组织型纤溶酶原激活剂cDNA表达质粒的构建及其在CHO细胞中的表达

人组织型纤溶酶原激活剂(t-PA)cDNA克隆片段。采用两种方式构建成真核表达质粒。第一:切除t-PA3＇端非编码区序列后插入SR启动子和SV_(40)晚期Poly(A)终止信号之间,形成pMGZ6001质粒;第二:将3＇端部分切除并带有Poly(A)加尾信号的t-PA片段插入由金属硫蛋白MT启动子调控的载体中,分别组建成含大T抗原与不含大T抗原的两个表达质粒pMGZ6002和pMGZ6003。这三种质粒用磷酸钙共沉淀法和电穿孔法转染CHO-dhfr细胞,阳性克隆细胞均能合成并分泌rt-PA。其分子量约68kD,并能与t-PA单克隆抗体特异结合,溶解纤维蛋白。阳性克隆经MTX选择培养扩增基因,培液中rt-PA表达水平可达3000IU/(10~6细胞·48h)。

Consturction of t-PA cDNA Clones and Expression in CHO Cells

Three different t-PA expression plasmids pMGZ6001, pMGZ6002 and pMGZ6003 were generated. ]. pMGZ6001 was constructed by inserting t- PA cDNA (without 3' UTR) between SR promoter and SV40 stopping signal. 2 . pMGZ6002 was generated by inserting t- PA cDNA ( with SV40 stopping signal) between MT promoter and T- antigen. 3. pMGZ6003 was from pMGZ6002 by deleting T-antigen. CHO-dhfr cells transacted with these three plasmids were able to synthesize and secrete rt-PA into medium. The properties of the rt-PA are:MW 68 kl). can bind to McAb against t- PA specifically, and has thromblytic effect. The positive clone was screen from the cells growing in increasing concentration of MTX. After MTX selective treatment, t PA cDNA copy was amplified and expression level of rt-PA reached about 30001U/106cells 48h.