携带EGFP基因的戊型肝炎病毒重组质粒的构建及其感染性的验证  

Construction of Recombinant Full-length Hepatitis E Virus Fused with EGFP and Assessment of Infectivity

作　　者：李云龙[1] 龙飞燕 杨臣臣 禹文海[2] 毕艳红[1] 王珏[1] 姜典财 彭福春 井申荣[1] 黄芬[1] 

机构地区：[1]昆明理工大学医学院,昆明650500 [2]中国医学科学院医学生物学研究所,昆明650118 [3]大连雅立峰生物制药有限公司,大连116000 [4]云南省洱源县右所镇畜牧兽医站,大理671200

出　　处：《病毒学报》2016年第5期529-537,共9页Chinese Journal of Virology

基　　金：国家自然科学基金(项目号:31360619;81660338);云南省自然科学基金(项目号:2013FB032);中国博士后科学基金(项目号:2014M562672;2015T81138)

摘　　要：为了构建携带增强型绿色荧光蛋白(Enhanced green fluorescent protein,EGFP)基因的戊型肝炎病毒(Hepatitis E virus,HEV)重组质粒,转染人肺癌细胞A549细胞验证其感染性,PCR法分两段扩增HEV全基因组序列和EGFP基因序列,将EGFP报告基因插入到HEV ORF2基因下游,并克隆到体外转录表达载体pGEM-7Zf(+)上。然后利用脂质体转染法将重组质粒转入A549细胞,24h后在荧光显微镜下观察EGFP的表达;转染72h后,利用免疫荧光法检测HEV ORF2蛋白的表达。转染7d后将发生病变的A549细胞收集作为接种物,接种A549细胞验证携带EGFP的HEV-EGFP重组病毒的感染性。结果显示经酶切和测序鉴定携带EGFP的HEV重组表达质粒pGEM-HEV-EGFP构建成功;EGFP基因与HEV在A549细胞中可融合表达;携带EGFP的HEV重组表达质粒转染A549细胞7d后出现病变,并且连续传代3代仍具有感染性。本研究成功构建了携带EGFP的HEV全基因组重组质粒pGEM-HEV-EGFP,并成功感染A549细胞,为进一步研究HEV的复制机制及致病机理奠定基础。The purpose of this study was to construct recombinant full-length hepatitis E virus（HEV）fused with enhanced green fluorescent protein（EGFP）,and assess its infectivity in A549 cells.Two fragments from the full-length HEV genome and the EGFP gene were amplified by PCR.The EGFP gene was inserted downstream of the HEV ORF2 and then cloned into the pGEM-7Zf（＋）vector containing the T7 and SP6RNA polymerase promoters,producing pGEM-HEV-EGFP.The construction of the pGEM-HEV-EGFP recombinant plasmid was confirmed by restriction enzyme digest and sequencing.The pGEM-HEV-EGFP recombinant plasmid was transfected into A549 cells to assess infectivity using Lipofectamine.EGFP expression was observed at 24hpost-transfection,and expression of the HEV ORF2 was detected by immunofluorescence,confirming the presence of the HEV ORF2 and EGFP fusion protein.Cytopathic effects were observed at day seven post-transfection.The infectivity of pGEM-HEV-EGFP was confirmed by the presence of fluorescence after three continuous passages.The recombinant pGEM-HEV-EGFP vector was successfully constructed and effectively infected A549 cells,which will facilitate future studies on the mechanisms of HEV infection and pathogenesis.

关 键 词：戊型肝炎病毒(HEV) 增强型绿色荧光蛋白(EGFP) 感染性 

分 类 号：R373.2[医药卫生—病原生物学]