莫氏巴贝斯虫trap基因的克隆表达及反应原性分析

发布时间：2018-04-28 01:28

本文选题：莫氏巴贝斯虫 + trap基因　；参考：《中国兽医科学》2017年03期

【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。
[Abstract]:In this study, the whole length of trap gene was successfully amplified from the genomic DNAA and c DNA of Babes Mori Lintan strain, and the structure of the gene and protein was verified by bioinformatics analysis software. The prokaryotic expression vector of p ET-30a-trap was constructed and transformed into Escherichia coli BL21(DE3)p Lys S receptive cells to induce expression. After ultrasonic fragmentation, the supernatant and precipitate were analyzed by SDS-PAG E. The reactivity of soluble rBm TRAPP W estern-blot was analyzed. The results showed that the trap gene of Babes Mori had 4 introns and 5 exons, and the open reading frame size was 2 100 BP. The first 23 amino acid is the signal peptide sequence, the 45t201 and 238H302 are the v WA and TSP1 domains of the TRAP protein family, respectively, and the recombinant protein r Bm TRAP exists in the form of soluble protein and inclusion body, and the Lintan strain of Babes motif. The positive serum of Tianzhu strain could specifically recognize r Bm TRAP, but had no cross reaction with the strain of Babes Mohlich Chongning county and Hebei strain, Xinjiang strain and Dunhuang strain of sheep Babes parasite, and the positive serum of Taylor lui and sheep without serosa. The results showed that rBm TRAP had the potential as a candidate antigen for serological diagnosis, which laid a foundation for the future immunological diagnosis of the infection of Babes Mori strain Lintan strain and Tianzhu strain.
【作者单位】：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;
【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)
【分类号】：S852.723

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