节点文献
抗呼吸道合胞病毒感染的新药物作用靶点的筛选
Screening of New Drug Targets against Respiratory Syncytial Virus Infection
【摘要】 目的为了筛选潜在的抗呼吸道合胞病毒药物作用靶标,以呼吸道合胞病毒感染相关的基因为靶标来构建抑制RSV感染的pSUPER RNAi表达载体,通过体外抗病毒实验进行验证,最后筛选出有效的靶基因。方法利用信使RNA(mRNA)差异显示法,获得特定细胞差异表达的mRNA差异显示谱,通过电子克隆获得这些EST片段的电子延伸产物,得到三十余条与RSV感染密切相关的基因。从中选取部分基因序列作为靶点,化学合成2条编码短发夹RNA序列的寡核苷酸链克隆到pSUPER载体上,构建若干靶向呼吸道合胞病毒感染细胞的相关基因的RNAi质粒。通过体外抗病毒实验来筛选有效的作用靶标结果。结果本研究设计的siRNA序列克隆到了pSUPER上相应位置,序列正确。针对RSV基因构建的重组质粒pshRNA-ZQ06/16/20/26均可减轻RSV所致的细胞病变,降低病毒滴度,重组质粒对细胞生长无明显细胞毒性作用,通过噻唑蓝比色法(MTT)从mRNA水平分析并确定了该RNAi效应对靶基因有特异性抑制作用。结论重组质粒pshRNA-ZQ06/20/26较pshRNA-ZQ16抗RSV作用要强。
【Abstract】 Objective To screen potential targets for anti- RSV drugs,we constructed the pSUPER RNAi vectors of targeting gene in infection of RSV on SPC- A1 cell in order to inhibit the replication of RSV at gene level. Methods Use DDRT- PCR method to obtain mRNA differential expression spectrum of SPC- A1 cell infected by RSV,then gain more than thirty genes which are close correlation in in- fection of RSV using electronic clone. We selected some gene sequences as target spot among the thirty genes. Sixty four base- pair oligos for hairpin RNA expression were chemically synthesized and annealed. PSUPER vector was linearized with BglII and HindⅢ. Finally,annealed oligos were inserted into the treated pSUPER to construct RNAi plasmid. Then active recombinant plasmids were selected primarily and the transfection efficiency,cytotoxicity were evaluated. We make use of Plaque assay and the MTT method to observe disturbing effection of four recombinant plasmids and specific inhibit effction of corresponding virus target gene. Results Four recombinant plasmids pshRNA- ZQ06 / 16 /20 /26 targeting the gene of RSV successfully constructed,and they could alleviated the CPE of RSV infectied SPC- A1cells. The recom- binant plasmids had not significant cytotoxicity on SPC- A1 cells. By the method of MTT,analysing and determing the effection of RNAi that can specific inhibit RSV target gene. Conclusions The pshRNA- ZQ06 /20 /26 showed a more potent inhibition than pshRNA- ZQ16.
- 【文献出处】 湖北中医药大学学报 ,Journal of Hubei University of Chinese Medicine , 编辑部邮箱 ,2013年06期
- 【分类号】R96
- 【被引频次】3
- 【下载频次】167