【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-19b的NcoⅠ-BamHⅠ或NdeⅠ-BamHⅠ位点内,共构建成6种人ZP3蛋白非融合表达质粒(pEZP3-1~pEZP3-6)和3种人ZP3蛋白融合表达质粒(pEZP3-7~pEZP3-9)。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Apr转化子,将Apr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,为进一步研究和应用人ZP3蛋白打下了基础。更多还原

【Abstract】 Objective: To preparation of recombinant human ZP3 protein by gene engineering.Methods: The cDNA 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 NcoⅠ-BamHⅠ sites or NdeⅠBamHⅠ sites of the expression vector pET-19b respectively,the six non-fusion expression recombinant plasmids(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~r transformants were selected and cultivated with shaking in the NZCYM medium contained Ap 100μg/mL at 35～37℃.In the logarithmic growth phase,the cultures were induced by added IPTG 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 Western 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.更多还原