霍乱毒素B亚单位与p277融合基因在大肠杆菌中表达及免疫分析

为了增强多肽表位的免疫原性,构建了霍乱毒素B亚单位(Cholera toxin B subunit,CTB)和p277的融合基因CTB-p277,将该融合基因克隆到pET28a( )中,获得原核表达载体pET28a-CTB-p277;并将该质粒转入大肠杆菌菌株BL21(DE3)中;重组菌株经2%的乳糖诱导5 h后的表达产物用12%的SDS-PAGE分析表明可以表达融合蛋白,融合蛋白占全菌蛋白约40%,且主要以包含体的形式存在。包含体经洗涤、裂解,用DEAE纤维素离子交换纯化,可以获得99.1%的重组蛋白。重组蛋白的包含体经复性后,在体外可以自组装成五聚体。重组蛋白免疫KK-Ay小鼠后,ELISA的分析表明可以产生抗p277的抗体。血糖和体重的测定结果显示,给药组小鼠的血糖有明显的降低,体重也有相应的减轻。同时GM1-ELISA的检测表明,CTB-p277在体外可以和神经节苷脂GM1(Monosialoganglioside)特异结合,具有生物活性。

Expression of CTB-p277 Fusion Gene in E. coli and Analysis of ITS Immunogenecity

To enhance the immunogenicity of polypepetide epitopes,a fusion gene CTB-p277,in which p277(from Shock heat protein 60 of human) was fused to the 3' end of CTB gene,was constructed and cloned into pET28a( ) to obtain a prokaryotic expression vector pET28a-CTB-p277.Subsequently the recombinant plasmid pET28a-CTB-p277 was transformed into E.coli strain BL21(DE3).After being induced with 2% lactose for 5h,the expression product was analyzed by sodium dodecyl sulphate-polyacrylamide gel(12%) electrophoresis(SDS-PAGE),and the results indicated that the recombinant protein CTB-p277 was expressed and accumulated as inclusion bodies.The recombinant CTB-p277 protein accumulated to the level of 40% total bacterial protein.About 99.1% purity of the recombinant protein was obtained following washing,denaturing and purifying via DEAE-cellulose chromatography.After the inclusion bodies were refolded in vitro,the recombinant protein represented assembled pentamers.After mucous immunity for KK-Ay mice with CTB-p277 fusion protein,the results of ELISA analysis demonstrated that the fusion protein could be recognized with the anti-p277 antibody;blood glucose concentration and body mass were decreased correspondingly.In addition,the results of the GM1-ELISA assay showed that the protein could bind to monosialoganglioside specifically.