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

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.