| 徐文婷,贺美玲,张 磊,黄 瑞,牛 华.嗜吞噬细胞无形体aph0653的克隆表达及重组蛋白的抗原性分析[J].现代生物医学进展英文版,2017,17(1):20-23. |
| 嗜吞噬细胞无形体aph0653的克隆表达及重组蛋白的抗原性分析 |
| Recombinant Expression of Anaplasma Phagocytophilum APH0653 and Determination of its Antigenicity |
| Received:May 20, 2016 Revised:June 12, 2016 |
| DOI：10.13241/j.cnki.pmb.2017.01.005 |
| 中文关键词: 人粒细胞无形体病 嗜吞噬细胞无形体 APH0653 抗原性 |
| 英文关键词: Human granulocytic anaplasmosis Anaplasma phagocytophilum APH0653 Antigenicity |
| 基金项目:国家自然科学基金项目（31470276） |
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| 中文摘要: |
| 摘要 目的：克隆表达嗜吞噬细胞无形体（Anaplasma phagocytophilum，AP）APH0653基因，并对表达产物进行抗原性分析。方法：以嗜吞噬细胞无形体基因组DNA为模板，使用特异性引物，PCR扩增APH0653基因并克隆入原核表达载体进行表达。使用镍柱亲和层析纯化重组蛋白，并应用免疫印迹方法检测APH0653与嗜吞噬细胞无形体感染血清的免疫反应性。结果：菌落PCR、DNA测序和SDS-PAGE蛋白凝胶电泳表明APH0653基因已成功克隆入原核表达载体，并可诱导表达重组蛋白。免疫印迹实验表明AP阳性血清可识别重组蛋白APH0653，并产生明显的特异性条带。结论：嗜吞噬细胞无形体APH0653蛋白可在原核表达系统中高效表达，且重组蛋白具有较好的抗原性。 |
| 英文摘要: |
| ABSTRACT Objective: Recombinant expression of Anaplasma phagocytophilum APH0653 and determination of its antigenicity. Methods: Bioinformatical analysis of APH0653 amino acid sequence was performed. DNA fragment encoding APH0653 was amplified by PCR using A. phagocytophilum genomic DNA as template, followed by ligation with prokaryotic vector pET28a(+) after double di- gestion with endonucleases. After the inserted DNA sequence was confirmed by colony PCR and sequencing analysis, the recombinant plasmid was transformed into Escherichia coli BL21(DE3) for protein expression. After purification by Nickel affinity chromatography, the recombinant APH0653 was subjected to western blot analysis to determine its antigenicity. Results: Bioinformatical analysis results showed that APH0653 is a hydrophilic protein with 5 antigenic determinants. The molecular mass of this protein was computed to be 18.5 kDa. The PCR product encoding APH0653 was cloned into pET28a(+) successfully as verified by colony PCR and sequencing anal- ysis. Recombinant APH0653 was highly expressed in E. coli BL21(DE3) after induction with IPTG, and purified with Nickel affinity chromatography. Western blot analysis showed that APH0653 strongly reacted with A. phagocytophilum positive sera. Conclusion: A. phagocytophilum APH0653 was highly expressed in a prokaryotic expression system. In addition, APH0653 is a protein with strong anti- genicity. |
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