【摘要】 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5×10⁷ 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。更多还原

【Abstract】 The present paper aimed to inhibit the expression of 3 D polymerase of coxsackievirus B by short hairpin RNAs(shRNAs) with lentiviral vector. Three pairs of RNA interference sequences targeting 3 D 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-3 DshRNA was constructed and co-transfected with psPAX2 and pMD2.G in 293 T cells. The packaged lentivirus(titer 5×10~7 TU) was subjected to functional evaluations in HeLa cells and mice. The inhibition of 3 D polymerase expression was observed.更多还原