摘要
目的构建针对柯萨奇病毒B4 2B基因的siRNA表达载体并检测该载体在体外培养细胞中对柯萨奇病毒B4的抑制作用。方法选择柯萨奇病毒B4的2B基因区21bp的基因片段作为靶序列,合成含有靶序列的DNA片段并克隆到pGCsi-U6/Neo/GFP/siNeGative载体中,构建pGCsi-U6/Neo/GFP/2B,使其可表达具有发夹结构的双链siR-NA,在转染该载体后用柯萨奇病毒B4攻击Hela细胞,检测该载体对病毒感染细胞的保护效应。结果通过酶切、电泳、序列分析及荧光显微镜观察证明载体构建成功,而且pGCsi-U6/Neo/GFP/2B转染组的柯萨奇病毒B4的滴度及TCID50都明显低于pGCsi-U6/Neo/GFP/siNeGative转染组。结论成功构建了柯萨奇病毒2B基因的siRNA表达载体,而且该载体在体外培养细胞中对柯萨奇病毒的复制具有抑制作用。
Objective Constructing the siRNA expressing plasmid pGCsi-U6/Neo/GFP/2B and test the inhibition effect of recombinant plasmid. Methods Chosed 21bp fragments of the Coxsaekie virus B4 2B gene as the target and synthesized target gene fragment, then cloned it into pGCsi-U6/Neo/GFP/siNeGative to get the recombinant plasmid pGCsi- U6/Neo/ GFP/2B; recombinant plasmids were transinfected into Hela ceil, then infected Hela cells by CVB4. Results the correct resuits showed that the recombinant plasmid has special fragments and DNA sequence by restrict nuelease digestion, electrophoresis and DNA sequencing; and the green fluorescent protein was also observed in Hela ceils under fluorescent microscope; the TCID50 of the Coxsaekie virus B4 in Hela ceils transfected with the plasmid pGCsi-U6/Neo/GFP/2B was lower than that transfected with the control negative sIRNA expressing vector pGCsi-U6/Neo/GFP/siNeGative. Conclusion Successful construction sIRNA expressing plasmid and inhibition the Coxsackie virus replicationin vitro.
出处
《中国实验诊断学》
2006年第1期52-55,共4页
Chinese Journal of Laboratory Diagnosis
基金
吉林大学创新基金资助项目(编号2002CX107)
美国FDA肠道疾病与性传播疾病预防与控制实验室徐德启教授合作项目
