摘要
目的:利用聚合酶链反应技术对清道夫受体SR-AⅡ基因进行定点突变的研究。方法:利用聚合酶链反应定点突变技术,首先设计4条(其中2条包含预定突变位点)引物,通过3轮PCR反应扩增出含有所需突变位点的片段,通过酶切连接方法将其重组到pEGFP-C1中形成pEGFP-SR-AⅡ-335质粒。结果:在真核表达载体pEGFP-SR-AⅡ-335基础上获得了SR-AⅡcDNAA1201G和A1202C的突变体,测序结果表明在序列中发生了预期的突变,使SR-AⅡ受体蛋白第335位的赖氨酸变为丙氨酸。结论:SR-AⅡ定点突变体的构建为进一步的功能研究奠定了基础。
Objective To investigate the site-directed mutagenesis of scavenger receptor SR-A Ⅱ gene in vitro. Methods The site-directed mutagenesis of scavenger receptor SR-A Ⅱ gene was made by PCR. Two sets of prinlers were designed according to the sequence of SR-A Ⅱ cDNA,and mismatch was introduced into primers, Mutagenesis was performed in a two-step PCR. The amplified fragments from the third PCR which contained the mutation site were subcloned into the T-vector pMD19,and then the fragments containing the mutation site was obtained from pMD19 with restriction enzyme digestion and was inserted into the same restriction site of pEGFP-SR-A Ⅱ . Results The sequencing analysis showed that the mutation site was correct, Mutation from A to G 1201 and A to C 1202 sites of SR-AⅡV eDNA were found. Conclusion The construction of SR-A Ⅱ site-directed mutant establishes the base of its further functional study.
出处
《实用医学杂志》
CAS
2008年第5期696-699,共4页
The Journal of Practical Medicine
基金
国家自然科学基金资助项目(编号:30671969)
关键词
受体
LDL基因
聚合酶链反应
定点突变
Receptors, LDL Genes Polymerase chain reaction Site-directed mutagenesis
