Construction of EV71 genome labeled by non-fusion enhanced green fluorescent protein

GENG Lei; DOU Ye; WANG Qinglu

doi:10.3969/j.issn.1002-266X.2017.42.006

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.