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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 vector containing HCV RNA-like particles capable of expressing ribonuclease (RNase). Methods After the expression vector pINCCL was digested with Hind Ⅲ, the amplified product was ligated with the non-coding region of HCVRNA (5 ’UTR) cut with the same endonuclease and ligated with T4 DNA ligase to construct a new expression vector pINCCL HCVRNA 5 ’UTR, and then transformed into BL21 DE3E. Coli for prokaryotic expression. Results A new expression vector pINCCL HCVRNA 5 ’UTR was successfully constructed. The prokaryotic expression plasmid was RNase-resistant. CONCLUSION: The pINCCL HCVRNA 5 ’UTR expression vector and prokaryotic expression system can serve as a platform for the construction and preparation of an RNA-resistant HCV RNA standard and control plasmid.