Construction of human nm23-H1 mutant and EGFP fusion genes using site-directed mutagenesis

ZHU Daxing, ZHOU Qinghua, WANG Yanping, ZHU Wen, CHEN Xiaohe, SUN Zhilin. Key Labora- tory of Lung Cancer Molecular Biology of Sichuan Province, West China Hospital, Sichuan University, Chengdu, Sichuan 610041, P.R.China  

Background and objective Previous researches have proven that nm23-H1 gene is a tumor metastatic suppressor gene, however, it is still unknown about its exact molecular mechanisms. Site-directed mutagenesis can correctly change the base sequence and get mutant proteins. The aim of this study is to construct nm23-H1 mutant and EGFP fusion genes by site-directed mutagenesis, and to provide basement for studying the functional and biochemical mechanisms of nm23-H1 gene. Methods Site-directed mutagenesis of nm23-H1 gene was performed by modified QuikChange TM Site-directed Mutagenesis Kit method. Pure plasmid containing fusion gene of nm23-H1 and EGFP (PLXSN-nm23-H1-EGFP) was mini-prepared. Four pairs of mutagenic primers were synthesized in vitro and the desired five mutations, S44A, P96S, H118F, S120G and P96S-S120G were introduced into nm23-H1-EGFP fusion gene by PCR. Results Five nm23-H1 mutant and EGFP fusion genes, nm23-H1 S44A-EGFP, nm23-H1 P96S-EGFP, nm23-H1 H118F-EGFP, nm23-H1 S120G-EGFP and nm23-H1 P96S-S120G-EGFP, were successfully constructed. The results of DNA sequencing confirmed that the base sequences of the mutant fusion genes were completely concordant with experiment design. Conclusion Five nm23-H1 mutant and EGFP fusion genes are successfully constructed, which can be used in further studies. QuikChange TM site-directed mutagenesis is a simple, rapid and efficient method.

【Key Words】： Site-directed mutagenesis nm23-H1 EGFP nm23-H1 mutant and EGFP fusion gene
【Fund】： 国家自然科学基金重点项目(No.30430300);; 教育部高等学校博士学科点专项基金(No.20040610060)资助~~
【CateGory Index】： R734.2

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