摘要
SARS冠状病毒是引起重症急性呼吸综合症的主要原因 ,目前尚没有特效药物或疫苗对抗这种新病毒。RNA干涉是指双链RNA可以特异地降解细胞内同源基因的mRNA。在哺乳动物细胞中 ,<30bp的小双链RNA能引起RNA干涉 ,又可以避免干扰素反应。通过体外转录得到SARS病毒 3种基因RNA依赖的RNA聚合酶、刺突蛋白及核衣壳蛋白部分片段的长双链RNA ,然后用RNaseⅢ有限切割成长度 <30bp的小干涉RNA。同时把上述 3种基因片段分别连接到质粒pGL3 Control中 ,得到的 3个质粒pGL R、pGL S和pGL N可以分别在细胞内转录出荧光素酶 RNA依赖的RNA聚合酶、 刺突蛋白、 核衣壳蛋白的杂合mRNA。上述质粒分别和相应的小干涉RNA共转染HEK2 93F细胞 ,测定荧光素酶活性 ,结果小干涉RNA使相应质粒表达荧光素酶的活性显著下降 ;用逆转录定量PCR反应测量mRNA丰度 。
SARS associated coronavirus has been identified for the cause of Severe Acute Respiratory Syndrome, for which there is no efficacious drugs or vaccines. RNA interference(RNAi) is a process in cell to degradation specific target mRNA by double stranded RNA. In mammalian cells, RNAi can be triggered by short interfering RNA (siRNA). RNA interference of virus specific genes has emerged as a potential antiviral mechanism. This work evaluated if RNase Ⅲ prepared short interfering RNAs can induce specific degradation of SARS coronavirus mRNAs in human cells.Three of SARS genes, RNA dependent RNA polymerase(RdRp), spike and nucleocapsid, were amplified with T7 promoter flanked primers. Long length double stranded RNA of these genes were transcribed in vitro and then were cleaved to <30bp length short interfering RNA with E.coli RNase Ⅲ. These siRNAs were termed esiRNA R,esiRNA S and esiRNA N respectively. RdRp, spike and nucleocapsid DNA fragments were inserted into the plasmid pGL3 Control, obtained plasmids pGL R, pGL S and pGL N can express hybrid mRNAs luciferase RdRp, spike and nucleocapsid in cells. Above plasmids and esiRNAs were co transfected to HEK293F cells with reference plasmid pRL TK. Firefly luciferase and Renilla luciferase activity were measured. Hybrid mRNAs' abundance was measured using reverse transcription real time PCR.Firefly luciferase expression of pGL R was reduced to 13% by esiRNA R. Expression of pGL S was reduced to 11% by esiRNA S. Expression of pGL N was reduced to 40% by esiRNA N. Control esiRNAs didn't affect luciferase expression; Hybrid mRNAs' abundance was dramatically reduced by corresponding esiRNAs.RNase Ⅲ prepared short interfering RNAs induce robust and specific degradation of SARS coronavirus mRNAs in HEK293F cells. These siRNAs could be used to inhibit SARS coronavirus in future research.
出处
《生物工程学报》
CAS
CSCD
北大核心
2004年第4期484-489,共6页
Chinese Journal of Biotechnology
基金
国家 973计划项目基金资助 (No .2 0 0 2CB5 13 2 0 4)~~
