Construction and Expression of nm23-H1 Gene with Different Enzyme Activities and Resistant Specific shRNA in Eukaryotic Expression Vector

• Main Authors: Zhansheng LU, Lili GUO, Lin LI, Zhihao WU, Qinghua ZHOU
• Format: Article
• Language: zho
• Published: Chinese Anti-Cancer Association; Chinese Antituberculosis Association 2014-03-01
• Series: Chinese Journal of Lung Cancer
• Subjects: Nm23-H1; Overlap extension PCR; Site-directed mutagenesis; Western blot
• Online Access: http://dx.doi.org/10.3779/j.issn.1009-3419.2014.03.01

Background and objective It has been proven that nm23-H1 gene was a tumor metastatic suppressor gene. However, it’s molecular mechanism of suppressing metastasis remains unexplored. There is a closely relationship between the abnormality of stucturs and functions of nm23-H1 gene, and cancer invasion and metastasis. We have constructed the vector with nm23-H1-shRNA and the vector with nm23-H1cDNA resistant to the specific shRNA. So, we plan to construct shRNA-resistant eukaryotic expression vector of nm23-H1 gene by site-directed mutagenesis, rescue experiment was performed to verify the nm23-H1 gene expression, and to provide basement for studying the biochemical mechanisms of nm23-H1 gene. Methods Site-directed mutagenesis of nm23-H1 gene was performed by overlap extension PCR method. Pure plasmid containing gene of nm23-H1 (shRNA-resistant) was prepared. The desired five mutations were constructed and cloned into the eukaryotic vector pcDNA3.1Hygro(+). The human lung adenocarcinoma cell A549/nm23-H1-shRNA (stable nm23-H1 gene silencing) was transfected with the five mutants, and the expression of the mutant proteins was determined by Western blot. Results Five eukaryotic expression vectors (shRNA-resistant) of nm23-H1, nm23-H1S44A, nm23-H1P96S, nm23-H1H118F, nm23-H1S120G, nm23-H1P96S-S120G, were successfully constructed. The results of DNA sequencing confirmed that the base sequences of the genes were completely concordant with experiment design. The expression of nm23-H1 mutant proteins was verified by Western blot. Conclusion Five eukaryotic expression vectors (shRNA-resistant) of nm23-H1 gene were successfully constructed, and the mutant proteins were verified. The site-directed mutagenesis technical of overlap extension PCR is a efficient, simple and economical method.