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目的表达香港海鸥菌(Laribacter hongkongensis)分子伴侣蛋白HdeA并鉴定其保护功能,为解析其抗酸机制奠定基础。方法通过与相关蛋白进行同源性比较,在香港海鸥菌中找出编码HdeA的基因。以HdeA基因序列为基础,设计特异性引物,PCR扩增HdeA基因,并将其连接到表达载体pET28a,构建的重组质粒pET28a-HdeA转化大肠埃希菌BL21(DE3),得到重组菌BL/pET28a-HdeA,用IPTG诱导表达目的蛋白并利用镍柱亲和层析纯化HdeA蛋白,采用光散射法分析HdeA蛋白的保护功能。结果通过Blast分析,从香港海鸥菌中找到编码HdeA的基因。以合成的特异性引物扩增出约315bp的HdeA片段,连接到原核表达载体pET28a后转化大肠埃希菌BL21,经IPTG诱导成功表达出HdeA蛋白,其分子质量单位为14.1ku,与理论值相符合。经His-tag亲和层析纯化,获得纯度较高的重组HdeA蛋白。光散射法检测分析HdeA蛋白能够减轻酸性条件下ADH蛋白聚集。结论纯化的重组HdeA蛋白在体外具有抗酸功能,为研究香港海鸥菌的抗酸机制奠定了基础。
Objective To express HdeA, a molecular chaperone of Laribacter hongkongensis, and to identify its protective function in order to lay the foundation for its anti-acid mechanism. Methods HedA gene was identified in Hong Kong seagull by homology comparison with related proteins. Based on the HdeA gene sequence, specific primers were designed, HdeA gene was amplified by PCR and ligated into the expression vector pET28a. The constructed recombinant plasmid pET28a-HdeA was transformed into E. coli BL21 (DE3) to obtain recombinant BL / pET28a -HdeA. The target protein was induced by IPTG and the HdeA protein was purified by nickel affinity chromatography. The protective function of HdeA protein was analyzed by light scattering. Results By Blast analysis, the gene encoding HdeA was found from Hong Kong seagull. The HdeA fragment of about 315bp was amplified by a synthetic specific primer and ligated into the prokaryotic expression vector pET28a and transformed into Escherichia coli BL21. The HdeA protein was successfully induced by IPTG, and its molecular mass unit was 14.1ku. Compared with the theoretical value meets the. Purified by His-tag affinity chromatography to obtain higher purity recombinant HdeA protein. Light scattering detection of HdeA protein can reduce ADH protein aggregation under acidic conditions. Conclusion The purified recombinant HdeA protein has antacid function in vitro, which lays the foundation for studying the antacid mechanism of Hong Kong’s sea cucumber.