具有不同酶活性并可抵抗特异shRNA降解的nm23-H1真核表达载体的构建和表达

摘要

背景与目的已有的研究证明nm23-H1基因是一个重要的肿瘤转移抑制基因，但其抑制肿瘤转移的生化机理尚不完全清楚。Nm23-H1基因结构和功能异常与肿瘤的侵袭转移有密切关系。我们前期已构建了nm23-H1的短发夹RNA（short hairpin RNA, shRNA）载体以及可抵抗此shRNA降解的nm23-H1的cDNA的表达载体，在此基础上我们欲应用基因定点突变技术构建具有不同酶活性并能抵抗此shRNA降解的nm23-H1cDNA真核表达载体，并通过恢复实验验证其表达，为进一步研究肿瘤抑制基因nm23-H1的分子机制提供理论基础和实验依据。方法以pcDNA3.1(+)-shRNA-resistant-nm23-H1质粒为突变模板，应用重叠延伸PCR方法引入nm23-H1基因四个单点突变和一个联合位点突变，并将突变基因片段克隆到真核表达载体pcDNA3.1Hygro(+)。将突变质粒转染人肺腺癌细胞株A549/nm23-H1-shRNA（稳定沉默nm23-H1基因），利用Western blot技术验证不同突变体nm23-H1蛋白的表达。结果成功构建了shRNA抵抗的nm23-H1S44A、nm23-H1P96S、nm23-H1H118F、nm23-H1S120G、nm23-H1P96S-S120G五个突变型真核表达载体，经DNA序列分析突变的碱基序列与实验设计完全一致，经Western blot验证nm23-H1蛋白表达正常。结论成功构建了五个具有不同突变位点的shRNA抵抗的nm23-H1基因真核表达载体，并且突变蛋白质nm23-H1表达正常，同时也表明重叠延伸PCR技术是一种高效、便捷、经济的DNA定点突变方法。%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. hTere is a closely relationship between the abnormality of stucturs and functions of nm23-H1 gene, and cancer invasion and metastasis. We have constructed the vec-tor with nm23-H1-shRNA and the vector with nm23-H1cDNA resistant to the speciifc shRNA. So, we plan to construct shR-NA-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 con-taining gene of nm23-H1 (shRNA-resistant) was prepared. hTe desired ifve mutations were constructed and cloned into the eukaryotic vector pcDNA3.1Hygro(+). hTe human lung adenocarcinoma cell A549/nm23-H1-shRNA (stable nm23-H1 gene silencing) was transfected with the ifve 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. hTe results of DNA sequencing conifrmed that the base sequences of the genes were completely concordant with experiment design. hTe expression of nm23-H1 mutant proteins was veriifed by Western blot. Conclusion Five eukaryotic expression vectors (shRNA-resistant) of nm23-H1 gene were successfully con-structed, and the mutant proteins were veriifed. hTe site-directed mutagenesis technical of overlap extension PCR is a effcient, simple and economical method.