摘要
为了构建抗表皮生长因子受体Ⅲ型突变体(epidermal growth factor receptor variant typeⅢ,EGFRvⅢ)的单链抗体PD0721的大肠杆菌(Escherichia coli)表达系统,作者研究了PD0721重组蛋白在大肠杆菌BL21中的最佳表达条件,并建立相应的重组蛋白质纯化方法。首先构建重组表达质粒PD0721-pET-22b(+)并转化至大肠杆菌BL21中,再利用SDS-PAGE研究不同诱导剂IPTG浓度、不同诱导温度、不同诱导时间和不同菌体浓度对PD0721重组蛋白表达的影响。利用镍柱亲和层析开发PD0721重组蛋白的纯化方法,并使用蛋白质印迹法以及N端测序法对纯化后的蛋白质进行鉴定。菌落PCR及琼脂糖凝胶电泳表明,PD0721-pET-22b(+)重组质粒及PD0721重组大肠杆菌BL21构建成功.PD0721重组蛋白在体外原核表达的最佳诱导剂浓度为0.6μmol/L,最佳温度为15℃,最佳诱导时间为12 h,最佳菌体浓度OD600约为0.6。经过Ni2+柱亲和层析后,当咪唑的浓度为150 mmol/L,可以得到纯度较高的PD0721重组蛋白。SDS-PAGE电泳和Western Blotting结果表明,重组蛋白质产物的相对分子质量约为31000,与理论相对分子质量一致,蛋白质的N端测序也与设计序列一致。作者成功构建了抗EGFRvⅢ抗体PD0721重组蛋白的体外原核表达体系,优化了最佳表达条件,并提供了纯化重组PD0721蛋白的可行方法。
This work aimed to construct the Escherichia coli expression system,obtain the optimum expression conditions and establish a robust purification method of single-chain antibody PD0721 against epidermal growth factor receptor variant typeⅢ(EGFRvⅢ).The recombinant plasmid PD0721-pET-22(+)was constructed and transformed into Escherichia coli BL 21.The optimum concentration of isopropyl-β-D-thiogalactoside(IPTG),induction temperature,induction time and cell concentration(OD600)of the PD0721 expression were investigated by SDS-PAGE method.Ni2+affinity chromatography was employed to purify PD0721 protein.Western Blotting and N-terminal sequencing were used to identify PD0721 protein.Colony PCR and agarose gel electrophoresis confirmed the successful construction of PD0721-pET-22b(+)recombinant plasmid and PD0721 recombinant Escherichia coli BL 21.The optimum IPTG concentration,temperature,induction time and OD600 of PD0721 expression were 0.6μmol/L,15℃,12 h and 0.6.PD0721 protein of high purity could be obtained when the concentration of imidazole was 150 mmol/L during the Ni2+affinity purification.SDS-PAGE and Western Blotting results showed that the molecular weight of the recombinant target protein was about 31000,consistent with the theoretical molecular weight.N-terminal sequencing of the protein was also consistent with the theoretical sequence of PD0721.In summary,the in vitro prokaryotic expression system of anti-EGFRvⅢantibody PD0721 was successfully constructed.The optimum expression conditions were obtained,and a feasible method for purifying PD0721 protein was established.
作者
张煜彬
叶路芬
吴忠秀
薛维娜
何彬
杨畅
李勇军
王永林
刘亭
ZHANG Yubin;YE Lufen;WU Zhongxiu;XUE Weina;HE Bin;YANG Chang;LI Yongjun;WANG Yonglin;LIU Ting(Guizhou Provincial Key Laboratory of Pharmaceutics/State Key Laboratory of Functions and Applications of Medicinal Plants,Guizhou Medical University,Guiyang 550004,China;Engineering Research Center for the Development and Application of Ethnic Medicine and TCM(Ministry of Education),Guizhou Medical University,Guiyang 550004,China;School of Medicine and Health Management,Guizhou Medical University,Guiyang 550025,China;School of Pharmacy,Guizhou Medical University,Guiyang 550025,China)
出处
《食品与生物技术学报》
CAS
CSCD
北大核心
2021年第7期42-49,共8页
Journal of Food Science and Biotechnology
基金
国家自然科学基金项目(81603189)
贵阳医学院院基金项目([2013]12)
贵州省教育厅大学生创新创业项目(20195200937)
黔科中引地项目([2018]4006)
黔科合平台人才项目([2016]5613/5677)。
