人血小板源性生长因子B基因的体外扩增以及克隆和鉴定  

In vitro proliferation, clone and identification of human platelet-derived growth factor B gene

作　　者：罗文娟[1] 王传富[1] 吕振华[2] 刘湘萍[2] 隋爱华[2] 

机构地区：[1]青岛大学医学院附属医院眼科,山东省青岛市266003 [2]青岛大学医学院附属医院中心实验室,山东省青岛市266003

出　　处：《中国临床康复》2006年第25期80-82,共3页Chinese Journal of Clinical Rehabilitation

基　　金：国家自然科学基金资助项目(30572011)~~

摘　　要：目的:自行构建人血小板源性生长因子B(PDGF-B)真核表达载体,为人血小板源性生长因子B基因转染真核细胞的实验研究及基因治疗奠定基础。方法:实验于2002-01/2005-01在青岛大学医学院附属医院中心实验室完成。①根据Genbank的人血小板源性生长因子B的全长基因序列,设计合成一对附加BamHⅠ,XhoⅠ两个限制性内切酶酶切位点的特异性引物。②采用反转录聚合酶链反应从人胎盘mRNA中扩增出人血小板源性生长因子B基因片段,将其插入真核表达载体PcDNA4构建成重组真核表达载体PcDNA4/PDGF-B。③经测序鉴定后,再用双酶切和聚合酶链反应对插入片段进行分析和验证。结果:反转录-聚合酶链反应产物经琼脂糖电泳结果显示在预期位置有相对分子质量为725bp的特异性扩增带。②序列分析、双酶切和聚合酶链反应结果证实插入片段序列正确。结论:成功扩增出完整的人血小板源性生长因子B基因,构建了基因重组真核表达载体PcDNA4/PDGF-B。为人及动物细胞的转基因研究,在基因水平改变其遗传性状打下基础。AIM： To self-construct eukaryotie expression vector of human plateletderived growth factor B （PDGF-B） so as to provide basis for experimental study and gene therapy of PDGF-B transfecting eukaryotie cell. METHODS： This experiment was conducted at the Central Laboratory, Affiliated Hospital of Qingdao Medical College from January 2002 to January 2005. （1） A pair of specific primers with restriction enzyme site of BamH I and Xho I were designed and synthesized;aeeording to gene sequence of human PDGF-B of Genbank. （2）Hman PDGF-B segment was sueeessfuUy obtained from human placent mRNA by reverse transeriptionpolymerase chain reaction（ RT-PCR）; Then the PDGF-B gene was inserted into PcDNA4 vector to construct recombinant eukaryotie expression plasmid PcDNA4/PDGF-B; （3） After sequenced and identified, the inserted segment was performed analysis and validation with double enzyme digestion and PCR. RESULTS： Gel electrophoresis result of product of RT-PCR showed that specific proliferative band with relative molecular weight of 725 bp appeared in the prospective place. （2） The inserted fragment was confirmed by sequencing ,double enzyme digestion and PCR analysis. CONCLUSION： The recombinant eukaryotie expression vector PeDNA4/PDGF-B was successfully constructed following suceessful amplification of complete PDGF-B gene. It provides basis for transgenie study and change of hereditary feature in gene level of the ceils of human being and animals.

关 键 词：血小板源性生长因子 基因 序列分析 

分 类 号：R329.29[医药卫生—人体解剖和组织胚胎学]