重组肠道病毒71型反向遗传系统构建

Construction of reverse genetics system for recombinant entervirus 71

目的利用反向遗传技术分别对肠道病毒71型(EV71)强毒株SDLY107和弱毒株SDLY1的3C和3CD编码区进行置换,拯救重组病毒。方法使用PCR技术得到重组片段3C(1)-3D-3'UTR(107)和3CD(1)-3'UTR(107),并克隆入p M D19-T。利用双酶切、T4连接酶将两种重组3CD-3'UTR分别置换入本室构建并保存的EV71的SDLY107株的感染性c DNA克隆p M D19-T-107,得到重组p M D19-T-107后,将其线性化并转染入横纹肌肉瘤(RD)细胞,盲传得到重组病毒107(1-3C)和107(1-3CD)。对收获病毒进行鉴定。结果构建了重组EV71全长c DNA克隆;RNA转染并盲传至第3代,36 h后RD细胞出现细胞病变(CPE),48 h出现明显的CPE,成功拯救重组病毒SDLY107(1-3C)和SDLY107(1-3CD);重组病毒产生的CPE更接近SDLY107。结论成功构建了重组EV71反向遗传系统,拯救了重组病毒SDLY107(1-3C)和SDLY107(1-3CD),为进一步研究3C与3D蛋白在EV71致病机制中的作用提供基础。

Objective To recombine 3C and 3CD region of virulent strain SDLY107 and low virulent strain SDLY1 of entervirus 71 （EV71）with reverse genetics, and to rescue recombinant viruses. Methods Recombinant fragments,3C （ 1 ） -3 D -3＇ UTR （ 107 ） and 3 CD （ 1 ） -3＇ UTR （ 107 ）, were obtained with PCR, and then cloned to pMD 19 -T. 3 CD -3＇ UTR of pMD19-T-107 constructed previously was replaced by recombinant 3CD-3＇ UTR through double digestion and ligase T4 to construct recombinant pMD19-T-107. Then the in vitro synthesized RNA transcripts were transfected into rhabdomyosarcoma （RD） cells to produce the rescued recombinant virus, SDLY 107 （1-3C） and SDLY 107 （1-3CD）. DNA sequences of the recombinant viruses were analyzed. Results The full length cDNA clone was constructed successfully. After 36 hours of third passages, cytopathic effect（CPE） was observed, and the CPE was observed obviously after 48 hours, suggesting the recombinant viruses were rescued successfully. There were a few differences between the CPE induced by recombinant viruses and SDLY107. Conclusion Reverse genetics system for recombinant EV71 was constructed successfully,and recombinant viruses were rescued successfully. The study lays a foundation for further research on pathogenesis of EV71.