大鼠D-双功能蛋白基因原核表达载体的构建及表达  

Construction and Expression of Prokaryotic Expression Vector of D-Bifunctional Protein Gene

作　　者：贺文[1] 赵雪彦[1] 姚敏[1] 史海水[1] 许丽辉[1] 刘昆[1] 姜玲玲[1] 

机构地区：[1]河北医科大学生化教研室石家庄市河东社区卫生服务中心,石家庄050017

出　　处：《生物技术通报》2011年第1期139-142,152,共5页Biotechnology Bulletin

基　　金：河北省科技支撑计划项目(062761387);河北省科学技术研究与发展指导计划项目(06275507)

摘　　要：利用原核表达系统构建大鼠D-双功能蛋白表达载体。设计基因拼接引物,通过RT-PCR合成DBP基因cDNA序列,将酶切、纯化的DBP基因与经相同处理的表达载体pET-28 a相连接,转化感受态大肠杆菌DH5α,筛选阳性重组子。将通过酶切及序列分析鉴定阳性的重组子质粒转入感受态大肠杆菌BL21-gold表达菌中,经IPTG诱导表达,通过SDS-PAGE分析目的蛋白表达情况。结果显示,成功获得了包含DBP基因的双链cDNA序列,酶切、序列分析及Western blotting证实成功构建了DBP基因的原核表达载体。通过原核表达系统,DBP蛋白以包涵体形式产生,复性后可获得高表达的目的蛋白。It was to construct prokaryotic expression vector of D-bifunctional protein（DBP） gene and express the protein in E.coli BL21.A pair of primers was designed according to DBP gene sequence published in GenBank.The total RNA of the rat liver was extracted and cDNA sequence of DBP gene was synthesized by RT-PCR.cDNA of DBP and the expression vector pET-28a were digested individually by restrictive endonuclease Nco I and Not I.After purified,the target fragment was linked to pET-28a.The recombinant plasmid was transferred into competent cell DH5α and the positive recombination was screened.The DBP fragment in vector pET-28a-DBP was conformed to the original sequence with restrictive endonuclease digesting and sequencing.The pET-28a-DBP plasmid was taken and transferred into E.coli BL21-gold for expression.Induced by 1 mmol/L IPTG,the expression product of DBP gene was analyzed by SDS-PAGE and Western blotting.Results showed that the prokaryotic expression vector of DBP gene was constructed and DBP protein had been expressed successfully in the form of inclusion bodies.The protein expression was induced with 1 mmol/L IPTG for 5 h.It proved that rat DBP protein was generated as the inclusion body in E.coli,which was refolded for DBP polyclone antibody.

关 键 词：D-双功能蛋白 原核表达 包涵体 大鼠 

分 类 号：Q78[生物学—分子生物学]