柯萨奇病毒B4 2B基因siRNA表达载体的构建及其对柯萨奇病毒B4抑制作用的研究

目的构建针对柯萨奇病毒B42B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pGCai-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siRNA，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DGCsi-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。

Inhibition of coxsackle virus B4 replication by small interfering RNA expression plasmid target to 2B gene

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.