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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。
OBJECTIVE: To clone and express APH0653 gene of Anaplasma phagocytophilum (AP), and to analyze its expression product. Methods: Using phagemid phage display genomic DNA as template, APH0653 gene was amplified by PCR and cloned into prokaryotic expression vector for expression using specific primers. The recombinant protein was purified by nickel column affinity chromatography and the immunoreactivity of APH0653 and phagocyte-free phage sera was detected by Western blotting. Results: The colony PCR, DNA sequencing and SDS-PAGE gel electrophoresis indicated that the APH0653 gene was cloned into the prokaryotic expression vector and induced the expression of the recombinant protein. Western blotting experiments showed that AP-positive serum can recognize the recombinant protein APH0653, and produce significant specific bands. CONCLUSION: APH0653, an asexual phagocyte-like protein, can be efficiently expressed in prokaryotic expression system, and the recombinant protein has good antigenicity.