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本研究根据Ct Ahpc基因序列设计特异性引物,以Ct DNA为模版PCR扩增Ahpc基因到大小为585 bp,将其连接到p ET28a获得重组质粒p ET28a-Ahpc,将该质粒转化到大肠杆菌BL21(DE3)获得重组菌BL/p ET28a-Ahpc,利用IPTG诱导重组Ahpc蛋白表达,目的蛋白分子量大小约为26 k D。利用Ni-NAT亲和层析法纯化得到的重组Ahpc蛋白。氧化铁二甲酚橙法检测发现Ahpc蛋白能够分解双氧水和叔丁基过氧化氢,具有分解过氧化合物的活性。测定重组大肠杆菌在百草枯所致氧化胁迫下的生长曲线,发现表达Ahpc蛋白的重组菌的生长情况比对照好。本研究成功表达和纯化得到沙眼衣原体Ahpc蛋白并测定其活性,为解析沙眼衣原体的抗氧化机制提供实验证据。
In this study, specific primers were designed according to the Ct Ahpc gene sequence. The Ahpc gene was amplified by Ct DNA as a template to a size of 585 bp and ligated into p ET28a to obtain a recombinant plasmid pET28a-Ahpc. The plasmid was transformed into Escherichia coli BL21 (DE3) to obtain the recombinant baculovirus BL / p ET28a-Ahpc. The recombinant protein Ahpc was induced by IPTG. The molecular weight of the target protein was about 26 kD. Recombinant Ahpc protein was purified by Ni-NAT affinity chromatography. Oxidation of xylenol orange assay found that Ahpc protein can decompose hydrogen peroxide and t-butyl hydroperoxide, has the activity of decomposition of peroxo compounds. The growth curve of recombinant Escherichia coli under oxidative stress induced by paraquat was determined and the growth of the recombinant bacteria expressing Ahpc protein was found to be better than that of the control. In this study, Ahpc protein of Chlamydia trachomatis was successfully expressed and purified, and the activity of Ahpc protein was determined. The results provide experimental evidence for the anti-oxidation mechanism of Chlamydia trachomatis.