应用聚合酶链反应技术构建人血小板反应素1基因第13外显子编码钙结合域突变体  

Establishing the mutant of coding calcium binding fragment of the 13th exon of human thrombospondin-1 gene with polymerase chain reaction

作　　者：戴健 马继政 刘剑南 陈相健 杨志健 杨笛 张寄南 

机构地区：[1]南京医科大学第一附属医院心内科暨心血管病研究所,江苏省135重点实验室,江苏省人类功能基因组重点实验室,江苏省南京市210029

出　　处：《中国临床康复》2005年第43期72-74,i0002,共4页Chinese Journal of Clinical Rehabilitation

基　　金：江苏省高新技术研究(BG2003033)~~

摘　　要：目的:利用聚合酶链反应定点突变技术构建人血小板反应素1基因第13外显子编码钙结合域突变体。方法:实验于2005-03/06在南京医科大学第一附属医院心血管病研究所完成。选择南京医科大学第一附属医院体检中心健康体检者血标本提取人基因组DNA,设计、合成两对引物,将突变位点设计在引物内,突变位点包含限制性内切酶BseNI酶切位点,采用聚合酶链式反应定点突变技术,应用高保真TaqDNA聚合酶,通过三轮聚合酶链扩增反应对血小板反应素1基因的第13外显子进行扩增,将第13外显子碱基8831A置换为G,对最终扩增产物进行酶切鉴定和测序。结果:获得人血小板反应素1第13外显子编码钙结合域突变体,经酶切鉴定和测序分析,除引入突变点产生预期A8831→G突变外,其余碱基序列无改变。结论:采用聚合酶链反应体外定点突变技术,成功构建人血小板反应素1基因第13外显子编码钙结合域突变体,为研究血小板反应素1基因该位点突变导致血小板反应素1蛋白的结构、功能变化奠定了基础。AIM： To establish the mutant of coding calcium binding fragment of the 13^th exon of human thrombospondin-1 （TSP-1） gene with polymerase chain reaction （PCR） site directed mutagenesis technology. METHODS： The experiment was conducted at the Institute of Cardiovascular Disease of First Affiliated Hospital, Nanjing Medical University from March to June 2005. DNA was extracted from blood samples of healthy individuals who were checked at center of health examination of First Affiliated Hospital of Nanjing Medical University. Two couple of primers were designed and synthesized. The fragment of mutation, which included BseNI enzyme digestion fragment of restriction endonuclease, was designed in the primer. Using the PCR site directed mutagenesis techniq.q..ue and Taq DNA polymerase, the 13^th exan of TSP- 1 was amplified through a third step PCR, and the base 8831A of the 134 exon was permuted into G. The ultimate amplified products were performed with restriction mapping and sequence analysis for confirmation. RESULTS： The mutant of coding calcium binding fragment of the 13^th exon of human TSP-1 was gained. After confirmation and sequence analysis of enzyme digestion, mutation fragment occurred expected A8831→G, mutation, and other base sequence had no change. CONCLUSION： The mutation of coding calcium binding fragment of the 13^th exon of human TSP-1 is successfully created with PCR site directed mutagenesis technique in vitro, which establish foundation for researching changes of structure and function of TSP-1 induced by mutation of TSP-1 gene.

关 键 词：血小板吸附蛋白1 聚合酶链反应 突变 序列分析 

分 类 号：R349.83[医药卫生—基础医学]