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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 reverse the 3D protein of the attenuated EV71 strain SDLY1 by reverse genetic technique into the virulent strain SDLY107, to rescue the recombinant virus and provide a platform for the study of EV71 3D protein. Methods The recombinant DNA of 3D coding region was obtained by overlapping PCR. The T-A clone was inserted into pMD19-T vector. It was double-digested and T4 ligated into plasmid pMD19-T-107 containing the full length of SDLY107 strain. Infected RNA was transcribed in vitro and transfected into Vero cells. The recombinant virus SDLY 107 (1-3D) was obtained by blind passage for 3 generations and the recombinant virus was identified by PCR and q RT-PCR. Results The 3D region with size of 600, 1 400 and 200 bp and its left and right fragments were amplified by PCR. The 1-3D fragments with the size of 2 100 bp were amplified by overlap PCR. The double digestion identified the recombinant plasmid with sizes of 7 400 and 2 700 bp fragment. Sequencing results confirmed that the 3D region was successfully transplanted. After transfection of infectious RNA into Vero cells, cell shrinkage became round and refraction increased when inoculated to the third passage. The size of the recombinant virus was 226 bp The amount of virus in cells started to increase slowly from 24 h after RT-PCR, and rapidly increased after 48 h. The amount of virus did not increase after 72 h. Conclusion The recombinant virus SDLY107 (1-3D) has been successfully rescued, providing the basis for further study on the virulence of 3D protein and its role in the pathogenesis of EV71.