曼氏裂头蚴cDNA文库的构建及诊断候选抗原筛选  

Construction of a cDNA library for Sparganum mansoni and screening of diagnostic antigen cadidates

作　　者：卢艳 陈家旭 宋鹏 李浩 艾琳 蔡玉春 储言红 陈韶红 LU Yan;CHEN Jia-Xu;SONG Peng;LI Hao;AI Lin;CAI Yu-Chun;CHU Yan-Hong;CHEN Shao-Hong(National Institute of Parasitic Diseases,Chinese Center for Disease Control and Prevention(Chinese Center for Tropical Diseases Research),NHC Key Laboratory of Parasite and Vector Biology,WHO Collaborating Centre for Tropical Diseases,National Center for International Research on Tropical Diseases,Shanghai 200025,China)

机构地区：[1]中国疾病预防控制中心寄生虫病预防控制所(国家热带病研究中心)、国家卫生健康委员会寄生虫病原与媒介生物学重点实验室、WHO热带病合作中心、国家级热带病国际联合研究中心,上海200025

出　　处：《中国血吸虫病防治杂志》2021年第4期380-386,共7页Chinese Journal of Schistosomiasis Control

基　　金：上海市卫计委科研课题面上项目(201740076);国家寄生虫资源库(NPRC-2019-194-30)。

摘　　要：目的构建曼氏裂头蚴cDNA文库,通过免疫筛选获得曼氏裂头蚴病诊断候选抗原基因。方法提取曼氏裂头蚴虫体总RNA,反转录合成cDNA,连接噬菌体载体,经体外包装后构建曼氏裂头蚴SMART cDNA文库。用曼氏裂头蚴病患者血清免疫筛选cDNA文库,获得阳性克隆,测定阳性克隆插入片段的DNA序列,进行同源性分析并预测编码蛋白的结构和功能。结果成功构建了曼氏裂头蚴cDNA文库,文库滴度为6.25×10^(6)pfu/mL,重组率为100%,文库插入片段的平均长度大于1100 bp。经免疫筛选获得12个阳性克隆,分为Sm-Ⅰ、Sm-Ⅱ、Sm-Ⅲ和Sm-Ⅳ4类,其代表克隆为Sm60-1、Sm58-1、Sm20-1、Sm22-3,插入片段长度分别为1134、1063、883、969 bp,编码蛋白分别与欧猥迭宫绦虫抗原多肽、胞质抗原、核糖体蛋白S4样蛋白和未命名蛋白具有较高同源性。结论成功构建了曼氏裂头蚴SMART cDNA文库,获得了4类阳性克隆,为曼氏裂头蚴病诊断抗原的进一步研究奠定了基础。Objective To construct a cDNA library of Sparganum mansoni and immunoscreen antigen candidates for immunodiagnosis of sparganosis mansoni.Methods Total RNA was extracted from S.mansoni,and reversely transcribed into cDNA,which was ligated into the phage vector.These recombinant vectors were packaged in vitro to construct the SMART cDNA library of S.mansoni.Then,the cDNA librarywas immunoscreened with sera from patients with sparganosis mansoni to yield positive clones.The inserted fragments of positive clones were sequenced and subjected to homology analyses,and the structure and functions of the coding proteins were predicted.Results The SMATR cDNA library of S.mansoni was successfully constructed.The titer of the cDNA librarywas 6.25×10^(6) pfu/mL,with a recombinant efficiency of 100%,and the mean length of the inserted fragments in the library was larger than 1100 bp.A total of 12 positive clones were obtained by immunoscreening,and were categorized into Sm-Ⅰ(Sm60-1),Sm-Ⅱ(Sm58-1),Sm-Ⅲ(Sm20-1)and Sm-IV(Sm22-3),with 1134,1063,883 bp and 969 bp long inserted fragments.Their coding proteins were highly homologous with the Spirometra erinaceieuropaei antigenic polypeptide,cytoplasmic antigen,ribosomal protein S4-like protein and unnamed protein product,respectively.Conclusions A SMART cDNA library of S.mansoni has been successfully constructed and 4 categories of positive clones have been identified,which provides a basis for further studies on diagnostic antigens for sparganosis mansoni.

关 键 词：曼氏裂头蚴 CDNA文库 免疫筛选 诊断抗原 SMART技术 

分 类 号：R383.35[医药卫生—医学寄生虫学]