摘要
目的:重组表达新型隐球菌Cap10蛋白并制备特异抗体,为新型隐球菌的检测和诊断提供特异抗原和抗体。方法:选择CAP10基因开放阅读框架(ORF)抗原表位集中、保守性高的片段,经过大肠杆菌偏嗜的密码子优化后进行基因合成,将目的片段克隆至原核表达载体pQE30中构建重组蛋白表达载体pQE30-CAP10并鉴定,IPTG诱导重组蛋白在M15中表达,并用SDS-PAGE和Western blot鉴定;镍柱纯化后,免疫家兔制备抗血清。结果:在大肠杆菌中成功表达Cap10,其纯度大于95%;二免后家兔血清抗体效价达到200万以上。结论:获得了高纯度的Cap10蛋白及高效价的多克隆抗体,为新型隐球菌的检测和致病机制研究奠定了基础。
Objective：To express capsule associated protein 10（Cap10） from Cryptococcus neoformans and prepare its polyclonal antibody for further application in the detection of Cryptococcus neoformans.Methods：The dominant B cell epitopes were analyzed by the DNAStar software and Goldkey software,and the conservative region was analyzed by NCBI Blast,then the selected gene fragment was optimized according to the tropism of the E.coli;the optimized gene was synthesized and cloned into the prokaryotic plasmid pQE30.Recombinant Cap10 was identified by SDS-PAGE and Western blot,and purified with Ni-NTA affinity chromatography;further,the purified Cap10 was immunized to the white rabbit to prepare the polyclonal antibody.Results：The recombinant Cap10 was highly expressed in the E.coli,and the purity of the protein was greater than 95%;the titer of the antibody in the rabbit serum was greater than 2,000,000.Conclusion：The high-purity recombinant Cap10 and high titer antibody was successfully prepared,which will be helpful for the detection of Cryptococcus neoformans and the study of pathogenic mechanism.
出处
《中国卫生检验杂志》
北大核心
2012年第6期1229-1231,1236,共4页
Chinese Journal of Health Laboratory Technology
基金
传染病重大专项(2008ZX10001-007)
