中介蝮蛇毒纤溶酶基因原核表达载体的构建及其在大肠 - 中国农学通报.pdf

中国农学通报 2013,29(2):27-30 Chinese Agricultural Science Bulletin 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 刘玉芬，于德涵，刘 鹏，陈 辉，赵文阁 （哈尔滨师范大学生命科学与技术学院，哈尔滨150025） 摘 要：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pMD 18-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。 关键词：中介蝮蛇；纤溶酶；原核表达；载体构建 中图分类号：Q78 文献标志码：A 论文编号：2012-2596 Expression in E. coli and Prokaryotic Expressive Vector Construction of Fibrinolytic Enzyme Gene from Gloydius intermedius LiuYufen,YuDehan,LiuPeng,ChenHui,ZhaoWenge (CollegeofLifeScienceandTechnology,HarbinNormalUniversity,Harbin150025) Abstract: Thefibrinolyticenzymeswhichwildlyexistedinmanykindsofsnakevenomshaveimportant medicineeffect,itcontributedtoopenupsnakevenomresourcesfromgeneengineering.TotalRNAwas extractedfromglandof Gloydius intermedius snakevenom,fibrinolyticenzyme(FLE)genewasamplifiedby 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 expressionvectorwastransformedintoE.coliandwasinducedtoexpressrecombinantproteinFLE.Thefusion protein was detected by SDS. The results indicated that prokaryotic expression vector ppPROEXHTb-FLE was constructed successfully, and expressed in E. coli, the molecular weight of recombinantproteinwasabout 30kD.Itwaseffectivetousetheprokaryoticexpressivevectortoexpress.All thatwouldprovidedbaseforgeneengineeringproductsofsnakevenomFLEinthefuture. Key words: Gloydiusinterm