异源表达的幽门螺杆菌cag4蛋白有溶菌糖基转移酶活性

Heterogenous expression and characterization of cag4 in Helicobacter pylori

摘要:

摘要【目的】构建融合基因原核表达载体pET-28a- cag4，并表达重组融合蛋白cag4，分析重组融合蛋白的酶活性，为新型抗生素（或是抗菌药物）的研发提供作用靶位。【方法】本研究利用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】在大肠埃希菌 BL21(DE3)中获得高效表达的重组蛋白; 经SDS-PAGE和Western Bolt鉴定表达后，采用Ni2＋-NTA柱在变性条件下纯化，并进行透析复性处理。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，表明目的蛋白具有明显的肽聚糖水解活性; 通过监测浊度下降速率，比较其在不同pH条件下活性的变化，即?A/（min?mg protein），结果表明，幽门螺杆菌cag4蛋白具有溶菌糖基转移酶活性。【结论】幽门螺杆菌cag4蛋白具有溶菌糖基转移酶活性。

Abstract:

Abstract:［Objective］To construct prokaryotic fusion gene expression vector pET-28a- cag pathogenicity island protein 4(cag4)，express the recombinant fusion protein cag4 and analyze the enzyme activity of recombinant protein.［Methods］We cloned cag4 gene, this encoded lytic transglycosylase in CagPAI of Helicobacter pylori NCTC11637 via PCR method. After TA cloning and restriction enzyme digested confirmed, we constructed prokaryotic expression plasmid pET-28a- cag4 and transformed the plasmid into E.coli BL21 (DE3) for heterogenesis expression after it is sequenced. We performed SDS-PAGE and Western blot. We purified and collected the recombinant protein by using Ni2＋-NTA columns under denaturation condition.We renatured the recombinant protein via dialysis. Besides, we isolated peptidoglycan from Micrococcus lysodeikticus by SDS boiling method.We detected the enzyme activity of recombinant protein by zymogram analysis and the effect of pH values by turbidimetric analysis.［Results］The recombinant protein has the enzyme activity of lytic transglycosylase.The enzyme activity, which defined as the rate of degraded (DA?min-1mg-1), was higher at pH 6.0 group than other groups (pH 5.0 and 7.0).［Conclusion］The cag4 of Helicobacter pylori NCTC11637 has the enzyme activity of lytic transglycosylase.