登革病毒2型型特异性抗原片段的鉴定及其抗体ELISA检测方法的建立  

Identification of Dengue virus type 2-specific antigen fragments and development of a rapid ELISA method for DV2 antibody

作　　者：李硕 李以圣 任瑞文[2] 官文升 陈迪佳 李春缘 杨柳[2] 陈月[2] 李鹏[1] LI Shuo;LI Yi-sheng;REN Rui-wen;GUAN Wen-sheng;CHEN Di-jia;LI Chun-yuan;YANG Liu;CHEN Yue;LI Peng(College of Animal Science and Technology,Heilongjiang Bayd Agricultural University,Daqing 163319,Heilongjiang Province,China)

机构地区：[1]黑龙江八一农垦大学,黑龙江大庆163319 [2]广州军区疾病预防控制中心,广东广州510507

出　　处：《中国生物制品学杂志》2020年第5期563-567,573,共6页Chinese Journal of Biologicals

基　　金：广东省科技计划项目(2014A020219007,2016A020219006);全军后勤科研项目(AWS16J020,BWS14J025)。

摘　　要：目的筛选、鉴定登革病毒(Dengue virus,DENV)2型(DV2)型特异性抗原片段;建立优化检测DV2抗体的ELISA方法,并进行临床应用验证。方法采用生物信息学方法分析1~4型(DV1~DV4)及其相近虫媒病毒基因组序列,选择预测得分较高且在不同DV2流行株中高度保守的表位;利用pMal-c2x表达系统制备其原核表达抗原,Western blot法分析其免疫原性;建立优化检测DV2抗体的ELISA方法,并验证方法的特异性及灵敏性;应用优化的方法检测33份各型登革热(Dengue fever,DF)确诊患者及30份健康体检人群血清样本。结果经生物信息学分析,预测获得DV2特异性抗原表位12段,预测得分值较高的8段(E123~128、E131~135、E143~149、E223~229、E286~294、E340~347、E383~387及E391~398)重组表达载体经双向测序证实读码框架准确,经Western blot分析,包含抗原位点E143~149区域的融合表达片段D2Ag3仅与DV2多克隆抗体发生免疫反应。ELISA优化条件为抗原包被浓度2μg/mL,4℃包被24 h,37℃封闭2 h;经该方法分析,D2Ag3原核表达抗原与DV2多克隆抗体反应的A450≥1.37,与其他20种多克隆抗体反应的A450均≤0.04,DV2多克隆抗体在40~20480倍稀释范围内,均可获得阳性检测结果。63份临床阳性样本A450>0.32,阴性样本A450<0.12,检测样本/阴性对照比值(sample/negative,S/N)>2.1。结论成功筛选鉴定了DV2型特异性抗原片段,初步建立了DV2 IgG抗体的ELISA检测方法,该方法具有良好的特异性及灵敏性,为DF的鉴别诊断提供了技术手段。Objective To identify Dengue virus(DENV)type 2(DV2)-specific antigen fragments,develop an ELISA method for rapid detection of DV2 antibody and apply in clinic.Methods The virus genome sequences of Dengue virus types 1~4,Japanese encephalitis virus(JEV)and yellow fever virus were analyzed by bioinformatic method and the DV2-specific antigen epitopes with high predicted scores,which were highly conserved in various epidemic DV2 strains,were selected and expressed in prokaryotic cells by using pMal-c2 x system,and determined for reactogenicity by Western blot.The ELISA for DV2 antibody was developed and optimized,and verified for specificity and sensitivity,by which the serum samples from 33 patients with Dengue fever of various types and 30 healthy peoples were determined.Results A total of 12 DV2-specific antigenic epitopes were obtained by bioinformatic analysis,from which 8 ones with high predicted scores(E123~128,E131~135,E143~149,E223~229,E286~294,E340~347,E383~387 and E391~398)were selected.Dual sequencing showed the correct reading frame of recombinant expression vector.The optimal antigen concentration,temperature and time for coating in the developed ELISA method were 2μg/mL,4℃and 24 h,while the temperature and time for blocking were 37℃and 2 h,respectively.Western blot showed that the expressed fragment D2Ag3 containing antigenic epitope E143~149 was only reacted with polyclonal antibody against DV2,with an A450 value of not less than 1.37.However,the A450 values of D2Ag3 with more than 20 kinds of other polyclonal antibodies were not more than 0.04.All the ELISA results of polyclonal DV2 antibody diluted by 40~20480 folds were positive.In the 63 clinical samples,the A450 values of positive samples were more than 0.32,while those of negative samples were less than 0.12,and the sample/negative(S/N)ratio was more than 2.1.Conclusion A DV2-specific antigen fragment was successfully screened.An ELISA method for rapid detection of DV2 IgG antibody was developed,which showed high specificity and sensitivit

关 键 词：登革病毒2型 型特异性抗原 原核表达 生物信息学方法 酶联免疫吸附测定 

分 类 号：R373.33[医药卫生—病原生物学]