摘要
目的:构建幽门螺杆菌cag4蛋白的原核重组体系,表达、纯化融合蛋白。方法:利用PCR技术从幽门螺杆菌NCTC11637中克隆了cag4基因;经T-A克隆,酶切鉴定,构建了原核表达载体pET-28a-cag4;经测序鉴定正确后,转化进入大肠埃希菌BL21(DE3)进行异源表达。利用IPTG体外诱导后,经SDS-PAGE和Western bolt鉴定目的蛋白表达后,采用Ni2+-NTA柱在变性条件下纯化目的蛋白,并对重组蛋白进行透析复性处理。将SDS煮沸法获得的溶壁微球菌肽聚糖掺入SDS-PAGE作为底物,进行酶谱分析。结果:成功扩增了cag4基因基因;重组质粒在大肠埃希菌BL21(DE3)中诱导表达出pET-28a-cag4融合蛋白;优化了pET-28a-cag4原核表达体系的最适条件;Western bolt分析结果显示表达的基因重组蛋白与目的蛋白相符;酶谱分析显示其具有明显的肽聚糖水解活性。结论:幽门螺杆菌cag4蛋白具有溶菌糖基转移酶活性。
Objective：To construct a recombination prokaryotic system of cag pathogenicity island protein 4（cag4） for expression and purifica-tion of the fusion protein.Methods：PCR technique was used to amplify cag4 gene encoding lytic transglycosylase in CagPAI of helicobacter pylori NCTC11637.By TA cloning and restriction enzyme digested,we constructed prokaryotic expression plasmid pET-28a-cag4 and transformed the plasmid into E.coli BL21（DE3） for heterogenesis expression after sequenced.SDS-PAGE and Western blot were performed after induced by IPTG in vitro.We purified and collected the recombinant protein by using Ni2＋-NTA columns under denaturation condition.The renaturation of the recombinant protein was performed via dialysis treatment.Besides these,we isolated peptidoglycan from Micrococcus lysodeikticus by SDS boiling method.The enzyme activity of recombinant protein was detected by zymogram analysis and the effect of pH values by turbidimetric analysis.Results：The cag4 gene was obtained successfully.E.coli BL21 strains which harbored the recombinant plasmid could express cag4 fusion protein.The inducing conditions of prokaryotic expression were optimized.The expressing protein was really identical to cag4 protein by Western blot.The enzyme activity,which defined as the rate of degraded（ΔA·min-1mg-1）,was higher at pH 6.0 group than other groups（pH5.0 and 7.0）.Conclusion：The cag4 in Helicobacter pylori possesses the enzyme activity of lytic transglycosylase.
出处
《中国免疫学杂志》
CAS
CSCD
北大核心
2011年第4期347-351,共5页
Chinese Journal of Immunology
基金
国家自然科学基金项目(30870096)
江苏省高校自然科学基金项目(08KJB310001)
江苏大学临床医学科技发展基金项目(JLY20080051)资助
关键词
幽门螺杆菌cag4蛋白
原核表达
纯化
融合蛋白
cag4 in Helicobacter pylori； Prokaryotic expression； Purification； Fusion protein
