细粒棘球蚴cDNA文库的构建和免疫筛选  

Construction and immunoscreening of cDNA library of Echinococcus granulosus

作　　者：朱慧慧[1] 许学年[1] 高春花[1] 汪俊云[1] 杨明涛[1] 石锋[1] 焦伟[2] 付承[2] 

机构地区：[1]中国疾病预防控制中心寄生虫病预防控制所,卫生部寄生虫病原与媒介生物学重点实验室世界卫生组织疟疾、血吸虫病和丝虫病合作中心,上海200025 [2]新疆疾病预防控制中心,乌鲁木齐830001

出　　处：《国际医学寄生虫病杂志》2012年第5期272-275,共4页International JOurnal of Medical Parasitic Diseases

基　　金：国家科技重大专项（2012ZX10004-220）和科技支疆课题（200891130）

摘　　要：目的构建细粒棘球蚴原头节的λZAPⅡcDNA文库，并对构建的文库进行免疫筛选。方法采集感染细粒棘球蚴的绵羊育囊中的原头节，用Trizol试剂抽提总RNA，用mRNA纯化试剂盒纯化mRNA作为模板合成eDNA，并对其进行末端平端化、EcoRI接头连接和磷酸化及XhoI酶切，然后应用SepharoseCL-2B分离柱对其进行分级分离，收集大于500bp的cDNA片段，使用GigapaekⅢGold试剂盒将合成的eDNA连接到λZAP-Ⅱ载体，并将重组载体包装到λZAP-Ⅱ噬菌体中，构建细粒棘球蚴eDNA文库。用囊型棘球蚴病患者混合血清对所构建的文库进行免疫筛选，并用NCBI中的Blast模块及其它生物信息软件对筛选出的阳性克隆进行生物信息学分析。结果成功构建了细粒棘球蚴原头节λZAPⅡeDNA文库，文库的滴度为1．8×105噬菌斑形成单位（plaqueformingunit，pfu）／ml，插入片段长度范围在1．0～4．0kb间，平均插入片段长度为2．6kb，插入效率为93．8％。对所构建的文库进行免疫筛选，共筛选到5个阳性克隆，其中有2个为细粒棘球蚴新发现基因，新发现基因存在众多B细胞表位。结论成功构建了细粒棘球蚴原头节λZAP-ⅡcDNA文库，初步免疫筛选得到2个新发现基因，生物信息学分析预测它们具有潜在的诊断价值。Objective To construct the λZAPⅡ eDNA library of Echinococcus granulosus for screening of immunodiagnostic candidate molecules. Methods The protoscoleees of Echinococcus granulosus （E. granulosus） were isolated from the fertile cysts of E. granulosus infected sheep, and total RNA of the protoscoleces was extracted using Trizol reagent, then mRNA was purified from the total RNA using mRNA purification kit. cDNA was synthesized using cDNA synthesis kit with the purified mRNA as template, then blunt-ended, linked with EcoR I adapter, phosphorylated, and cut by Xho I enzyme using the λZAP-Ⅱ EcoR I/CIAP-kit. The cDNA obtained was then separated by Sepharose CL-2B colum, and cDNA longer than 500 bp was collected and cloned into λZAP-Ⅱ vector. The recombinant vectors were packaged into λZAP-Ⅱ phage thus to constitute cDNA library. The cDNA library was immunoscreened by mixed patient sera of cystic echinococcosis and the positive clones obtained were sequenced and analysed by Blast module in NCBI and other informatics softwares. Results The λZAP-Ⅱ cDNA library was constructed with the titer of l. 8 x 105 plaque forming unit（pfu） /ml, the length range of the inserts 1.0 - 4.0 kb, the average length of 2.6 kb, and the insert efficiency of 93.8%. Five positive clones were obtained through immunoscreening of the eDNA library, and 2 of them are new genes of E. granulosus with multiple B cell epitopes. Conclusion The λZAP-Ⅱ cDNA library of E. granulosus was successfully constructed and positive clones were obtained through immunoscreening, among them 2 clones are new genes which could become candidate molecules for diagnosing cystic echinococcosis.

关 键 词：细粒棘球蚴 原头节 CDNA文库 免疫筛选 

分 类 号：R[医药卫生]