重组人ZP3蛋白在大肠杆菌中的高效表达  

High-level Expression of Recombinant Human ZP3 Protein in E.coli

作　　者：叶志华[1] 梁建庆[1] 

机构地区：[1]广东省微生物研究所广东省菌种保藏与应用重点实验室,广东广州510070

出　　处：《生物技术》2006年第6期3-7,共5页Biotechnology

基　　金：广东省科技攻关项目资助("重组人卵透明带-3蛋白的高效表达及其分离制备的研究";2004B10401004)

摘　　要：目的：用基因工程方法制备重组人ZP3蛋白。方法：以全长人ZP3 cDNA片段为模板，通过PCR扩增出编码人ZP3蛋白不同肽段的cDNA片段，然后将这些cDNA片段分别插入到表达载体pET-I9b的Nco I-BamH I或Nde I-BamH I位点内，共构建成6种人ZP3蛋白非融合表达质粒（pFZP3-1-pEZP3-6）和3种人ZP3蛋白融合表达质粒（pFZP3-7-Pfzp3-9）.将这9种表达质粒分别转化大肠杆菌Rosetta2（DE3）感受态细胞并选择出Ap转化子，将Ap转化子接种到NZCYM培养基中（含AP 100μg/mL），在35-37℃振荡培养到对数生长期，加入IPTG至1.0-1.5mmol/L浓度诱导培养3h，离心收集细胞进行SDS-PAGE电泳检测和Western Blot杂交分析。结果：这9种人ZP3蛋白表达质粒在大肠杆菌Rosetta2（DE3）中得到高效表达，目的蛋白占总细胞蛋白的10-25%，表达产物均以包涵体形式存在。结论：成功构建了重组人ZP3蛋白原核表达系统，为地一步研究和应用人ZP3蛋白打下了基础。Objective： To preparation of recombinant human ZP3 protein by gene engineering. Methods： The eDNA fragments coding the different region of human ZP3 protein were amplified by PCR using the template of full - length human ZP3 gene cDNA. After the cDNA fragments were inserted into the Nco Ⅰ - BamH I sites or Nde Ⅰ - BamH Ⅰ sites of the expression vector pET - 19b respectively, the six non - fusion expression recombinant plasnfids（pEZP3 - 1 - pEZP3 - 6）and the three fusion expression recombinant plasmids（pEZP3 - 7 - pEZP3 - 9）were resulted. The competent cells of E. coli strain Rosetta2（DE3） were transformed with the nine recombinant plasmids respectively, and the Ap transformants were selected and cultivated with stroking in the NZCYM medium contained Ap 100μg/mL at 35 - 37℃. In the logarithmic growth phase, the cultures were induced by added WIG to the medium to 1.0 -1.5mmoL/L, and were continued to cultivate for another three hours. The culture cells were collected by centrifugation, and the cell lysates were analysed by SDS - PAGE and by Westem blot. Results： The recombinant human ZP3 protein had been expressed in a high level in E. coli Rosetta2（DE3）/pEZP3 - 1 - pEZP3 - 9, and the products were existed as a form of inclusion body and occupied about 10 - 25% of the total cell proteins. Conclusion： The proharyotic expression system of recombinant human ZP3 protein had been constructed successfully, and it will be the foundation for the preparation of studying and applying human ZP3 protein.

关 键 词：人ZP3蛋白 表达 大肠杆菌 

分 类 号：Q786[生物学—分子生物学] Q781