幽门螺杆菌VacA蛋白的纯化

Purification of VacA protein of Helicobacter pylori

目的对幽门螺杆菌(Hp)相对分子质量(Mv)95×103的VacA蛋白进行分离纯化,为进一步研究VacA的作用机制奠定基础.方法采用经硫酸铵沉淀及透析后得到的Hp CCUG17874菌株浓缩上清,应用吸附层析、疏水层析、阴离子交换层析及凝胶过滤等液相层析方法进行VacA蛋白的纯化.每步层析后得到的样品均检测蛋白浓度及空泡毒素活性并行SDS-PAGE电泳检测蛋白成分.结果纯化蛋白的Mr为95×103,电泳纯,具有明显的空泡毒性,比活力达20480,与上清原液相比纯化倍数提高了4550倍,产率约为5 μg/L.结论采用吸附层析、疏水层析、离子交换层析及凝胶过滤的纯化流程,可以成功分离纯化Hp液体培养上清液中的VacA,纯化的VacA蛋白可用于Hp致病机理的研究.

Objective To separate and purify the VacA protein (molecular weight of 950000)of H.pylori for the research on machanism of vacuolating toxicity. Methods The purification of VacA involved ammonium sulfate precipitation of proteins present in broth culture supernatant of CCUG17874 strains, followed by hydroxyapatite, hydrophobic interactive, anion exchange and gel filtration chromatography. After every step the protein concentration and toxicity were detected, and SDS-PAGE was also performed. Results The 5ml dialyzed sapmple from one liter culture was applied to a hydroxyapatite column. After washing with 10mmol/L phosphate buffer at a flow rate of 0.35ml/min, VacA was eluted in the first peak, the max absorbance of which was 278mAu.Protein in the sample was then precipitated with a 50%saturated solution of ammonium sulfate, and dialyzed in Tris buffer. Hydrophobic interactive chromatography was performed on a phenyl-Sepharose CL-4B column with the same buffer containing 0.5mol/L(NH_4)_2SO_4, and proteins containing VacA were eluted with 60mmol/L Tris. Anion exchange chromatography was performed on a DEAE-sepharose FF column. All the five samples have vacuolating activity after anion exchange were collected and concentrated, then applied to Superdex 200 column.Purified VacA was eluted and collected with a volume of 200 μ1(124.5μg/ml).The specific activity ratio of purified VacA reached 20480(2560/0.125), and the purification fold was 4551. The recovery of purified toxin was 5μg per liter of culture supernatant Conclusion VacA can be purified through hydroxyapatite, hydrophobic interactive, anion exchange and gel filtration chromatography from the supematants of H.pylori.