中介蝮蛇毒纤溶酶基因原核表达载体的构建及其在大肠杆菌中的表达

为了从中介蝮蛇毒腺中提取总RNA，研究其具有重要药用价值的纤溶酶，从基因工程的角度进行研究开发蛇毒资源。通过RT-PCR法扩增纤溶酶基因与pMD18-T载体连接进行TA克隆，得到FLE基因，再亚克隆到pPROEXHTb原核表达载体上，构建重组表达载体；将阳性重组表达载体转化到大肠杆菌中诱导表达，采用SDS-PAGE检测该重组蛋白的分子量。结果表明：成功的构建了重组原核表达载体ppPROEXHTb-FLE，并在大肠杆菌中获得表达，重组蛋白分子量约为30 KDa。说明可以采用该方法进行中介蝮蛇毒纤溶酶的原核表达，该研究为进行蛇毒纤溶酶的开发奠定了分子基础。

Expression in E. coli and Prokaryotic Expressive Vector Construction of Fibrinolytic Enzyme Gene from Gloydius intermedius

The fibrinolytic enzymes which wildly existed in many kinds of snake venoms have important medicine effect, it contributed to open up snake venom resources from gene engineering. Total RNA was extracted from gland of Gloydius intermedius snake venom, fibrinolytic enzyme (FLE) gene was amplified by RT-PCR. The gene was ligased with pMD18-T vector, then FLE was obtained; It was subcloned into pPROEXHTb prokaryotic expressive vector to construct expressive vector; The positive recombinant expression vector was transformed into E. coli and was induced to express recombinant protein FLE. The fusion protein was detected by SDS-PAGE. The results indicated that prokaryotic expression vector ppPROEXHTb-FLE was constructed successfully, and expressed in E. coli, the molecular weight of recombinant protein was about 30kD. It is effective to use the prokaryotic expressive vector to express. All that would provided base for gene engineering products of snake venom FLE in the future.