色素上皮细胞衍生因子表达载体的构建  

Gene cloning of human pigment epithelium-derived factor in E.coli DH5α

作　　者：杨楠[1] 姚密河 张桂荣[3] 杨翰仪[3] 

机构地区：[1]解放军210医院眼科,大连116021 [2]第二炮兵51基地531医院,吉林通化134002 [3]吉林大学基础医学院生物化学与分子生物学教研室,长春130021

出　　处：《免疫学杂志》2006年第1期98-100,103,共4页Immunological Journal

摘　　要：目的构建色素上皮细胞衍生因子(PEDF)的表达载体。方法从孕8周的胚胎绒毛中提取总RNA,经RT-PCR扩增,获得编码人PEDF的全基因序列,采用T-A克隆法,将PEDF基因克隆入PMD18-T中间载体并转化入大肠杆菌E.coliJM109中,提取重组质粒并酶切鉴定,将鉴定正确的质粒命名为PMD18-T/PEDF,DNA测序,测序正确后构建重组表达载体pET28a/PEDF,转化大肠杆菌E.coliDH5α并酶切鉴定。结果重组表达载体pET28a/PEDF在大肠杆菌E.coliDH5α中克隆表达。结论构建了PEDF重组表达载体pET28a/PEDF,为重组PEDF的进一步表达及纯化奠定了基础。Objective To clone the recombinant human pignent epithehum-derived factor （PEDF） in E. coli DH5α. Methods Total RNA was extracted from a human embryo and human PEDF was obtained by RT-PCR. The cDNA was cloned into vector PMD18-T by T-A methods, and then transformed into E. coli JM109. The recombinant plasmid PMD18-T/PEDF was extracted and double digested. The pMD18-T/PEDF and pET28a were hgated. The recombinant plasmid pET28a/PEDF was constructed and transfected into E, coli DH5α. Results The recombinant plasmid pET28a-PEDF was obtained and expressed in E. coli DH5α, Conclusion The recombinant plasmid pET28a/PEDF is constructed successfully, which provides a basis for purification and expression of PEDF.

关 键 词：色素上皮细胞衍生因子 克隆 表达 载体 

分 类 号：R346[医药卫生—基础医学]