摘要
目的原核表达炭疽杆菌PA63蛋白,以此为包被抗原建立检测相应抗体的间接ELSIA方法,用于炭疽血清免疫学诊断。方法选择炭疽杆菌PA63为目的基因,合成全长序列,构建pCzn1-PA63质粒,克隆至E.coli ArcticExpress表达菌株,优化IPTG表达条件,通过包涵体变性复性,Ni柱纯化获得可溶性PA63蛋白。利用棋盘滴定法确定包被PA63蛋白的最佳浓度及血清最佳稀释度建立间接ELISA方法,对炭疽阳性血清和阴性血清进行检测,计算ROC曲线下面积,确定Cut-off值,灵敏度与特异度。结果成功构建了pCzn1-PA63质粒,IPTG 37℃诱导表达蛋白主要以包涵体形式存在。包涵体经变性、复性,纯化后获得PA63蛋白。用5μg/ml PA63包被酶标板,血清稀释度为1∶50建立的ELISA方法;ROC曲线下面积为0.969(P<0.01),Cut-off值为0.2865时的灵敏度为91.18%(95%可信区间为76.32%-98.14%),特异度为94.64%(95%可信区间为85.13%-98.88%)。结论成功表达具有生物活性的重组炭疽杆菌PA63抗原蛋白,以此为包被抗原建立的间接ELISA检测炭疽PA抗体,具有较高的灵敏度与特异度,可用于炭疽的血清学诊断。
Objective To express the PA63 protein of Bacillus anthracis in a prokaryotic expression system and to use that protein as a coating antigen to create a method of indirect ELISA to detect the corresponding antibodies for the sero- logical diagnosis of anthrax.Methods Bacillus anthracis PA63 was selected as the target gene and its full-length sequence was sequenced.The plasmid pCzn1-PA63 was constructed and cloned into the E.coli Arctic Express strain for expression.Conditions were optimized for expression with IPTG.The soluble PA63 protein was purified using an Ni column after denaturation and renaturation of inclusion bodies.Indirect ELISA was performed using checkerboard titration to determine the optimal concentration of PA63 protein and the optimal dilution of serum.Anthrax-positive and-negative sera were tested,and the area under the ROC curve was calculated to determine the cut-off value,sensitivity,and specificity.Results The pCzn1-PA63 plasmid was successfully constructed,and protein expression induced with IPTG at 37℃ was mainly evident in the form of inclusion bodies.The inclusion bodies were denatured and renatured,and PA63 protein was obtained after purification.The optimum coating concentration of PA63 in indirect ELISA was 5μg/ml,serum dilution was 1:50,the area under the ROC curve was 0.969(P<0.01),the cut-off value was 0.2865,and sensitivity was 91.18%(95%).The confidence interval was 76.32-98.14%,and specificity was 94.64%(95% confidence interval: 85.13-98.88%).Conclusion Biologically active recombinant B.anthracis PA63 protein was successfully obtained and used as a coating antigen in indirect ELISA to detect anthrax PA antibody.This technique has a high level of sensitivity and specificity and can be used for serological diagnosis of anthrax infection.
作者
刘东立
马琳
张恩民
石一
李文涓
LIU Dong-li;MA Lin;ZHANG En-min;SHI Yi;LI Wen-juan(Shaanxi Provincial Center for Disease Prevention and Control,Xi'an ,China 710054;Chinese Center for Disease Control and Prevention)
出处
《中国病原生物学杂志》
CSCD
北大核心
2018年第11期1227-1231,共5页
Journal of Pathogen Biology
基金
陕西省卫生计生科研基金项目(No.2016D094)
作者简介
通讯作者:刘东立(1974-),男,陕西西安人,本科副主任医师。研究方向:传染性疾病预防控制。E-mail:ldl029@foxmail.com.
