幽门螺杆菌双组分系统耐酸相关蛋白ArsS的原核表达及纯化  

Prokaryotic Expression and Purification of Acid-resistance-associated Two Component System Protein ArsS from Helicobacter pylori

作　　者：张晓丽[1] 章金勇[1] 邹全明[1] 

机构地区：[1]第三军医大学临床微生物与免疫学教研室重庆市生物制药工程技术研究中心,重庆400038

出　　处：《中国生物制品学杂志》2010年第8期813-815,820,共4页Chinese Journal of Biologicals

摘　　要：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 express the acid-resistance-associated two component system protein ArsS of Helicobacter pylori in prokaryotic cells, purify the expressed product and lay a foundation of further study on its function as well as its role in acid-resistance mechanism of H. pylori. Methods ArsS gene was amplified by PCR using the genome of H. pylori isolate 26695 as a template and inserted into vector pET-22b（＋）. The constructed recombinant plasmid pET-22b（＋）-ArsS was transformed to E. coli BL21（DE3）for expression under induction of 0. 5 mmol / L IPTG at 25℃. The expressed product was purified by affinity chromatography and molecular sieve chromatography. Results Both restriction analysis and sequencing proved that recombinant plasmid pET-22b（＋）-ArsS was constructed correctly. The expressed product, in a soluble form, contained more than 30% of total somatic protein, and was effectively purified by molecular sieve chromatography using a buffer containing 150 mmol / L sodium chloride. The purified protein, at a concentration of about 10 mg / ml, reached a purity of more than 95%. Conclusion ArsS protein was successfully expressed in prokaryotic cells and purified.

关 键 词：幽门螺杆菌 耐酸 双组分系统 基因表达 

分 类 号：R378[医药卫生—病原生物学] Q78[医药卫生—基础医学]