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使用基因工程方法构建了霍乱毒素B亚单位(Cholera toxin B subunit,CTB)与谷氨酸脱羧酶65(glutamic acid decarboxylase65,GAD65)串联三肽GADⅢ(包括p217—236,p524—538,p290—306)的融合基因CTB-GADⅢ。将融合基因克隆到大肠杆菌表达载体pET-28a中,获得的重组质粒转化大肠杆菌BL21(DE3)。重组菌株经乳糖诱导后,其表达产物经过15%SDS-PAGE分析表明该菌株可以以包涵体形式表达融合蛋白,Mr约为17.6k。含有CTB-GADⅢ重组蛋白的包涵体经过变性、复性、纯化后,可以得到五聚体结构的CTB-GADⅢ。神经节苷脂GM1(monosialoganglioside)结合实验表明重组CTB-GADⅢ蛋白可以与GM1特异性结合,表明该融合蛋白保持了CTB形成五聚体的生物活性。使用该重组蛋白在NOD小鼠8周龄、10周龄和12周龄时滴鼻免疫小鼠共3次,可以显著降低小鼠的发病率,达到治疗1型糖尿病的作用。
The cholera toxin B subunit (CTB) and glutamic acid decarboxylase 65 (GAD65) tandem tripeptide GADⅢ (including p217-236, p524-538, p290-306 ) Fusion gene CTB-GADIII. The fusion gene was cloned into E. coli expression vector pET-28a, and the recombinant plasmid obtained was transformed into E. coli BL21 (DE3). After the recombinant strain was induced by lactose, its expressed product was analyzed by 15% SDS-PAGE. The result showed that the recombinant strain could express the fusion protein in the form of inclusion body. Mr was about 17.6k. Inclusion bodies containing CTB-GADIII recombinant protein can be denatured, renatured and purified to obtain pentameric CTB-GADIII. GM1 (monosialoganglioside) binding assay showed that the recombinant CTB-GADIII protein can specifically bind to GM1, indicating that the fusion protein retains the biological activity of CTB forming pentamers. Immunization of mice with NOD mice at 8 weeks, 10 weeks and 12 weeks of age with NO 3 mice for 3 times in total can significantly reduce the incidence of mice and achieve the goal of treating type 1 diabetes.