Site-directed Mutagenesis Based on Overlap Extension PCR

[Objective] To establish an efficient, convenient and economical method for site-directed mutagenesis. [Method] The target mutation was introduced into primers designed by DNAMAN5.0 software. Through overlap extension PCR for twice obtained the mutation gene which of the full length of the recombinant Human Tissue type plasminogen activator （Reteplase）. The mutation gene cloned it into pEASY- blunt simple cloning vector for sequencing. [Result] The sequencing results showed that three site mutations were fully consistent with the expected results （10~ site had been added a base-pair of A, C had been changed into G at 137~ site, G had been changed into A at 686~ site）.Three site mutations were introduced by using overlap extension PCR on one-step. The overall rate of obtaining the mutant sites was 100%. Site-directed mutagenesis will clone the recombinant Human Tissue type plas- minogen activator and laid the basis for the functional study. [Conclusion] Site-directed mutagenesis was successfully implemented based on the overlap extension PCR which is an efficient, convenient and economical DNA-directed mutagenesis method.

人胰岛素样生长因子Ⅰ在毕赤氏酵母中的表达研究

利用PCR定点突变技术对人胰岛素样生长因子（huIGF-I）基因进行改造,克隆到pGEMT载体测序。并构建酵母表达载体pGaAIGF-I,转化毕赤氏酵母GS115。摇瓶发酵4d后,检测表达水平占可溶性总蛋白的15％以上,且表现出较好细胞增殖活性。