一种谷氨酸脱羧酶65相关肽融合蛋白的制备及其治疗1型糖尿病的药效研究

Genetic Fusion of Glutamic Acid Decarboxylase 65 Derived Peptides to the B-Subunit of Cholera Toxin and Its Retardation Effect of Diabetes in NOD Mice

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。

Mucosally induced tolerance is an attractive strategy for immunotherapy of autoimmune diseases. In this study, A recombinant expression plasmid pET28a-CTB-GADⅢ was construcated in which glutamic acid decarboxylase 65 derived peptides （p217 -236, p524- 538, p290 306） genes were fused to the 3＇ end of cholera toxin B subunit gene. Subsequently the recombinant plasmid was transformed into E. coli strain BL21 （DE3）. The recombinant protein CTB GADⅢ was expressed and accumulated as inclusion bodies after being induced with 5 mmol/L lactose for 5 h. Purificated recombinant protein was obtained after washing, renaturing and purifying via CMS2-cellulose and sephadex G100 chromatography. The protein was further analyzed for GM1 binding activity in a GM1-ELISA. It showed good GMl-binding, although not as strong as cholera toxin B subunit. In addition, the CTB-GADⅢ fusion protein induces efficient immunosuppression after nasal administration of nonobese diabetic mice, raising the possibility of using such construct in the treatment of type 1 diabetes.