多房棘球蚴主要卵抗原p40-1的原核表达、多克隆抗体制备及其组织定位  

Prokaryotic expression,polyclonal antibodies preparation and tissue localization for MEAp40-1 of Echinococcus multilocularis metacestodes

作　　者：何学东 郭小腊 孙晓林[1] 曾巧英[1] 郑亚东[2] HE Xue-dong;GUO Xiao-la;SUN Xiao-lin;ZENG Qiao-ying;ZHENG Ya-dong(College of Veterinary Medicine,Gansu Agricultural University,Lanzhou 730070,China;State Key Laboratory of Veterinary Etiological Biology/Key Laboratory of Animal Parasitology of Gansu Province/Lanzhou Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Lanzhou 730046,China)

机构地区：[1]甘肃农业大学动物医学院,甘肃兰州730070 [2]中国农业科学院兰州兽医研究所家畜疫病病原生物学国家重点实验室甘肃省动物寄生虫病重点实验室,甘肃兰州730046

出　　处：《中国兽医科学》2020年第8期982-988,共7页Chinese Veterinary Science

基　　金：国家重点基础研究发展计划(973)项目(2015CB150300);国家自然科学基金项目(U1703104,31472185)。

摘　　要：初步确定MEAp40-1的基因特征及其组织分布特征。根据WormBase数据库中多房棘球蚴MEAp40-1序列设计特异性引物,利用RT-PCR技术扩增MEAp40-1编码基因并构建重组表达质粒pET-28a-MEAp40-1,IPTG诱导表达并用His-Ni亲和层析柱纯化,制备多克隆抗体。通过间接ELISA方法检测多克隆抗体效价,应用免疫荧光技术定位MEAp40-1在原头蚴中的分布。通过生物信息学分析发现,多房棘球绦虫表达3种不同的MEAp40(MEAp40-1~3),分别分布在不同染色体上。系统进化树显示,每个多房棘球绦虫MEAp40基因与其相应的细粒棘球绦虫的同源基因组成一支。多房棘球绦虫MEAp40-1编码基因全长945 bp,编码314个氨基酸。7种绦虫的MEAp40-1蛋白很保守,均存在α晶体结构域,并且位于该结构域中的协同分子伴侣结合位点也非常保守。MEAp40-1重组蛋白大小约为37 ku,其多克隆抗体效价可达1∶409 600,可识别虫体中的MEAp40-1天然蛋白。MEAp40-1蛋白主要分布在多房棘球蚴后端,而头节、囊壁等其他部位均未见表达。结合以上研究结果,推测MEAp40-1蛋白在绦虫中的生物学功能保守,可能与多房棘球绦虫原头蚴的发育有关。The aim of study was to determine the characteristics and tissue distribution of MEAp40 gene.Specific primers were designed and synthesized according to the sequence of Echinococcus multilocular metacestodes MEAp40 gene retracted from Worm Base.MEAp40-1 gene was amplified using RT-PCR and a recombinant expression plasmid p ET-28 a-MEAp40 was constructed.The expression of recombinant plasmid p ET-28 a-MEAp40 was induced with IPTG and purified by His-Ni affinity chromatography.The purified protein was used to immunize rabbits for preparing polyclonal antibodies.The titer of polyclonal antibodies was detected by indirect ELISA.MEAp40-1 localization in E.multilocularis metacestodes was observed by immunofluorescence staining.Bioinformatics analysis showed that E.multilocularis contained three different MEAp40 genes(MEAp40-1 to 3),which were distributed on different chromosomes.The phylogenetic tree showed that each E.multilocularis MEAp40 and its corresponding Echinococcus granulosus homologous genes constituted a branch,indicating that three MEAp40 genes may have formed before the speciation of E.multilocularis and E.granulosus.The full length of E.multilocularis MEAp40-1 was 945 bp,encoding 314 amino acids.All the MEAp40-1 proteins of 7 tapeworms had a conservative alpha domain,and the synergistic chaperone binding sites in this domain were also very conserved.The size of recombinant MEAp40-1 protein was about 37 ku.The titer of prepared polyclonal antibodies reached at1 ∶409 600.The prepared polyclonal antibodies could specifically recognize the native E.multilocularis MEAp40-1 protein.MEAp40-1 protein was mainly distributed in the posterior part of E.multilocularis metacestodes,but not expressed in the anterior part,cyst wall and other parts of metacestodes.Taken together,it is speculated that MEAp40-1 protein may have a conserved biological functions in tapeworms,possibly being related to the development of E.multilocularis protoscolex.

关 键 词：多房棘球蚴 MEAp40-1基因 原核表达 多克隆抗体 免疫荧光 

分 类 号：S852.734[农业科学—基础兽医学]