凝血因子Ⅸ无义突变真核表达载体的构建及其在COS-7细胞的表达  

Construction of Nonsense-mutated Eukaryotic Expression Vector of Factor Ⅸ Gene and Its Expression in COS-7 Cells

作　　者：聂欣[1] 杨林花[1] 柴宝峰[2] 申泉[2] 张媛[1] 张耀方[1] 陈剑芳[1] 

机构地区：[1]山西医科大学第二医院血液科,山西太原030001 [2]山西大学生物技术研究所,山西太原030006

出　　处：《中国实验血液学杂志》2010年第3期749-752,共4页Journal of Experimental Hematology

基　　金：山西省归国留学人员科研基金(编号2009重点7);山西省科技攻关项目(编号022073-1)

摘　　要：本研究以含有fⅨ cDNA的pcDNA3.1质粒为模板构建4种包含不同类型无义突变的fⅨ真核表达载体并进行鉴定,实现其在COS-7细胞中的表达。采用以PCR为基础的定点突变法体外构建fⅨ基因无义突变体,并通过DNA序列分析进一步鉴定证实;应用脂质体转化技术将野生型、突变型fⅨ表达载体分别瞬时转染COS-7细胞,采用实时定量PCR鉴定fⅨ mRNA在各组的相对表达水平。结果表明,4个突变型质粒除相应位点的无义突变外,未见其它突变,提示无义突变载体构建成功。实时定量PCR鉴定表明,各突变组真核表达载体已成功转染COS-7细胞株并能转录为mRNA。结论:采用以PCR为基础的定点突变法可在体外构建fⅨ真核表达载体,并可在COS-7细胞中表达,其转录过程中不引发mRNA降解,这为进一步研究无义突变引起FⅨ功能丧失及表达量降低的机制和治疗研究提供了物质基础。The purpose of this study was to construct 4 types of nonsense-mutated eukaryotic expression plasmids of fⅨ gene, using peDNA3.1 plasmid containing fiX cDNA as template, and to indentify, then to perform their expression in COS-7 cells. These stop mutants constructed by site-directed mutagenesis based on PCR, and further confirrned by DNA sequencing. COS-7 ceils were transfected with either the wild-type or mutated fiX expression constructs, then the relative expression levels of fiX mRNA were detected by real time fluorescent quantitative PCR. The result showed that except the designed sites, there were no other nucleotide mutation in the sequences of four nonsense mutants. The results of real time PCR proved that the nosense-mutated vectors can be effectively expressed in COS-7 cells. It is concluded that the nonsense-mutated eukaryotic expression vectors of fiX gene have been successfully constructed and can express in COS -7 cells, which provides the material basis for further researches on mechanism and treatment of FiX deficiency and the function defects caused by nonsense mutation.

关 键 词：凝血因子IX 真核表达载体 血友病B 提前终止翻译密码子 

分 类 号：R544.1[医药卫生—心血管疾病] Q789[医药卫生—内科学]