靶向呼吸道合胞病毒M2基因pshRNA载体质粒的构建及意义

Construction and significance of the short hairpin RNA recombinant plasmid targeting M2 gene of respiratory syncystial virus

目的:构建针对呼吸道合胞病毒(RSV)M2基因m RNA的短发卡结构状RNA(shRNA)重组载体质粒,为利用RNA干扰技术抗RSV感染的深入研究奠定基础;方法:按shRNA设计原则,选择RSV-M 2基因作为靶基因,设计19bp长的能产生短发卡结构的两段DNA反向重复序列,中间以9bp序列间隔,经退火形成互补双链,克隆至转录载体pgenesil-1上,重组质粒经酶切和测序鉴定,然后将构建成功的重组质粒转染H EP2细胞,荧光显微镜下观察绿色荧光细胞发生率以评估转染效率。结果:将设计合成的DNA片段成功克隆至载体上,经酶切及序列鉴定为目的序列,并且荧光显微镜下观察细胞有较多的绿色荧光蛋白表达。结论:成功构建了针对RSV M 2基因m RNA的shRNA重组载体质粒,可利用重组质粒进一步研究其对RSV感染的抑制,从而为抑制RSV感染寻找新的基因治疗手段。

Objective： To construct the recombinant plasmid targeting M2 gene of respiratory syncystial virus （RSV） for further study of RNA interference （RNAi） technology in inhibiting RSV infection. Methods： According to the designing principles of shRNA, The M2 gene of RSV was selected as the target gene. A 19bp reverse repeated sequences with 9bp spacer were designed and synthesized. The complement strain were obtained by annealing and inserted into the vector pgenesil-1 containing green fluorescenet protein （GFP） sequence and U6 promotor. Finally the recombinant plasmid were identified by enzyme digesion and DNA sequencing, then transfected into HEP2 cells and observed by fluorescence microscope. Results： The recombinante plasmid targeting the mRNA of RSV M2 gene was successfully constructed and the good efficents of transfection were found by fluorescence microscope. Conclusion： The successful construction of the pshRNA recombinant plasmid targeting the M2 gene of RSV will help futher study on application of the RNAi technology to anti-RSV infection.