短发卡结构状RNA体外抑制呼吸道合胞病毒M2基因mRNA水平和病毒蛋白表达的研究

Inhibition of Respiratory Syncytial Virus M2 Gene mRNA and Viral Protein Expresson by Short Hairpin RNA in vitro

目的观察针对人类呼吸道合胞病毒(RSV)M2基因mRNA构建的短发卡结构状RNA重组质粒在细胞水平对RSV复制的影响,为利用RNA干扰技术抑制RSV感染的研究奠定基础。方法将已成功构建的重组质粒pshRNA7816/8330转染HEp-2细胞,通过显微镜观察pshRNA7816/8330对RSV感染细胞病变(CPE)的抑制效率,利用四甲基偶氮唑盐比色实验检测pshRNA7816/8330的细胞毒性,经RT-PCR方法检测pshRNA7816对RSVM2mRNA表达的影响,免疫细胞化学染色法检测pshRNA7816对细胞内RSV蛋白的影响。结果pshRNA7816/8330对HEp-2细胞的正常生长没有明显的细胞毒性作用,2个重组质粒均能抑制RSV所致的CPE,但pshRNA7816对CPE抑制作用较pshRNA8330要强,RT-PCR和免疫细胞化学染色法结果显示pshRNA7816能明显降低RSVM2基因mRNA和RSV蛋白表达水平。结论针对RSVM2-1基因所构建的短发卡结构状RNA重组质粒pshRNA7816在细胞水平能有效抑制RSV的CPE形成降低RSVM2mRNA和病毒蛋白表达水平,从而干扰RSV的复制。

Objective To investigate the influence of short hairpin RNA （shRNA） reeombinated plasmid targeting M2 gene of respiratory syncytial virus （RSV） on inhibiting viral replication in cell culture system. Methods The reeombinated plasmid pshRNA7816/ 8330 was transfected into HEp 2 cells. MTT assay was used to detect the cytotoxicity of recombinated plasmid on HEp- 2 cells. The effects of pshRNA7816 and pshRNA8330 on change of cytopathogenic effect （CPE） of HEp- 2 cell induced by RSV infection were observed microscopically. The reverse transeriptive PCR（RT- PCR） was performed to detect expression of RSVM2 mRNA and protein level of RSV was measured by immunocyteoehemistry. Results The results of MTT demonstrated that pshRNA7816/8330 had no significant toxicity on growth of HEp- 2 cells. The recombinant plasmids pshRNA7816 and pshRNA8330 targeting RSVM2 gene could alleviateCPE of HEp 2 cells induced by RSV infection, but the pshRNA7816 showed a more potent inhibition than pshRNA8330. RT PCR and immunocytochemistry showed that pshRNA7816 could efficiently inhibit expression of M2 mRNA and protein of RSV. Conclusion The recombinate plasmid pshRNA7816 targeting the mRNA of RSVM2 1 gene can alleviate the CPE of HEp- 2 cells induced by RSV infection and reduce expression of mRNA and protein of RSV.