尿激酶型纤溶酶原激活因子cDNA的克隆与原核表达

目的构建尿激酶型纤溶酶原激活因子（uPA）原核表达质粒，并在大肠埃希菌BL21中表达，为进一步研究uPA奠定基础。方法应用逆转录RT—PCR，从人肝细胞cDNA中扩增人尿激酶型纤溶酶原激活因子基因序列，与原核表达质粒pET32a重组，获得表达质粒uPA—pET32a。用氨苄青霉素平板筛选转化子。双酶切与DNA测序进行鉴定，用IPTG诱导表达，并用Westernblotting进行鉴定。结果从肝细胞cDNA中扩增的uPA基因片段长1296bp，酶切及DNA测序证实uPA-pET32a重组质粒构建正确，表达融合蛋白分子量约为68900Mr，经Western blotting证实为目的蛋白。结论成功构建了重组原核表达质粒uPA—pET32a。并能在大肠埃希菌内表达，所表达的融合蛋白分子量大小与预期的相一致．为进一步的研究奠定了基础。

Cloning and Expression of Urokinase Type Plasminogen Activator cDNA

Objective To clone the Urokinase Type Plasminogen Activator （uPA） cDNA and express the uPA as a fusion protein in E.coli. Method A eDNA fragment of uPA was amplified by RT-PCR, and cloned into pET32a vector.The reeombinan plasmid uPA-pET32a was digested with restriction endonuelease Hind m and Xhol I . E.coli BL21 cells were transformed by the recombinant plasmid. The expression of the recombinant protein was induced by IPTG and the fusion protein was analyzed by SDS-PAGE and western blotting. Result A 1296bp uPA eDNA fragment was PCR amplified and cloned into pET32a vector. The fusion protein （about 68900 Mr） was expressed in E.coli. Conclusion The recombinant plasmid uPA-pET32a have been constructed successfully and the fusion protein can be expressed in E.coli BL21 cells.