莫氏巴贝斯虫trap基因的克隆表达及反应原性分析  

Cloning,expression and immunoreactivity analysis of Babesia motasi trap gene

作　　者：何欣[1] 刘军龙[1] 刘爱红[1] 王锦明[1] 牛庆丽[1] 李有全[1] 殷宏[1] 罗建勋[1] 关贵全[1] 

机构地区：[1]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》2017年第3期328-333,共6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(31072130);农业科技创新工程项目(ASTIP);甘肃省青年科技基金项目(145RJYA272)

摘　　要：本研究以莫氏巴贝斯虫临潭株为研究对象,分别从其基因组D N A和c DNA中成功扩增trap基因全长,应用生物信息学分析软件分析验证了该基因和蛋白的结构;构建p ET-30a-trap原核表达载体,转入大肠杆菌BL21(DE3)p Lys S感受态细胞进行诱导表达;表达产物超声破碎后,对上清和沉淀进行SDS-PAG E分析。纯化可溶性的r Bm TRAP,W estern-blot分析其反应原性。结果显示,莫氏巴贝斯虫trap基因具有4个内含子和5个外显子,开放阅读框大小为2 100 bp。第1~23位氨基酸为信号肽序列,第45~201和第238~302位分别为TRAP蛋白家族的v WA和TSP1结构域;重组蛋白r Bm TRAP以可溶性蛋白和包涵体两种形式存在,莫氏巴贝斯虫临潭株、天祝株阳性血清可特异性识别r Bm TRAP,而与莫氏巴贝斯虫宁县株和河北株、羊巴贝斯虫未定种新疆株和敦煌株、吕氏泰勒虫和羊无浆体阳性血清无交叉反应。结果表明,r Bm TRAP具有作为血清学诊断方法候选抗原的潜力,为将来建立鉴别诊断莫氏巴贝斯虫临潭株、天祝株感染的免疫学诊断方法奠定了基础。In the present study,focused on the Babesia motasi Lintan,the full length of trap gene was amplified from genomic DNA and c DNA.The structures of the gene/protein were analyzed and verified by using bioinformatic softwares.A prokaryotic expression vector,p ET-30a-trap,was constructed and transformed into Escherichia coli BL21（DE3） p Lys S to induce protein expression.Supernatant and precipitate were analyzed by SDS-PAGE after the expression product was lysed by ultrasonication.Soluble r Bm TRAP was purified and then its immunoreactivity was determined by western-blot.The result showed that the trap gene contained 4 introns and 5 exons with an open reading frame（ORF） of 2100 bp.The 1-23 animo acids were a signal peptide sequence,45-201 and 238-302 were vWA and TSP1 motifs of TRAP protein family,respectively.The rBmTRAP was expressed as two forms,soluble protein and insoluble inclusion bodies.The r Bm TRAP could be specifically recognized by positive sera from sheep infected by B.motasiLintan/Tianzhu.No cross-reactions was present with the sera of B.motasi Hebei/Ningxian,Babesia sp.Xingjiang/Dunhuang,Theileria luwenshuni and Anaplasma ovis.The result suggests that rBmTRAP has the potetial for considering as a candidate of antigen used in sero-diagnostic methods.It was established a foundation for developing immulogical diagnostic methods for differentiating infection of B.motasi Lintan/Tianzhu from other ovine parasites in future.

关 键 词：莫氏巴贝斯虫 trap基因 原核表达 反应原性分析 

分 类 号：S852.723[农业科学—基础兽医学]