植原体免疫主导膜蛋白Imp基因原核表达载体构建及表达  

Construction of Immunodominant Membrane Protein Gene Prokaryotic Expression Vectors to Phytoplasma and Its Expression

作　　者：柴化建[1] 赵海泉[1] 张丽君 罗焕亮[3] 

机构地区：[1]安徽农业大学生命科学学院,合肥230036 [2]深圳市职业技术学院,深圳518055 [3]深圳出入境检验检疫局,深圳518045

出　　处：《生物技术通报》2012年第6期106-110,共5页Biotechnology Bulletin

基　　金：深圳市科技计划项目(JC200903180710A)

摘　　要：旨在构建植原体免疫主导膜蛋白Imp基因原核表达载体,并进行初步表达。以重组克隆质粒pMD18-T-Imp为模板,PCR扩增Imp基因片段。构建表达载体pET-28a(+)-Imp,转化宿主菌E.coliBL21(DE3)。筛选阳性克隆,提取重组质粒作PCR鉴定、酶切鉴定及IPTG诱导表达鉴定。PCR及双酶切结果显示,重组质粒pET-28a(+)-Imp构建成功。经IPTG诱导BL21(pET-28a(+)-Imp)表达约20 kD的蛋白,与预期的携带6×His-Tag的目的蛋白(19.5 kD)大小相符,主要以包涵体形式存在。结果显示,构建的表达载体pET-28a(+)-Imp在E.coliBL21(DE3)中能够达一定量表达,为进一步纯化Imp蛋白奠定基础。To construct expression vector of phytoplasma immunodominant membrane protein gene （ Imp ） , and to induce expression in E. coli BL21 （ DE3 ） . The gene of Imp was amplified by PCR from the recombinant colony vector pMD18-T-Imp. The Imp gene was inserted into plasmid pET-28a （ ＋ ） , the recombinant plasmid pET-28a （ ＋ ） -Imp was transformed into E. coli BL21 （ DE3 ） . The recombinant plasmid was extracted and purified, and was analyzed by PCR and restricted enzyme assay. Recombinant protein was expression being induced by IPTG, and was identified by SDS-PAGE. PCR and double enzyme digestion assay confirmed that the recombinant plasmid pET-28a （ ＋ ） -Imp was constructed successfully. By IPTG inducing, the recombinant protein was obtained in E. coli BL21 （ DE3 ） . SDS-PAGE analysis indicated the fusion protein Imp with 6 × His-Tag was about 20 kD, mainly expressed in the inclusion body. The recombinant expression vector pET-28a （ ＋ ） - Imp was able to express of Imp in E. coli BL21 （ DE3 ） , lays a foundation for further research on producing purification of Imp.

关 键 词：植原体 免疫主导膜蛋白 原核表达 

分 类 号：S432.42[农业科学—植物病理学]