摘要
目的 利用真核系统对E1蛋白进行表达并纯化,建立一种以重组E1蛋白为包被抗原的风疹病毒抗体ELISA检测方法。方法 通过生物信息学预测E1优势抗原表位,根据真核系统表达的特点进行序列优化,全基因合成获得风疹病毒E1优势片段核酸序列,构建真核表达载体,在真核系统内进行诱导表达并纯化,用Western blot证实E1蛋白的抗原性。建立ELISA检测系统,并初步检测100份临床血清标本。结果 SDS-PAGE证实重组E1蛋白在酵母中高表达,Western blot证实该E1蛋白具有良好的抗原性,成功建立了检测IgM间接ELISA方法,对临床血清标本的初步检测显示该试剂盒具有较好的敏感性和特异性。结论 本ELISA检测试剂有较高敏感性和较强的特异性,可进一步开发用于检测风疹病毒的早期感染的试剂盒。
Objective To express and purify E1 protein by eukaryotic system,and to establish the ELISA detection method for rubella virus antibody with recombinant E1 protein as coating antigen.Methods The E1 dominant antigen epitopes were predicted by bioinformatics.The nucleic acid sequence of the rubella virus E1 dominant fragment was obtained by whole gene synthesis according to the expression characteristics of eukaryotic system,and the eukaryotic expression vector was constructed.The expression of E1 protein was induced in eukaryotic system and purified.The antigenicity of E1 protein was confirmed by Western blot.ELISA detection system was established,and 100 clinical serum samples were preliminarily detected.Results SDS-PAGE results confirmed that the recombinant E1 protein was highly expressed in yeast.Western blot results confirmed that the E1 protein had good antigenicity.A method for detecting IgM by indirect ELISA was successfully established.The kit had both the good sensitivity and specificity showed by the results of preliminary detection of clinical serum samples.Conclusion Our self-built ELISA detection system has the high sensitivity and specificity,and it can be used to detect the early infection of rubella virus.
作者
李秀义
高冬梅
张守柱
潘继文
温和
Li Xiuyi;Gao Dongmei;Zhang Shouzhu(Dept of Clinical Laboratory,The Third Affiliated Hospital of Anhui Medical University,The First People's Hospital of Hefei,Hefei 230061)
出处
《安徽医科大学学报》
CAS
北大核心
2019年第8期1210-1214,共5页
Acta Universitatis Medicinalis Anhui
基金
国家自然科学基金(编号:81101273)
合肥市科技局科技计划项目(编号:2010-014)
关键词
风疹病毒
E1蛋白
酵母表达
rubella virus
E1 protein
yeast expression
作者简介
李秀义,男,副主任检验技师,硕士研究生;温和,女,副主任检验技师,副教授,责任作者,E-mail:2488207025@qq.com
