肠道病毒71型3D重组病毒的拯救

Rescue of 3D recombinant enterovirus 71

目的利用反向遗传技术将肠道病毒71型(EV71)弱毒株SDLY1的3D蛋白置换入强毒株SDLY107,拯救重组病毒,为EV71 3D蛋白的研究提供操作平台。方法利用重叠PCR的方法得到3D编码区的重组片段,T-A克隆插入p MDl9-T载体。通过双酶切、T4连接将其置换到含有SDLY107株全长的质粒p MD19-T-107中。体外转录获得感染性RNA,转染Vero细胞,盲传3代得到重组病毒SDLY 107(1-3D),通过PCR和q RT-PCR对重组病毒进行鉴定。结果 PCR扩增得到大小为600、1 400和200 bp的3D区及其左右片段;重叠PCR扩增得到大小为2 100 bp的1-3D片段;双酶切鉴定重组质粒得到大小为7 400和2 700 bp的片段,测序结果证实3D区置换成功;感染性RNA转染Vero细胞后,接种至第3代时观察到细胞皱缩变圆,折光性增强;PCR鉴定重组病毒得到大小为226 bp的片段;q RT-PCR检测到细胞中病毒量于24 h开始缓慢增加,48 h后快速增多,至72 h后病毒量不再增长。结论成功拯救了重组病毒SDLY107(1-3D),为进一步研究3D蛋白的毒力和其在EV71致病机制中的作用提供基础。

Objective To replace 3D region of enterovirus 71（ EV71） high virulent strain SDLY107 with low virulent strain SDLY1 by reverse genetics technology,and to rescue recombinant virus,providing a platform for investigation on EV71 3D protein. Methods The recombine fragment of 3D protein was obtained by overlap PCR,and then inserted into p MD19-T vector. 3D region of p MD19-T-107 containing the full-length gene of SDLY107 was replaced with recombine fragment through double digestion and ligase T4 to construct p MD19-T-107（1-3D）. The infectious RNA was obtained by in vitro transcription,then transfected into Vero cells and gained recombinant virus particles after three passages. PCR and q RT-PCR were performed for recombinant virus identification. Results Apa I-3D anterior region（600 bp）,3D region（1 400 bp） and 3’UTR-Xba I region（200 bp） were obtained by PCR,and 1-3D（2 100 bp） was obtained by overlap PCR. The recombinant plasmid was identified by double enzyme digestion（7 400 and 2 700 bp）,and sequencing results further confirmed the successful replacement of 3D region. Cell shrinkage,roundness and refractive index enhancement were observed after transfecting the infectious clone into Vero cells and cultivating to the third generation. Identification of recombinant virus by PCR gained fragment of 226 bp; q RT-PCR detected the amount ofvirus increased slowly from 24 h to 48 h and increased rapidly from 48 h to 72 h,and the amount of virus no longer growed after 72 h. Conclusion The recombinant virus SDLY107（1-3D） is rescued successfully. The study provids the basis for further research on the virulence of 3D protein and its role in the pathogenesis of EV71.