人ZnT-8作为1型糖尿病新型免疫调节剂的研究

Study of human ZnT-8 as novel immunomodulator for type 1 diabetes mellitus

目的:构建人锌转运体8(Zinc transporter 8,Zn T-8)羧基端原核表达载体,表达和纯化人Zn T-8羧基端(Zn T-8-COOH)重组蛋白,为寻找1型糖尿病新型免疫调节剂创造条件。方法:人Zn T-8-COOH c DNA克隆入p ET-32a载体,在大肠杆菌BL21中利用异丙基-硫代-β-D-半乳糖苷(isopropyl-thio-β-D-galactoside,IPTG)诱导表达,经十二烷基硫酸钠-聚丙烯酰胺凝胶电泳和Western blot鉴定表达产物,镍离子亲和层析纯化重组蛋白。40只4周龄的雌性非肥胖型糖尿病(non obese diabetic,NOD)小鼠随机分为Zn T-8重组蛋白预处理组及生理盐水对照组,分别腹腔注射Zn T-8重组蛋白和生理盐水,每周测量各组NOD小鼠体质量,检测血糖、血清C肽水平。结果:经PCR扩增后得到306 bp的DNA片段,成功构建到p ET-32a表达载体上,经测序分析序列正确;Zn T-8重组蛋白在大肠杆BL21中获得成功表达,并得以有效纯化。Zn T-8重组蛋白预处理组NOD小鼠与生理盐水对照组相比,28周龄时Zn T-8重组蛋白预处理组的体质量高于生理盐水对照组(P<0.05)。28周龄时Zn T-8重组蛋白预处理组的血糖低于生理盐水对照组(P<0.05);28周龄时Zn T-8重组蛋白预处理组的血清C肽水平高于生理盐水对照组(P<0.05)。结论:成功制备人Zn T-8-COOH重组蛋白,并初步证实了其对1型糖尿病的免疫调节作用。

Objective：To lay a foundation for screening novel immunomodulator for type 1 diabetes mellitus by generating human Zinc transporter 8（of Zn T-8,a novel type 1 diabetes autoantigen） recombinant protein using a prokaryotic expression system. Methods：The COOH-terminal c DNA of the human Zn T-8 gene was subcloned into the p ET-32 a vector and transformed into competent Escherichia coli（E.coli） BL21 cells. After transformation E.coli was induced using isopropyl β-D- 1-thiogalactopyranoside;human recombinant of Zn T-8 protein was extracted,purified by Nickel sepharose affinity chromatography,and was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot. Forty female non obese diabetic（NOD） mice of four weeks old were divided into 2 groups in random. In Zn T-8 recombinant protein group,mice were given recombinant protein of Zn T-8;in control group,mice were given saline. The body mass,blood glucose levels and serum C-peptide levels were recorded every week. Results：The c DNA of the carboxyl terminal of Zn T-8（306 bp） was successfully amplified by PCR. The sequence matched completely with the sequence of Zn T-8-COOH in Gene Bank. The recombinant Zn T-8-COOH was successfully expressed in a soluble form in E.coli BL21 and purified. Compared with those of control group,the body mass in Zn T-8 recombinant protein group was higher at 28 weeks old（P0.05）;the glucose levels in Zn T-8 recombinant protein group were lower at 28 weeks old（P0.05）;the serum C-peptide levels in Zn T-8-COOH group were higher at 28 weeks old（P0.05）. Conclusion：Human recombinant of Zn T-8-COOH protein is successfully prepared. The recombinant protein can be used as immunomodulator in type 1 diabetes mellitus.