表达海肾荧光素酶的重组柯萨奇病毒B组3型质粒的构建及鉴定  

Constructions and identification of recombinant Coxsackievirus B3 with Renilla luciferase expression

作　　者：王慧[1] 武帅钦[2] 陈阳[2] 钟照华[2] 赵文然[3] 佟雷[2] 

机构地区：[1]哈尔滨医科大学病原学实验教学中心,哈尔滨150081 [2]哈尔滨医科大学微生物学教研室 [3]哈尔滨医科大学细胞生物学教研室

出　　处：《国际病毒学杂志》2015年第3期149-153,共5页International Journal of Virology

基　　金：国家自然科学基金（81101234,81271825,31270198）;黑龙江省卫生厅课题（2009-213）

摘　　要：目的 构建能表达荧光素酶的重组柯萨奇病毒B组3型（coxsackievirus B3,CVB3）,以实现定量检测CVB3.方法 将荧光素酶基因克隆至CVB3基因组开放读码框起始位置,重组病毒基因组DNA转染HeLa细胞以恢复病毒、测序,评价重组病毒的荧光素酶表达,扩增重组病毒并测定重组病毒株毒力.结果 成功构建2个重组病毒质粒,转染HeLa细胞,表达海肾荧光素酶（humanizedform of Renilla Luc,hRLuc）、荧火虫荧光素酶（firefly luciferase,Luc）的pCVB3-hRLuc和pCVB3-Luc转染后第1代均可检测到hRLuc和Luc活性,但只有pCVB3-hRLuc转染细胞有明显细胞病变.扩增和纯化的CVB3-hRLuc病毒感染HeLa细胞可见明显细胞病变及hRLuc活性.扩增的CVB3-Luc没有观察到明显的细胞病变,且从第2代病毒开始就未能检测到荧光素酶活性.用HeLa细胞经空斑形成试验进行毒力测定,CVB3-hRLuc的毒力为1.4（107 pfu/ml）.结论 成功构建了表达hRLuc的重组CVB3毒株CVB3-hRLuc,为定量研究CVB3感染打下基础.Objective To develop recombinant coxsackievirus B3 （CVB3） with humanized form of luciferase expression for quantitative analysis of CVB3 infection.Methods The luciferase gene was cloned at the beginning of the open reading frame of CVB3 genome.The recombinant viruses were recovered by HeLa cell transfection.The sequences of these recombinants were confirmed.The expression of hRLuc and virulence had been evaluated.Results Two recombinant plasmids contained modified CVB3 genome were successfully constructed.hRLuc or Luc could be detected in the HeLa cells transfected with pCVB3-hRLuc and pCVB3-Luc by transfecting HeLa cells,however,cytopathic effect （CPE） could only be observed in HeLa cells transfected with pCVB3-hRLuc.The newly constructed CVB3-hRLuc viruses had been cloned by plaque forming assay.Infected with the cloned CVB3-hRLuc viruses,hRLuc could be detected consistently.However,CVB3-Luc failed to show the luciferase expression from the second passage.The virulence of CVB3-hRLuc was 1.4（107 pfu/ml as determined by plaque forming assay）.Conclusions The recombinant CVB3 viruses with expression of hRLuc were successfully constructed and enable us to quantitatively study CVB infection.

关 键 词：柯萨奇病毒B组3型 海肾荧光素酶 基因重组 

分 类 号：R373.23[医药卫生—病原生物学]