重组人组织型纤溶酶原激活剂突变体基因的克隆、表达与生物活性鉴定

Cloning,Expression and Identification of Activity of Recombinant Tissue-type Plasminogen Activator Mutant

目的 :研究重组人组织型纤溶酶原激剂突变体 (reteplase,r -PA)基因的克隆和表达 ,并对表达产物进行活性鉴定。方法 :通过PCR的方法从t-PA基因上扩增得到r-PA基因的编码序列 ,并克隆到pUC19载体中 ,经DNA测序正确后 ,将该基因克隆到含有T7启动子的高效表达质粒pJZ - 10 0中 ,转化E .coliBL2 1(DE3) ,得到含有r-PA基因的工程菌。工程菌经IPTG诱导表达 ,SDS -PAGE检测表达情况。提取包涵体 ,经过体外稀释法复性 ,亲和色谱层析纯化 ,测定产物的活性。结果 :表达的重组蛋白约占可溶性总蛋白的 2 0 % ,复性并纯化后测定活性为 5 80 0 0IU mg。结论 :成功构建了重组r-PA的表达质粒 ,表达产物具有良好的生物活性。

Objective:Study on cloning and expression of recombinant tissue-type plasminogen activator mutant.Identify the activity of recombinant protein.Method:Amplification r-PA coding sequence from human t-PA gene and then cloned it into pUC19 vector.The sequence is identical with which reported.This sequence is cloned into pJZ-100 expression vector which includes T7 promoter.Being induced by IPTG,reteplase were expressed in E.coli BL21(DE3).The result of expression was identified by SDS-PAGE.The inclusion boby refold by dilution in vitro.The protein of renaturation was purified by affinity chromatograph.Results:Expressed reteplase were above 20% of total bacterial protein.After renaturation and purification,reteplase specific activity was 580000IU/mg.Conclusion:An expression plasmid for reteplase was successfully constructed.The recombinant protein showes effective fibrinolysis.