fibroblasts （CEF） infected with IBDV at 24 hours post transfecting with pchU6-shRNA12, the virus titer was less than 10 TCIDsJ0.1 mL, in the contrast, the IBDV control was 108.75 TCID^0.1 mL. The t0-day old SPF chicken embryos also showed growing normal and the virus titers was less 10 ELDM0.1 mL post 96 hours when inoculated with mixture of pchU6-shRNA12 and IBDV via the allantoic cavity （in vivo）, whereas the mortality rate was 100% and the virus titer reached 107 ELD50/0.1 mL in control group. Additionally, the inhibition rates for IBDV replication were from 92% to 95% in embryos inoculated with pchU6-shRNA12, compared to IBDV control group detected using real-time quantitative RT-PCR, suggesting that chicken U6 promoter effectively drove the transcription of the shRNAs, and the transcribed siRNAs were able to effectively inhibit IBDV replication in both of in vitro and in vivo.