新疆出血热病毒基因组抗原表位及其实验室检测价值的评价  

Genomic epitopes of Xinjiang hemorrhagic fever virus and the evaluation of its laboratory testing value

作　　者：张花花 赵国玉[2] 李阳 史深 安冉 袁江玲[2] 刘东亮[4] 窦景锐 雒涛[2] 孙素荣[4] 张渝疆[2] Zhang Huahua;Zhao Guoyu;Li Yang;Shi Shen;An Ran;Yuan Jiangling;Liu Dongliang;Dou Jingrui;Luo Tao;Sun Surong;Zhang Yujiang(Department of Epidemiology and Health Statistics,School of Public Health,Xinjiang Medical University,Urumqi 830054,China;Xinjiang Uygur Autonomous Region Center for Disease Control and Prevention Disinfection and Infection Control Center,Urumqi 830002,China;Urumqi Maternal and Child Health Care Hospital,Urumqi 830001,China;Xinjiang Key Laboratory of Biological Resources and Genetic Engineering,College of Life Science and Technology,Xinjiang University,Urumqi 830046,China)

机构地区：[1]新疆医科大学公共卫生学院流行病与卫生统计学教研室,乌鲁木齐830054 [2]新疆维吾尔自治区疾病预防控制中心消毒与感染控制中心,乌鲁木齐830002 [3]乌鲁木齐市妇幼保健院,乌鲁木齐830001 [4]新疆大学生命科学与技术学院新疆生物资源与基因工程重点实验室,乌鲁木齐830046

出　　处：《中华检验医学杂志》2023年第2期127-136,共10页Chinese Journal of Laboratory Medicine

基　　金：国家自然科学基金(81960369);新疆维吾尔自治区高校科研计划重点项目(XJEDU2019I002);国家重点研发计划(2016YFC1200101)。

摘　　要：目的掌握克里米亚-刚果出血热病毒(CCHFV)核蛋白(NP)和糖蛋白(GP)片段的精细抗原表位谱分布,明确优势抗原表位在克里米亚-刚果出血热(CCHF)实验室检测中的价值。方法2014—2021年在新疆大学生命科学院实验室将CCHFV YL04057株采用改良生物肽合成方法分段表达出由8个氨基酸组成的最小合成短肽,以CCHFV多克隆抗体或单克隆抗体14B7(IgM)或CCHFV阳性羊血清为抗体,用免疫印迹方法鉴定出NP和GP片段上具有抗原活性的最小抗原表位(BCE),并采用邻接法将获得的具有序列多态性的BCE与不同地区的CCHFV进行空间聚类,确定BCE组合方式与地理区域分布的相关性,探讨其在建立血清学诊断中的应用价值。采用原核表达质粒(pET-32a、pGEX-KG)和带有不完整谷胱甘肽(GST188)标签的原核表达质粒(pXXGST-ST-1)构建和表达NP片段上6个不同肽段长度的优势抗原表位,建立间接酶联免疫吸附测定(ELISA)检测方法,以经免疫荧光法(IFA)鉴定的CCHF羊血清为对照,统计分析不同肽段长度的抗原表位重组蛋白与IFA检测结果的特异度、敏感度和总符合率。结果CCHFV NP和GP片段共有30个具有抗原活性的BCE,其中NP片段抗原表位集中的核心中间片段NP_(2)(aa 170~305)有6个BCE,两端的NP1(aa 1~200)和NP3(aa 286~482)有9个BCE;GP片段的Gc(aa 1~558)和Gn(aa 533~708)片段有14个BCE和1个包含15个氨基酸的长抗原肽(AP),NP片段BCE氨基酸序列的同源性为97.1%,GP片段对应的同源性为89.1%。在GP片段9个具有序列多态性的BCE中,由GnEc1、GnE2、GnE4、GcE3、GcE6和GcAP-4(Ap)6个组合BCE可将15株来自全球不同地区的CCHFV聚类成亚洲Ⅰ、亚洲Ⅱ、非洲Ⅰ、非洲Ⅱ和欧洲5个地理类群。构建表达的PET-32a-NP(全长)、PGEX-KG-NP_(2)(aa 170~305)、pGEX-KG-NP_(2-1)(aa 235~275)、PGEX-KG-NP_(2-1-1)(aa 237~256)、pXXGST-1-NP_(2-1-2)(aa 250~265)和PGEX-KG-NP_(2-1-3)(aa 260~276)6个重组蛋白CCHFV NP兔多克隆抗血清(pAb)免疫Objective To grasp the distribution of fine antigenic epitope profiles of nucleoprotein(NP)and glycoprotein(GP)fragments of Crimean-Congo hemorrhagic fever virus(CCHFV)and to clarify the value of dominant antigenic epitopes in laboratory testing of Crimean-Congo hemorrhagic fever(CCHF).Methods In a minimal synthetic short peptide consisting of 8 amino acids was segmentally expressed by CCHFV YL04057 strain using a modified bio-peptide synthesis method from 2014 to 2021 in the laboratory of Xinjiang University,College of Life Sciences.Using CCHFV polyclonal antibody or monoclonal antibody 14B7(IgM)or CCHFV-positive sheep serum as antibodies,the minimal antigenic epitopes(BCEs)with antigenic activity on NP and GP fragments were identified by immunoblotting,and the obtained BCEs with sequence polymorphism were spatially clustered with CCHFV from different regions using the neighbor-joining method to determine the combination mode of BCEs with geographical correlation of regional distribution,to explore its application in establishing serological diagnosis.A prokaryotic expression plasmid(pET-32a),an E.coli expression plasmid(pGEX-KG)and a prokaryotic expression plasmid with an incomplete glutathione(GST188)tag(pXXGST-ST-1)were used to construct and express six dominant antigenic epitopes of different peptide lengths on NP fragments,and an indirect Enzyme-linked immunosorbent assay(ELISA)was established.CCHF sheep serum identified by immunofluorescence assay(IFA)was used as a control,and the specificity,sensitivity and overall compliance of the recombinant proteins with different peptide lengths of antigenic epitopes with IFA assay results were statistically analyzed.Results CCHFV,NP and GP fragments had a total of 30 antigenically active BCEs,among which the core intermediate fragment NP_(2)(aa 170^(th)-305^(th)),which had a concentration of antigenic epitopes in the NP fragment,has 6 BCEs,and the NP1(aa 1^(st)-200^(th))and NP3(aa 286^(th)-482^(nd))at both ends have 9 BCEs;the Gc(aa 1^(st)-558^(th))and Gn(aa 533^(th

关 键 词：出血热病毒 克里米亚-刚果 表位 酶联免疫吸附测定 核蛋白 糖蛋白 

分 类 号：R512.8[医药卫生—内科学] R446.6[医药卫生—临床医学]