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目的提高带某些限制酶切位点聚合酶链反应(PCR)扩增产物克隆入表达载体的效率。方法将带HindⅢ酶切位点的PCR扩增产物与T载体连接,转化BL21DE3EColi,提取质粒后,用HindⅢ酶切,电泳分离并提取纯化的目的片段。然后,与经过相同的限制性酶酶切的表达载体pNCCL1连接。结果经T载体克隆酶切构建的内含严重急性呼吸综合征冠状病毒(SARSCoV)核酸的病毒样颗粒表达载体,方法简单快速,每次均可获得成功。而采用直接酶切扩增产物的构建方法,未获得成功构建的表达载体。结论T载体克隆可用于对直接酶切效果不好(如HindⅢ)的PCR扩增片段的表达载体的连接构建,方法简便高效。
Objective To improve the efficiency of cloning PCR products with some restriction enzyme sites into expression vector. Methods The PCR amplification product with Hind Ⅲ restriction site was ligated with T vector and transformed into BL21 DE3 E coli. The plasmid was extracted and digested with Hind Ⅲ. The purified target fragment was isolated by electrophoresis. Then, it was ligated with the expression vector pNCCL1 which had undergone the same restriction enzyme digestion. Results The virus-like particle expression vector containing the SARS CoV (SARSCoV) nucleic acid was successfully constructed by T vector cloning. The method was simple and rapid, and was successful every time. However, the direct digestion amplification products were not constructed successfully. Conclusion The T vector cloning can be used to construct the expression vector of the PCR amplification fragment which is not directly digested by Hind Ⅲ. The method is simple and efficient.