靶向3D基因shRNA慢病毒载体的构建及功能鉴定

Construction and functional verification of a shRNA targeting 3D polymerase of coxsackievirus B

本研究旨在筛选抑制B组柯萨奇病毒(coxsackievirus B,CVB)3D聚合酶表达的短发卡RNA(short hairpin RNA,shRNA)并构建其慢病毒表达载体,以抑制CVB的3D聚合酶表达。设计并合成3对针对3D基因的RNA干扰序列及相应的对照组序列,通过定量反转录-聚合酶链反应(quantitative reverse transcription-polymerase chain reaction,RT-qPCR)和蛋白免疫印迹法筛选出干扰效率最高的一对,合成shRNA序列。退火后,应用基因重组技术构建pLVTHM-3DshRNA重组质粒,经酶切鉴定及DNA测序后,将重组正确的pLVTHM-3DshRNA与psPAX2、pMD2.G共转染293T细胞,包装慢病毒并鉴定其对CVB3 3D基因表达的干扰效果。测序结果显示,成功构建了pLVTHM-3DshRNA重组慢病毒载体,并收获了包装后的慢病毒,病毒滴度为5×10^(7 )TU。感染HeLa细胞和BALB/c小鼠后,3D聚合酶表达量下降。本研究成功构建了慢病毒Lenti-sh3D,其能有效下调CVB 3D聚合酶的表达,为进一步抗CVB致病机制的研究奠定了基础。

The present paper aimed to inhibit the expression of 3D polymerase of coxsackievirus B by short hairpin RNAs (shRNAs) with lentiviral vector.Three pairs of RNA interference sequences targeting 3D gene and corresponding control sequences were designed and synthesized.The pair with the highest interference efficiency was screened by quantitative reverse transcription-polymerase chain reaction (RT-qPCR) and Western blotting.The recombinant plasmid pLVTHM-3DshRNA was constructed and co-transfected with psPAX2 and pMD2.G in 293T cells.The packaged lentivirus (titer 5×10 7 TU) was subjected to functional evaluations in HeLa cells and mice.The inhibition of 3D polymerase expression was observed.