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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, the full-length trap gene was amplified from genomic DNA and c DNA respectively using the Lactobacillus casei M Babel as a research object, and the structure of the gene and protein was verified by bioinformatics analysis software. The construction of p ET-30a-trap prokaryotic expression vector was transformed into Escherichia coli BL21 (DE3) p Lys S competent cells induced expression; the expression product was sonicated, the supernatant and the precipitate was analyzed by SDS-PAGE. Purified soluble rBm TRAP was analyzed by Western-blot. The results showed that trap gene Mpbbacterium spp. Had 4 introns and 5 exons, the open reading frame was 2 100 bp. The amino acids at positions 1 to 23 are signal peptide sequence, and the 45 WA and 238 to 302 are respectively the v WA and TSP1 domains of the TRAP protein family. The recombinant protein r Bm TRAP exists in both soluble forms and inclusion bodies, Mobic Babes insects Lintan strain, Tianzhu positive serum can be specifically identified r Bm TRAP, and Mbuzai Babei Chongning County and Hebei strains, sheep Babesia undetermined Xinjiang strains and Dunhuang Strain, Lu Tailor Insects and sheep without serum positive cross-reaction. The results showed that rBm TRAP has the potential as a candidate antigen for serodiagnosis and laid the foundation for the future establishment of an immunological diagnosis method for the differential diagnosis of Mott-Babesia lintan strain and Tianzhu strain.