耐核糖核酸酶内含HCV RNA病毒样颗粒的表达

Expression of HCV RNA containing and RNase-resisting virus-like particles

目的 构建可以表达耐核糖核酸酶 (RNase)的内含HCVRNA病毒样颗粒的载体质粒。方法 将表达载体pINCCL用HindⅢ酶切后 ,与用相同内切酶酶切的HCVRNA非编码区 (5′ UTR)扩增产物 ,在T4DNA连接酶的存在下连接 ,构建一新的表达载体pINCCL HCVRNA 5′ UTR ,再转化BL2 1 DE3E .coli进行原核表达。结果 成功构建得到了新的表达载体pINCCL HCVRNA 5′ UTR ,经原核表达为耐RNase的内含HCVRNA病毒样颗粒。结论 得到的pINCCL HCVRNA 5′ UTR表达载体及原核表达系统 ,可作为一个耐RNase的HCVRNA标准品和质控品的构建和制备表达载体平台。

Objective To construct a expressing plasmid carrier platform that can be used for preparation of RNase-resistant HCV RNA standards and controls. Methods A cDNA fragment of HCV RNA genome 5′-UTR and expression vector pI NCCL DNA were ligated together with T4 DNA ligase after being digested with HindⅢ restriction nucleases. Then, a new expression plasmid carrier pI NCCL-HCV RNA 5′-UTR was constructed. The prokaryotic expression was carried out by transformation of pI NCCL-HCV RNA 5′-UTR into BL21-DE3 E.coli . Results A new expression plasmid carrier pI NCCL-HCV RNA 5′-UTR was successfully constructed. RNase-resistant hepatitis C virus-like particles were obtained after prokaryotic expression of pI NCCL-HCV RNA 5′-UTR. Conclusion The expression plasmid carrier pI NCCL-HCV RNA 5′-UTR is constructed and prokaryotic expression system can be used as an expressing plasmid carrier platform for preparation of RNase-resistant HCV RNA standards and controls for HCV RNA reverse transcription-polymerase chain reaction (RT-PCR) detection.