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

Abstract:

Objective：To lay a foundation for screening novel immunomodulator for type 1 diabetes mellitus by generating human Zinc transporter 8（of ZnT-8，a novel type 1 diabetes autoantigen） recombinant protein using a prokaryotic expression system. Methods：The COOH-terminal cDNA of the human ZnT-8 gene was subcloned into the pET-32a vector and transformed into competent Es－cherichia coli（E.coli） BL21 cells. After transformation E.coli was induced using isopropyl β-D- 1-thiogalactopyranoside；human re－combinant of ZnT-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 di－vided into 2 groups in random. In ZnT-8 recombinant protein group，mice were given recombinant protein of ZnT-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 cD－NA of the carboxyl terminal of ZnT-8（306 bp） was successfully amplified by PCR. The sequence matched completely with the se－quence of ZnT-8-COOH in GeneBank. The recombinant ZnT-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 ZnT-8 recombinant protein group was higher at 28 weeks old（P<0.05）；the glucose levels in ZnT-8 recombinant protein group were lower at 28 weeks old（P<0.05）；the serum C-peptide levels in ZnT-8-COOH group were higher at 28 weeks old（P<0.05）. Conclusion：Human recombinant of ZnT-8-COOH protein is successfully prepared. The recombinant protein can be used as immunomodulator in type 1 diabetes mellitus.