重组人组织型纤溶酶原激活剂缺失变体基因的克隆及工程菌的构建与鉴定  

Gene Cloning, Construction and Characterization of the Engineered Strain of Recombinant Human K_2tPA

作　　者：陈于红[1] 朱镇华[1] 刘蓓钫[1] 张菁[1] 傅一工[1] 王石泉[1] 张巍[1] 杨庆[1] 刘建宁[1] 

机构地区：[1]南京大学分子医学研究所,南京210093

出　　处：《南京大学学报（自然科学版）》2004年第1期1-6,共6页Journal of Nanjing University（Natural Science）

基　　金：国家重大科技专项"创新药物和中药现代化"(2002AA2Z3451)

摘　　要：　利用RT PCR技术,从人脐带静脉上皮细胞中克隆人组织型纤溶酶原激活剂基因,将其接入TA克隆载体PCR2.1中,经DNA序列测定后,以该重组质粒DNA为模板,用PCR方法获得了人组织型纤溶酶原激活剂缺失变体(K2tPA)基因,将其转入pET29a,构建了重组表达质粒pET29a/K2tPA,并转化至大肠杆菌BL21(DE3)中,构成工程菌.经IPTG诱导表达,在40kDa处有一明显表达条带,表达量为约占菌体总蛋白的20%.该菌种在贮存与复苏及传代过程中具有良好的质粒稳定性和表达稳定性.K_2tPA is one of the third generation drugs. It is a deletion mutation of the wild type tPA molecule, where the domains of finger, epidermal growth factor and Kringle 1 domains have been removed. We have engineered recombinant human K_2tPA. The human tPA gene was obtained from endothelial cells of human umbilical vein by RT PCR, from which the cDNA of K_2tPA was then cloned by PCR. The pET29a/K_2tPA plasmid was constructed and expressed in E.coli BL21 (DE3) by induction of IPTG. The expression product with 40 kDa was identified by SDS PAGE, and the protein was found in the inclusion body and accounted for 20% of the total bacterial proteins.It was shown that the engineered strain was very stable for plasmid maintenance, resuscitation, and expression efficiency during storage and regeneration.

关 键 词：组织型人纤溶酶原激活剂变体基因 工程菌 质粒稳定性 表达稳定性 克隆 

分 类 号：Q785[生物学—分子生物学] Q55