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目的 为耐核糖核酸酶 (RNase)的RNA标准品和质控品的表达制备提供 1个通用载体平台。方法 将MS2 噬菌体基因组中成熟酶蛋白和包膜蛋白基因编码序列的 1 7kbcDNA目的片段 ,用HindⅢ和EcoRⅠ酶切后 ,用于相同内切酶酶切的表达载体质粒pET2 8b中 ,并在T4 DNA连接酶的存在下连接 ,构建一新的表达载体pINCCL ,再转化BL2 1 DE3E Coli进行原核表达。结果 成功构建出新的表达载体pINCCL ,经原核表达为耐RNase的病毒样颗粒。结论 本研究得到的pINCCL表达载体及原核表达系统 ,可作为 1个耐RNase的RNA标准品与质控品的构建和制备表达通用载体平台 ,以促进有关标准品和质控品的研究。
Aim To provide a universal vector platform for the preparation of RNase-resistant RNA standards and controls. Methods The 17kbcDNA fragment of the coding sequence of mature enzyme and envelope protein in MS2 bacteriophage genome was digested with HindⅢ and EcoRⅠand used in the expression vector plasmid pET2b of the same restriction enzyme digestion. Ligase to construct a new expression vector pINCCL, then transformed into BL21 DE3E Coli for prokaryotic expression. Results The new expression vector pINCCL was successfully constructed and prokaryotic expressed as RNase-resistant virus-like particles. Conclusion The pINCCL expression vector and prokaryotic expression system obtained in this study can be used as a universal vector platform for the construction and preparation of an RNase-resistant RNA standard and control product to promote the research on standards and quality control products.