肠道病毒71型3D重组病毒的拯救

发布时间：2018-01-09 23:13

本文关键词：肠道病毒71型3D重组病毒的拯救　出处：《山东大学学报(医学版)》2017年08期 　论文类型：期刊论文

【摘要】：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。
[Abstract]:Objective to replace the 3D protein of attenuated enterovirus 71 strain EV71 into the virulent strain SDLY107 by reverse genetic technique to save the recombinant virus. Methods the recombinant fragment of 3D coding region was cloned and inserted into p MDl9-T vector by using overlapping PCR. T4 ligation was inserted into the plasmid p MD19-T-107 containing the full length of SDLY107 strain. Infectious RNAs were obtained by transcription in vitro and transfected into Vero cells. The recombinant virus was identified by PCR and Q RT-PCR. The result of PCR amplification was 600. 3D region of 1 400 BP and 200 BP and its left and right fragments; A 1-3D fragment of 2100bp was amplified by overlapping PCR. The fragments of 7 400 BP and 2 700 BP were identified by double enzyme digestion. The results of sequencing confirmed that 3D region replacement was successful. After transfection of Vero cells with infectious RNA, it was observed that the cells were crumpled and rounded at the third passage, and the refraction was enhanced. A 226bp fragment of the recombinant virus was identified by PCR. Q RT-PCR detected that the amount of virus in the cells began to increase slowly at 24 h and then increased rapidly after 48 h. Conclusion the recombinant virus SDLY107 (1-3D) was successfully saved. It provides a basis for further studying the virulence of 3D protein and its role in the pathogenesis of EV71.
【作者单位】：山东大学公共卫生学院病毒学研究室;山东省医疗器械产品质量检验中心微生物室;
【基金】：国家自然科学基金面上项目(81371833) 山东省重点研发计划(2015GSF118162)
【分类号】：R373
【正文快照】： 肠道病毒71型(enterovirus 71,EV71)是手足口病(hand-foot-mouth disease,HFMD)的主要病原体之一,主要感染5岁以下的婴幼儿[1],其临床表现轻重不一,轻者仅表现为发热和手、足及口腔等处的斑丘疹和疱疹等,严重者可伴发无菌性脑膜炎、脑脊髓炎和神经源性肺水肿等中枢神经系统并

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