• 中文核心期刊
  • CSCD来源期刊
  • 中国科技核心期刊
  • CA、CABI、ZR收录期刊

副结核分枝杆菌85A、85B蛋白生物信息学特征及其原核表达

Bioinformatics and Prokaryotic Expressions of 85A and 85B in Mycobacterium paratuberculosis

    摘要
  • 摘要:
    目的 探究副结核分枝杆菌（Mycobacterium avium subsp. paratuberculosis, MAP）85A 和 85B 蛋白的生物信息学结构特征，通过原核表达技术获取两种蛋白，并评估其反应原性，从而为后续该菌 ELISA 检测试剂盒的研发及疫苗研究奠定基础。
    方法 运用生物信息学在线分析平台对85A 和 85B 蛋白的氨基酸序列展开分析，去除跨膜区和信号肽结构，进行密码子优化，成功构建 pET-30a-MAP-85A 和 pET-30a-MAP-85B 重组质粒。以 BL21（DE3）作为表达菌株进行原核表达，优化诱导表达的温度、IPTG 浓度及诱导时间；对诱导表达蛋白进行可溶性分析，利用镍柱进行纯化；用 Western blotting 技术评估重组蛋白的免疫反应性。
    结果 成功获得重组 85A 蛋白，其分子量约为 31.3 kDa，在 37 ℃、IPTG 终浓度 1.0 mmol·L−1诱导 4 h，可达到最佳表达效果；重组 85B 蛋白分子量为 31.8 kDa，在 37 ℃、IPTG 终浓度 0.8 mmol·L−1 诱导 6 h 表达量最好。两种重组蛋白均以包涵体形式表达，经镍离子柱纯化后获得高纯度目标蛋白。以 MAP 阳性牛血清为一抗，对纯化后蛋白进行 Western blotting 检测，结果显示特异性条带，充分证实 85A 和 85B 重组蛋白具备良好的免疫反应性。
    结论 研究成功实现副结核分枝杆菌重组 85A 和 85B 蛋白的高效表达，且有较好的免疫反应性，为该菌免疫学检测技术的开发提供重要的理论基础。

     

    Abstract:
    Objective Bioinformatics of 85A and 85B of Mycobacterium avium subsp. paratuberculosis (MAP) were studied, and the proteins obtained by prokaryotic expression technology to determine the reactogenicity for the development of an ELISA detection kit and vaccine.
    Methods The amino acid sequences of 85A and 85B were secured using an online bioinformatics platform. The transmembrane region and signal peptide structure were removed, and the codons optimized. pET-30a-MAP-85A and pET-30a-MAP-85B recombinant plasmids were successfully constructed. Using BL21 (DE3), the induction time, temperature, and IPTG concentration for the prokaryotic expression were optimized. The solubility of the proteins with the induced expression was analyzed, the purification conducted on nickel columns, and the immunoreactivity evaluated by western blotting.
    Results The recombinant 85A was successfully obtained showing a molecular weight of approximately 31.3 kDa. An optimal expression was achieved after 4 h of induction at 37 ℃ with a final IPTG concentration of 1.0 mmol·L−1. The recombinant 85B had a molecular weight of 31.8 kDa exhibiting an optimal expression after 6 h of induction at 37 ℃ with 0.8 mmol·L−1 of IPTG. Both recombinant proteins were expressed in the form of inclusion bodies and high-purified on nickel ion columns. Using the MAP-positive bovine serum as primary antibody, the immunoreactivity of the purified proteins was verified by western blot.
    Conclusion The recombinant 85A and 85B were successfully expressed with confirmed immunoreactivity for the development of immunological detection on M. avium subsp. paratuberculosis.

     

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