定点突变法构建人尼古丁受体α5亚单位突变体(Asn398)慢病毒表达载体  

Construction of lentiviral expression vector of human nicotinic receptor α5 subunit mutant (Asn398) by site-specific mutagenesis

作　　者：赵祝香[1] 彭芳[2] 何桦[1] 李裕军[1] 赵子文[1] 

机构地区：[1]广州市第一人民医院呼吸内科,广东广州510180 [2]广州医科大学附属第三医院呼吸内科,广东广州510150

出　　处：《广州医科大学学报》2017年第2期6-10,共5页Academic Journal of Guangzhou Medical University

基　　金：广东省科技厅项目(2013B021800069)

摘　　要：目的:克隆人尼古丁受体α5亚单位(CHRNA5),以野生型CHRNA5为模板,通过定点突变法构建CHRNA5突变体(Asn398)慢病毒真核表达载体,为Asn398功能研究提供基础。方法:RT-PCR克隆CHRNA5基因片段,运用重叠延伸PCR法进行定点突变,定向克隆至EX-Q0146-LV201,序列分析鉴定野生及突变重组子,将CHRNA5突变体Asn398及未突变体定向克隆到EX-Q0146-LV201慢病病毒表达质粒上。并转染至HEK293T细胞中,RT-PCR检CHRNA5 mRNA表达,Westernblot检测其蛋白表达。结果:重组子目的基因的序列分析结果显示成功克隆CHRNA5 cDNA基因,并成功将CHRNA5第398位氨基酸天冬氨酸(Asp)转变为天冬酰胺(Asn),突变体及未突变在转染细胞中均获得CHRNA5 mRNA及蛋白表达。结论:成功构建CHRNA5突变体Asn398慢病毒表达载体,证实其在293细胞中有mRNA转录及其蛋白表达,为下一步进行功能研究奠定基础。Objective ：To clone human nicotine receptor α5 subunit （CHRNA5） with wild-type CHRNA5 as template, and to construct lentiviral eukaryotic expression vector of CHRNA5 mutant （Asn398） by site-specific mutagenesis,and to provide the basis for the study of Asn398 function. Methods：The CHRNA5 gene fragment was cloned by RT-PCR and directed cloned into EX-Q0146-LV201 by overlap-extension PCR for site-specific mutagenesis. The sequence analysis was used to identify wild and mutant recombinants. The CHRNA5 mutant Asn398 and the non-mutant were directed cloned into the EX-Q0146-LV201 lenfiviral expression plasmid, and transfected to HEK293T cells. The mRNA expression of CHRNA5 was examined by RT-PCR, and the protein expression was determined by Western blot. Results： The sequence analysis of the recombinant subunit target gene showed that the CHRNA5 cDNA gene was successfully cloned and the amino acid, aspartic acid （Asp） at position 398 in CHRNA5 was successfully transformed into asparagine （Asn）. The mutant and non-mutant obtained CHRNA5 mRNA and protein expression in the transfected cells. Conclusion： The CHRNA5 mutant Asn398 lentiviral expression vector is successfully constructed, confirming its mRNA transcription and protein expression in 293 cells, which provides the basis for further functional study.

关 键 词：尼古丁受体 定点突变 慢病毒 突变 

分 类 号：R563[医药卫生—呼吸系统] R593.28[医药卫生—内科学]