山东医药2017，Vol.57Issue(42)：20-24,5.DOI:10.3969/j.issn.1002-266X.2017.42.006
非融合绿色荧光蛋白标记EV71型人肠道病毒的构建
Construction of EV71 genome labeled by non-fusion enhanced green fluorescent protein
摘要
Abstract
Objective To construct the Enterovirus 71 (EV71)labeled by non-fusion enhanced green fluorescent pro-tein (EGFP)so as to provide a method for studying the anti-viral mechanism of EV71 and screening of antiviral drugs. Methods The EV71 genome labeled by EGFP was constructed by connecting non-fusion link-EGFP sequence in the back of 3D sequence and front of 3'-UTR,and then new EV71 virus was obtained by transferring EV71-link-EGFP plasmid into RD cell line. The location of link and EGFP sequence were identified by PCR. Virus titer was estimated with TCID50 methods at the 24,48,72,96,120,144,and 168 h after RD cells were infected,respectively. The mRNA level was an-alyzed by RT-PCR. The recombinant virus was used to infect the RD cells and RD cells lysed after 72 h. The GFP and P1 protein expression was detected by Western blotting. After the recombinant virus infected RD cells,and after 13 rounds of passage,the EGFP gene stability was determined by Western blotting. Results EV71-link-EGFP recombinant virus was obtained via analyzing mRNA expression level,observing GFP fluorescence and locating link and EGFP site. The curve peak in recombinant virus was the same as the wild type virus. The GFP fluorescence value in recombinant virus was the highest at 48 h. GFP and P1 proteins were stably expressed in recombinant virus. GFP protein level in 13th generation re-combinant virus had no significant difference as compared with that of the first generation. Conclusion EV71-Link-EGFP recombinant virus is constructed successfully via adding link sequence between 3D and EGFP sequence,which can grow well,effectively express EGFP gene in RD cells and has stable heredity.关键词
人肠道病毒/肠道病毒71型/绿色荧光蛋白/荧光标记/基因重组Key words
human enterovirus/Enterovirus 71 type/green fluorescent protein/fluorescence labeling/genetic recom-bination分类
生物科学引用本文复制引用
耿磊,窦烨,王清路..非融合绿色荧光蛋白标记EV71型人肠道病毒的构建[J].山东医药,2017,57(42):20-24,5.基金项目
山东省高校中医药抗病毒协同创新中心基金资助项目(XTCX2014B01-07). （XTCX2014B01-07）
