摘要
背景与目的以前的研究已经证明nm23H1基因是肿瘤转移抑制基因,但其抑制肿瘤转移的分子机制目前还不完全清楚。基因定点突变技术可以精确地改变基因的特定碱基序列,获得突变蛋白质。本研究的目的是应用基因定点突变技术构建突变型nm23H1增强型绿色荧光蛋白(EGFP)融合基因,为进一步研究肿瘤抑制基因nm23H1的功能、生化作用机制提供理论基础和实验依据。方法以逆转录病毒PLXSN野生型nm23H1EGFP质粒为突变模板,应用QuikChangeTM定点突变方法,简单、快速、高效地引入nm23H1S44A、nm23H1P96S、nm23H1H118F、nm23H1S120G四个点突变和一个联合位点突变nm23H1P96SS120G,构建了突变型nm23H1EGFP融合基因。结果成功构建了nm23H1S44AEGFP、nm23H1P96SEGFP、nm23H1H118FEGFP、nm23H1S120GEGFP、nm23H1P96SS120GEGFP五个突变型nm23H1EGFP融合基因,经DNA序列分析突变的碱基序列与实验设计完全一致。结论成功构建了五个具有不同突变位点的突变型nm23H1EGFP融合基因。QuikChangeTM定点突变技术是一种简单、快速、高效的基因定点突变方法。
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, D44A, 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-H1^96S-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.
出处
《中国肺癌杂志》
CAS
2006年第2期117-122,共6页
Chinese Journal of Lung Cancer
基金
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~~
作者简介
通讯作者：周清华，E-mail：zhouqh1016@yahoo．com．cn
