CAJ | 학술논문

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.
목적 구건능표체형광소매적중조가살기병독B조3형(coxsackievirus B3,CVB3),이실현정량검측CVB3.방법 장형광소매기인극륭지CVB3기인조개방독마광기시위치,중조병독기인조DNA전염HeLa세포이회복병독、측서,평개중조병독적형광소매표체,확증중조병독병측정중조병독주독력.결과 성공구건2개중조병독질립,전염HeLa세포,표체해신형광소매(humanizedform of Renilla Luc,hRLuc)、형화충형광소매(firefly luciferase,Luc)적pCVB3-hRLuc화pCVB3-Luc전염후제1대균가검측도hRLuc화Luc활성,단지유pCVB3-hRLuc전염세포유명현세포병변.확증화순화적CVB3-hRLuc병독감염HeLa세포가견명현세포병변급hRLuc활성.확증적CVB3-Luc몰유관찰도명현적세포병변,차종제2대병독개시취미능검측도형광소매활성.용HeLa세포경공반형성시험진행독력측정,CVB3-hRLuc적독력위1.4(107 pfu/ml).결론 성공구건료표체hRLuc적중조CVB3독주CVB3-hRLuc,위정량연구CVB3감염타하기출.
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.