多房棘球绦虫RNA结合蛋白28的鉴定  

Molecular characterization of RNA binding protein 28 of Echinococcus multilocularis

作　　者：寇永杰 李瑞 郑亚东 夏天奇 时恒枝 王璞 杨兴 孙晓林[1] KOU Yong-jie;LI Rui;ZHENG Ya-dong;XIA Tian-qi;SHI Heng-zhi;WANG Pu;YANG Xing;SUN Xiao-lin(College of Veterinary Medicine,Gansu Agricultural University,Lanzhou 730070,China;Key Laboratory of Applied Technology on Green-Eco-Healthy Animal Husbandry of Zhejiang Province,Zhejiang Provincial Engineering Laboratory for Animal Health Inspection&Internet Technology,Zhejiang International Science and Technology Cooperation Base for Veterinary Medicine and Health Management,College of Animal Science and Technology&College of Veterinary Medicine of Zhejiang A&F University,China-Australia Joint Laboratory for Animal Health Big Data Analytics,Hangzhou 311300,China;College of Basic Medical Sciences,Dali University,Dali 671000,China)

机构地区：[1]甘肃农业大学动物医学院,兰州730030 [2]浙江省绿色生态健康畜牧业应用技术重点实验室,浙江省动物卫生检验与互联网技术工程实验室,浙江省兽药与健康管理国际科技合作基地,浙江农林大学动物科技学院,动物医学院,中澳动物健康大数据分析联合实验室,杭州311300 [3]大理学院基础医学院,大理671000

出　　处：《中国人兽共患病学报》2022年第6期490-495,共6页Chinese Journal of Zoonoses

基　　金：国家自然科学基金资助项目(No.32160843)。

摘　　要：目的通过扩增多房棘球绦虫(Echinococcus multilocularis)RNA结合蛋白28(RBP28)基因并构建重组表达质粒,诱导表达并纯化重组RBP28蛋白,探索多房棘球绦虫RBP28的分子特征。方法根据WormBase数据库RBP28基因序列设计特异性引物;RT-PCR扩增RBP28基因并构建重组表达质粒,诱导表达并纯化重组RBP28蛋白,免疫新西兰大白兔,制备多克隆抗体;用Western blot检测多克隆抗体特异性,并通过免疫荧光观察RBP28在多房棘球蚴及其细胞中的分布;利用免疫共沉淀技术,获得与RBP28互作的蛋白沉淀产物后,对二者进行蛋白银染和质谱鉴定。结果RBP28编码基因大小约为1245bp;重组RBP28相对分子质量约为M_(r)50000;Western blot结果表明,多克隆抗体能够特异性识别重组蛋白RBP28及多房棘球蚴全蛋白中的天然RBP28蛋白,但与猪带绦虫、泡状带绦虫和细粒棘球绦虫无明显的抗原交叉反应;免疫荧光定位表明,RBP28主要分布在多房棘球蚴的生发层,在多房棘球蚴细胞则主要分布于细胞质中;对银染和质谱鉴定结果比较分析,获得了7种可能与RBP28相互作用的蛋白质。结论本研究确定了RBP28蛋白编码基因的大小、分子量及其多克隆抗体的特异性。通过免疫荧光定位,发现RBP28主要分布在多房棘球蚴的生发层,并利用质谱鉴定获得了7种与RBP28有互相作用的蛋白质,为研究多房棘球绦虫病的致病机制奠定了基础。By expanding the RNA binding protein 28(RBP28)gene of Echinococcus multilocularis and constructing recombinant expression plasmid,inducing expression and purifying recombinant RBP28protein,the molecular characteristics of E.multilocularis RBP28were explored.Specific primers were designed according to the RBP28gene sequence retrieved from the WormBase database.The RBP28gene was amplified by RT-PCR and a recombinant expression plasmid was constructed.The recombinant RBP28protein was induced and purified.New Zealand white rabbits were immunized to prepare polyclonal antibodies.Western blot was used to detect the specificity of polyclonal antibodies,and immunofluorescence was used to check the distribution of RBP28in E.multilocularis metacestodes and their cells.After obtaining protein samples that could interact with RBP28by immunoprecipitation,they were identified by silver staining and mass spectrometry.The coding fragment of the RBP28gene was 1245bp in length.The molecular weight of recombinant RBP28was about M_(r)50000.The Western blot results showed that the polyclonal antibodies could specifically recognize the recombinant protein RBP28and the natural RBP28protein among the whole proteins of E.multilocularis metacestodes,but there was no obvious antigenic crossreaction with Taeniasolium,Taenia alveolata and E.granulosus.Immunofluorescence localization showed that RBP28was mainly distributed along the germinal layer and in the cytoplasm of the cells.Seven proteins that may interact with RBP28were obtained by comparing the results of silver staining and mass spectrometry.In this study,the size and molecular weight of the RBP28protein encoding gene and the specificity of the polyclonal antibody were determined.By immunofluorescence localization,it was found that RBP28was mainly distributed in the germinal layer of Echinococcosis multilocularis,and seven proteins interacting with RBP28were identified by mass spectrometry,which has laid a foundation for studying the pathogenesis of E.multilocularis.

关 键 词：多房棘球蚴 RNA结合蛋白28 免疫荧光定位 免疫共沉淀 质谱 

分 类 号：R383.3[医药卫生—医学寄生虫学]