摘要
目的:制备重组可溶性截短型马尔堡病毒包膜糖蛋白(MAGP),并验证其抗原性。方法:PCR扩增MAGP截短片段MAGP_(40-289)、MAGP_(148-526)、MAGP_(240-526)和MAGP_(258-526),克隆至原核表达载体pET-32a并转化至大肠杆菌进行诱导表达,经亲和层析、阴离子交换层析获得纯度良好的可溶性截短蛋白抗原,利用ELISA试验对该蛋白的抗原性进行验证。结果:截短抗原MAGP_(240-526)在大肠杆菌中可溶性表达良好,表达量约17 mg/L;Western印迹显示该抗原能与Ad5-MAGPopt免疫后的小鼠血清发生特异反应。结论:经原核系统表达的截短型MAGP具有良好的抗原性,可用于检测相关疫苗激发的抗MAGP抗体水平或评价抗MAGP的结合活性。
Objective: To prepare the soluble truncated recombinant glycoprotein(GP) of Marburg virus(MARV)in Escherichia coli and verify its antigenicity. Methods: A total of 4 DNA sequences of truncated GP genes,MAGP_(40-289), MAGP_(148-526), MAGP_(240-526) and MAGP_(258-526) were amplified by PCR, then were inserted into p ET-32 a vector respectively. The recombinant clones were selected and the proteins of interest were expressed by IPTG induction and purified by affinity chromatography and ion exchange chromatography. The recombinant protein's antigenicity was assayed by ELISA. Results: The truncated antigen MAGP_(240-526) was solubly expressed in E.coli with a yield of ～17 mg/L. Western blotting showed that MAGP_(240-526)could react specifically with the sera from mice and rhesus monkeys immunized by Ad5-MAGPopt. Conclusion: The soluble truncated protien GP from E.coli has good antigenicity, and it can be used in detecting MARV GP antibodies.
出处
《生物技术通讯》
CAS
2018年第1期13-16,共4页
Letters in Biotechnology
关键词
马尔堡病毒
包膜糖蛋白
原核表达
Marburg virus
envelope glycoprotein
prokaryotic expression
