PcDNA3.1(+)/CYP11B2真核表达质粒的构建及其在NCI-H295R细胞中的表达  

The construction of eukaryotic expression vector PcDNA3.1(+)/CYP11B2 and the expression in NCI-H295R cells

作　　者：蔡辉 黄文利 孙朝辉 Cai Hui;Huang Wenli;Sun Zhaohui(Yunnan Provinical Hospital of Traditional Chinese Medicine,Kunming 615000)

机构地区：[1]云南省中医医院云南中医药大学第一附属医院老年病科

出　　处：《中国现代医药杂志》2019年第9期21-25,共5页Modern Medicine Journal of China

摘　　要：目的构建人醛固酮合成酶基因(CYP11B2)的真核表达质粒,并在人肾上腺上皮细胞(NCI-H295R)中表达,为下一步定点突变研究奠定基础。方法从人的肾上腺组织中提取总的RNA,采用RT-PCR扩增法扩增人的CYP11B2全长cDNA并克隆到PMD18-T载体中,然后再亚克隆于PcDNA3.1(+)真核表达载体,通过菌液PCR和酶切鉴定重组真核表达质粒,并以DNA测序证实。利用阳离子脂质体介导体外转染技术瞬时转染NCI-H295R,RT-PCR和放射免疫法(RIA)检测CYP11B2基因的表达。结果菌液PCR扩增和酶切证实真核表达质粒构建成功,测序表明CYP11B2基因cDNA全长(1 509bp)和GenBank上公布的序列(D13752)相比较,存在一个碱基差异A3323G,这个变异使得第173位的赖氨酸变为精氨酸,核苷酸序列的同源性为99.9%,氨基酸序列的同源性为99.8%。真核表达质粒转染人肾上腺上皮细胞后CYP11B2 mRNA表达增加,细胞上清中的醛固酮含量显著增加。结论①通过查阅国外SNP数据库得知K173R为一个SNP位点,编号为rs4539。②成功构建了人CYP11B2基因的真核表达质粒,阳离子脂质体是NCI-H295R有效的体外转染体系,本实验为进一步研究该基因的结构与功能关系奠定了基础。Objective To construct the eukaryotic expression vector of human aldosterone synthease and detect their expression in human NCI-H295 R cells, which will make foundation for site-directed mutagenesis. Methods The full-length c DNA of human aldosterone synthease gene (CYP11 B2) was amplified by RT-PCR and inserted into the PMD18-T vector. The recombinant plasmid was digested by restriction endonucleases and subcloned into eukaryotic expression vector Pc DNA3.1(+). The recombinant plasmid was identified by PCR and double digestion with restriction endonucleases, and confirmed by DNA sequencing. Then the recombinant plasmid was transfected into NCIH295 R cells by lipofectamine method. The expression of CYP11 B2 gene mRNA in NCI-H295 R cells was assayed by RT-PCR, and the aldosterone in medium was detected by RIA. Results The eukaryotic expression vector was constructed successfully. DNA sequencing showed that the length of cDNA was 1509 bp. There was a gene variant A3323 G compared with the standard sequence (D13752) in GenBank. The protein sequence change was K173 R. The homology was 99.9% in nucleotide acid and 99.8% in amino acid. The expression of CYP11 B2 mRNA in human adrenal cortical cells increased after transfection and the production of aldosterone in cells increased too. Conclusion ①K173 R is a SNP which had been registered in dbSNP database, the code number is rs4539.②The eukaryotic expression plasmid of CYP11 B2 has been successfully constructed. The expression plasmid can express in NCI-H295 R cells. It will lay the foundation for study in the relationship between structure and function.

关 键 词：PcDNA3.1(+)/CYP11B2 NCI-H295R 转染 

分 类 号：R346[医药卫生—基础医学]