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目的构建铜绿假单胞菌凝集素PA-ⅠL基因的重组表达质粒,在大肠杆菌BL21(DE3)中高效表达并评价其生物学活性。方法以铜绿假单胞菌PAO1基因组为模板设计引物,构建p ET-28a(+)-PA-ⅠL重组表达质粒,转化至大肠杆菌中诱导表达,用Ni亲和层析法对目的蛋白进行纯化。流式细胞术评价重组表达的PA-ⅠL蛋白对细胞表面Gb3/CD77的结合活性,菌落平板计数法评价重组蛋白在细菌黏附宿主细胞过程中的功能。结果与结论 SDS-PAGE电泳显示,纯化后的PA-ⅠL蛋白具有较高的纯度,重组表达的PA-ⅠL蛋白能与细胞表面的糖鞘脂Gb3/CD77结合,且呈浓度依赖性抑制PA的PAO1对宿主细胞的黏附。
Objective To construct a recombinant plasmid expressing Pseudomonas aeruginosa agglutinin PA-ⅠL and express it in E. coli BL21 (DE3) and evaluate its biological activity. Methods The PAO1 genome of Pseudomonas aeruginosa was used as a template to design the recombinant plasmid p ET-28a (+) - PA-ⅠL. The recombinant plasmid was transformed into E. coli and induced to express. The target protein was purified by Ni affinity chromatography . Flow cytometry was used to evaluate the binding activity of the recombinant PA-I protein to Gb3 / CD77 on the cell surface. Colony-counting assay was used to evaluate the function of the recombinant protein in bacterial adhesion to host cells. RESULTS AND CONCLUSION SDS-PAGE electrophoresis showed that purified PA-ⅠL protein had high purity. Recombinant PA-ⅠL protein could bind to glycosphingolipid Gb3 / CD77 on cell surface and inhibit PA in a concentration-dependent manner PAO1 adhesion to host cells.