摘要
目的探究Rv1985c、rv3807c、rv1981c基因编码蛋白的生物学特征,建立间接ELISA方法并对其诊断效能进行分析和评价。方法从NCBI数据库中获取rv1985c、rv3807c、rv1981c的全基因序列;应用生物信息学分析软件SOPMA预测蛋白的二级结构;采用SWISS-MODEL建立rv1985c、rv3807c和rv1981c 3个蛋白的3D模型;采用TMHMM Server v.2.0分别预测并分析蛋白的跨膜结构;采用IEDB在线软件预测分析蛋白质线性B细胞抗原表位。以H37RV标准株基因组为模版,PCR扩增目的基因序列并克隆至表达载体中,经IPTG诱导表达后采用Ni柱纯化得到高纯度的目的蛋白,方阵滴定法确定其在间接ELISA中的最佳反应条件。结果rv1985c、Rv1981c为亲水性蛋白且无跨膜结构域,rv3807c为疏水性蛋白且存在3次跨膜结构,3个蛋白均无信号肽,Rv1985c、Rv1985c的不稳定系数高于rv3807c,通过B细胞表位分析各筛选出3个优势表位区。成功构建3个重组表达载体,亲和层析纯化后Western blot验证3个蛋白均能与结核病患者血清反应,具有良好的反应原性,3个蛋白间接ELISA检测血清中IgM敏感性分别为37.20%、54.70%、40.60%,特异性分别为63.70%、71.30%、67.50%,检测IgG时敏感性为:47.60%、61.60%、46.50%,特异性分别为49.70%、74.80%、74.30%。结论生物信息学分析rv1985c、rv3807c、rv1981c均含优势B细胞表位,原核表达的3个蛋白均具有作为ELISA诊断抗原的潜力,为结核病快速诊断候选抗原筛选提供了理论基础。
Objective To explore the biological characteristics of the proteins encoded by the rv1985c,rv3807c,and rv1981c genes and evaluate their value as protein antigens in serological diagnosis and potential applications.Methods The genomic sequence of rv1985c,rv3807c,and rv1981c was obtained from the NCBI database,the bioinformatic analysis software SOPMA was used to predict the secondary structure of the proteins,SWISS-MODEL was used to create 3 D models of Rv1985 c,Rv3807 c,and Rv1981 c,TMHMM Server v.2.0 was used to predict and analyze the transmembrane structure of the proteins,and the online software IEDB was used to predict and analyze linear B cell epitopes of the proteins.Using the H37 RV standard strain genome as a template,the target gene sequences were amplified with PCR and cloned into an expression vector.Expression of the proteins was induced with IPTG,and the proteins were purified on a Ni column to obtain highly pure target proteins.Square matrix titration was used to determine the optimal reaction conditions for indirect ELISA,to devise a method of detection with indirect ELISA and to analyze and evaluate its diagnostic effectiveness.Results rv1985c and rv1981c are hydrophilic proteins with no transmembrane domains.Rv3807 c is a hydrophobic protein with three transmembrane structures,and none of the three proteins have signal peptides.The instability coefficients of rv1985c and rv1985c are higher than that of rv3807c.Three predominant epitope regions were identified in B cell epitope analysis.Three recombinant expression vectors were successfully constructed and purified using affinity chromatography.When detecting serum IgM with indirect ELISA,rv1985c had a sensitivity of 37.20% and a specificity of 63.70%,rv3807c had a sensitivity of 54.70% and a specificity of 71.30%,and rv1981c had a sensitivity of 40.60% and a specificity of 67.50%.When detecting IgG,rv1985c had a sensitivity of 47.60% and a specificity of 49.70%,rv3807c had a sensitivity of 61.60% and a specificity of 74.80%,and rv1981c had a sensitivity of 46.50% and a specificity of 74.30%.Conclusion The basic structural features of rv1985c,rv3807c,and rv1981c were analyzed using analysis software online,and the dominant B cell epitopes were screened.Results verified that all three proteins have the potential to serve as diagnostic antigens and are also candidates for rapid diagnosis of tuberculosis.These findings provide a theoretical basis for antigen screening.
作者
杨航
努尔塞力克·努素甫
杨亚军
姬祥
马忠臣
张萍
曹旭东
陈创夫
YANG Hang;NuErSaiLiKe Nusufu;YANG Ya-jun;JI Xiang;MA Zhong-chen;ZHANG Ping;CAO Xu-dong;CHEN Chuang-fu(College of Animal Science and Technology,Shihezi University,Shihezi,Xinjiang,China 832000;Collaborative Innovation Center for Prevention and Treatment of Infectious Zoonoses Highly Prevalent in Western China;Agriculture and Rural Bureau of Xinyuan County;Xinjiang Kashi Prefecture Tuberculosis Control Institute)
出处
《中国病原生物学杂志》
CSCD
北大核心
2021年第2期143-149,共7页
Journal of Pathogen Biology
基金
国家重点研发计划项目(No.2018YFD050090202,No.2017YFD0500304)。
作者简介
通讯作者:陈创夫,E-mail:ccf-xb@163.com;通讯作者:曹旭东,E-mail:cxd100@sohu.com;杨航(1995-),男,新疆石河子人,硕士研究生。主要研究方向:病原分子诊断。E-mail:923959312@qq.com。
