多房棘球绦虫丝氨酸蛋白酶基因的克隆、表达及抗原性鉴定  

Cloning and characterization of Echinococcus multilocularis serine protease

作　　者：臧小燕 田梦潇 郭刚 齐文静 郭宝平 李军 李林[1] 张文宝 ZANG Xiao-yan;TIAN Meng-xiao;GUO Gang;QI Wen-jing;GUO Bao-ping;LI Jun;LI Lin;ZHANG Wen-bao(College of Basic Medicine,Xinjiang Medical University,Urumqi 830054,China;State Key Laboratory of Pathogenesis,Prevention and Treatment of Diseases Highly Endemic to Central Asia,Clinical Medicine Institute,The First Affiliated Hospital of Xinjiang Medical University)

机构地区：[1]新疆医科大学基础医学院,新疆乌鲁木齐830054 [2]中亚高发病成因与防治国家重点实验室,临床医学研究院,新疆医科大学第一附属医院

出　　处：《中国病原生物学杂志》2020年第10期1151-1156,共6页Journal of Pathogen Biology

基　　金：国家自然科学基金重点项目(No.81830066)。

摘　　要：目的克隆多房棘球绦虫(Echinococcus multilocularis,Em)丝氨酸蛋白酶(SP)基因,通过原核系统表达Em-SP并对其抗原性进行鉴定。方法PCR扩增获得Em-SP片段;构建重组质粒pET30a-Em-SP,NdeI/HindIII双酶切并测序,将测序正确的重组质粒转化至E.coli BL21(DE3)菌株中,用异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达,采用SDS-PAGE电泳分析表达蛋白的分子质量单位和表达丰度。表达产物经亲和层析(Ni-IDA树脂)纯化后采用Western blot鉴定蛋白的反应原性。结果PCR扩增SP基因片段为1374 bp,重组质粒pET30a-Em-SP酶切后电泳和测序表明阅读框架序列与设计一致。IPTG诱导重组质粒转化菌表达蛋白主要以包涵体形式存在,表达蛋白的相对分子质量约为51×103,与预期值大小相符,且能被鼠抗His-Tag抗体识别,用包虫患者血清Western blot鉴定呈阳性。结论克隆的Em-SP基因在原核中成功表达,表达产物具有良好的反应原性,可作为诊断试剂的候选抗原。Objective To clone the serine protease(SP)gene of Echinococcus multilocularis and express E.multilocularis SP using a prokaryotic expression system and to determine its antigenicity.Methods An E.multilocularis SP gene fragment was amplified with PCR using primers designed based on the sequence in GenBank.The recombinant plasmid vector pET30 a-Em-SP was constructed with Nde I/Hind III restriction enzyme cleavage and then transformed into E.coli BL21(DE3).Recombinant clones were verified with PCR and sequencing was used to verify open reading frames.Expression of the recombinant protein was induced with IPTG on LB medium containing antibiotic kanamycin at a concentration of 50 g/ml.The recombinant protein was purified using an Ni-IDA affinity column.The molecular weight was determined using SDS-PAGE and Western blotting.Results The SP gene fragment was amplified using PCR,which indicated that it was 1,374 bp in length.Restriction enzyme digestion indicated that the fragment was the right size,and sequencing indicated that the inserted fragment open reading frame was correct,indicating that the recombinant plasmid pET30 a-Em-SP was successfully constructed.E.multilocularis SP was successfully expressed in E.coli with IPTG on LB medium.SDS-PAGE indicated that the protein was insoluble and in the form of inclusion bodies with a relative molecular weight of about 51×10~3.Western blot analysis indicated the purified band of recombinant protein was recognized by both anti-His-Tag antibodies and pooled sera from patients with alveolar echinococcosis.Conclusion The cloned E.multilocularis SP gene was successfully expressed in a prokaryotic expression system.The recombinant protein was recognized by sera from patients with alveolar echinococcosis,indicating the protein can be a potential antigen for development of a diagnostic kit.

关 键 词：多房棘球绦虫 丝氨酸蛋白 原核表达 Western blot 抗原性 

分 类 号：R383.33[医药卫生—医学寄生虫学]