柯萨奇病毒B4双siRNA表达载体的构建及表达

Construction of double siRNA expressing vector of Coxsackie virus B4

目的:构建以柯萨奇病毒B4的1A和2A基因为靶基因并带有绿色荧光蛋白标志的可表达发夹结构的双链siRNA的表达载体pU6/double-siRNA/Neo/GFP/1A/2A。方法:选择柯萨奇病毒B4的1A和2A区基因21 bp为靶序列分别合成65 bp的互补片段并克隆到pSilencer2.1U6 Neo和pGCsi-U6/Neo/GFP质粒中,经限制性内切酶消化、琼脂糖凝胶电泳分离、回收DNA片段,及连接酶连接构建双siRNA表达质粒pU6/double-siRNA/Neo/GFP/1A/2A;将构建的载体转染Hela细胞后,荧光显微镜观察荧光的表达。结果:限制性内切酶消化、琼脂糖凝胶电泳检测重组质粒pU6/double-siRNA/Neo/GFP/1A/2A,有正确的特异性片段,DNA测序也证明其具有正确序列;将该重组质粒转染入Hela细胞后,重组质粒构建成功并在真核细胞内表达GFP,表达时间可达15 d以上。结论:针对柯萨奇病毒B4的1A和2A基因的双siRNA表达载体pU6/double-siRNA/Neo/GFP/1A/2A构建成功,并可在真核细胞内持续表达15 d以上。

Objective To construct the double hairpin siRNA and green fluorescent protein （GFP） expressing vector pU6/double-siRNA/Neo/GFP/1A/2A to interfere 1A and 2A gene of Coxsackie virus B4. Methods 21 bp fragments of the Coxsackie virus 134 2A and 1A gene were chosen as the targets and 65 bp complimentary fragments were synthesized, then the target gene fragments were cloned into pSilencer2.1U6 Neo and pGCsi-U6/Neo/GFP/ siNeGative, respectively, then the double siRNA expressing vector pU6/double-siRNA/Neo/GFP/1A /2A was constructed by restrict endonuclease digestion, elctrophoresis isolation and reclaimer, ligatied by T4 DNA ligase; then the double siRNA expressing plasmid was transfected into Hela cells, and the GFP was observed under fluorescent microscope. Results The correct results showed that the recombinant plasmid had the correct special fragments and DNA sequence detected by restrict endonuclease digestion, electrophoresis and DNA sequencing; and GFP was also observed in Hela cells tansfected with pU6/double-siRNA/Neo/GFP/1A /2A under fluorescent microscope more than 15 d after transfection. Conclusion The double siRNA expressing vector pU6/double- siRNA/Neo/GFP/1A/2A is constructed successfully; it has the correct target viral gene sequences and can express GFP gene in Hela cells more than 15 d after transfection.