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目的原核表达并纯化幽门螺杆菌双组分系统耐酸相关蛋白ArsS,为深入研究其功能及其在幽门螺杆菌耐酸机制中的作用奠定基础。方法以幽门螺杆菌菌株26695基因组为模板,采用PCR法扩增ArsS基因,插入pET-22b(+)载体,构建重组原核表达质粒pET-22b(+)-ArsS,转化E.coliBL21(DE3),0.5mmol/LIPTG25℃诱导表达,并采用亲和层析与分子筛层析对重组蛋白进行纯化。结果重组原核表达质粒pET-22b(+)-ArsS经双酶切及测序鉴定,证明构建正确;重组蛋白以可溶性形式表达,表达量占菌体总蛋白的30%以上;分子筛层析图谱显示,在150mmol/LNaCl条件下,目的蛋白层析效果较好,纯化后的重组蛋白纯度可达95%以上,浓度约为10mg/ml。结论已成功原核表达并纯化获得了高纯度的ArsS蛋白。
Objective To prokaryotic express and purify Helicobacter pylori two-component acid-tolerant protein ArsS in order to further study its function and its role in Helicobacter pylori acid resistance. Methods The Helicobacter pylori strain 26695 was used as a template to amplify ArsS gene by PCR and inserted into pET-22b (+) vector to construct recombinant prokaryotic expression vector pET-22b (+) - ArsS. The recombinant was transformed into E. coli BL21 The recombinant protein was induced by 0.5mmol / L IPTG at 25 ℃. The recombinant protein was purified by affinity chromatography and molecular sieve chromatography. Results The recombinant plasmid pET-22b (+) - ArsS was confirmed by double enzyme digestion and sequencing. The recombinant protein was expressed in soluble form and accounted for more than 30% Under the condition of 150mmol / L NaCl, the target protein was better, and the purity of purified recombinant protein was more than 95% with a concentration of about 10mg / ml. Conclusion High purity ArsS protein was obtained by prokaryotic expression and purification.