吸血蝙蝠纤溶酶原激活剂α2的原核表达及抗体制备  

Prokaryotic expression of Desmoudus rotundus salivary plasminogen activator alpha 2 and preparation of its polyclonal antibodies

作　　者：魏昭荣[1] 李刚强[1] 李校堃[2] 刘德虎[1] 

机构地区：[1]中国农业科学院生物技术研究所,北京100081 [2]吉林农业大学生物反应器与药物开发教育部工程研究中心,长春130118

出　　处：《高技术通讯》2008年第3期294-298,共5页Chinese High Technology Letters

基　　金：863计划(2001AA212127);中国农业科学院人才基金资助项目

摘　　要：利用人工合成的吸血蝙蝠唾液纤溶酶原激活剂α2(DSPAa2)基因,研究了其在细菌中的表达及表达产物的纯化和抗体制备。首先人工合成 DSPAa2基因,并构建原核表达载体转化大肠杆菌。经 IPTG 诱导后,DSPAa2在细菌中得到了高效表达。SDS-PAGE 结果显示,以包涵体的形式存在的 DSPAce2重组蛋白占到细菌总蛋白的32%。包涵体裂解后经亲合层析柱纯化,100mL 菌液中能得到2.2mg 纯的重组蛋白。用纯化的 DSPAa2分别免疫大鼠和小鼠,经 ELISA 检测,获得了效价达到1:12800以上的高质量的抗血清。West-ern blot 结果显示抗体能与 DSPAα2特异性地结合。This study was conducted to express the synthesized Desmoudus rotundus salivary plasminogen activator alpha 2 （DSPAα2） in E. coli and prepare high effective antiserum using purified expression products. The DSPAα2 was synthesized and cloned into prokaryotic vector for expression in bacteria. DSPAα2 was effectively expressed in E. coli after IPTG induction. The result of SDS-PAGE analysis indicated that the recombinant protein, produced in the form of inclusion body, occupied 32% of total bacterial proteins. 2.2 mg pure recombinant protein was obtained in 100 mL culture broth after purification by the affinity chromatography. The purified protein was used to immune rats and mouse, and high-effective antiserums （over 1：12800） were obtained. Western blot analysis showed that the antiserum could specifically combine with the recombinant DSPAα2.

关 键 词：吸血蝙蝠 纤溶酶原激活剂α2 重组蛋白 基因表达 免疫应答 急性心脑血栓 溶栓类蛋白药物 

分 类 号：Q789[生物学—分子生物学]