Expression Of Human Globular Adiponectin-glucagon-like Peptide-1 Analog Fusion Protein And Its Hypoglycemic Effect Assay

Preface:Adiponectin is a protein produced and secreted by adipose tissue that associated with insulin sensitivity, antiinflammatory, metabolic and cardiovascular disease. C-terminal globular domain (the globular domain of adiponectin, Globular Adiponectin, gAd,108-244aa) has been confirmed to have greater potency than full-length adiponectin. Glucagon-like peptide-1 (GLP-1) is a hormone secreted by pancreatic islet cells and intestinal L cells. It is demonstrated that the N-terminal region is important for the biological activity by binding with GLP-1 receptor in a glucose-dependent insulinotropic manner to promote insulin synthesis and release. It enhances glucose-dependent insulin secretion, promotesβ-cell proliferation, suppresses glucagon secretion, reduces hepatic glucose production and food intake, lowers postprandial glucose excursions and so on. However, GLP-1 has a short half-life in vivo. It's cleared by two forms:enzymatic removal and organ removal.By gene amplication and recombination, prokaryotic expression vector PET28a- gAdiponectin-GLP-1-A was constructed by connecting two genes coding globular adiponectin and GLP-1-A (the eighth amino acid alanine codon GCT is replaced by glycine codon GGT) together. Based on the preliminary studies, a further study to obtain gAdiponectin-GLP-1-A (gAd-GLP-1-A) fusion protein is carried out. The protein was expressed in BL21 (DE3), purified by High-Affinity Ni-IDA Resin, refolded in urea gradient (6,4,2,1 and 0 mol/L) refolding buffer, digested by enterokinase and at last its hypoglycemic activity was demonstrated.Materials and methodsFirst, acquisition of human globular adiponectin-glucagen-like peptide-1 analogues fusion proteinThe recombinant plasmids were checked by Sequencing (Nanjing Ginster Technology Co, Ltd, Nanjing China). The correct ones were transformed into E. coli BL21 (DE3) to express globular adiponectin-glucagen-like peptide-1 analogues fusion protein. The supernatant and precipitate was separated by ultrasound. The precipitate was collected, washed, dissolved and then purificated by Ni-IDA resin. The protein was refolded in bag filter in the urea solution by the gradient method. The histidine tag in the protein was removed by enterokinase. At last, the protein were collected and analyzed by SDS-PAGE and Western Blot.Second, diabetic mice model construction and gAd-GLP-1-A fusion protein hypoglycemic activity assayStreptozotocin was used to establish diabetic mice model. Mice were divided into normal control group, diabetes control group and diabetes treatment group. The diabetes treatment group was injected by gAd-GLP-1-A fusion protein. The normal control group and diabetic control group were injected by the same amount of saline respectively. At last, the blood glucose was observed at 30min, 1h,1.5h,2h,2.5h and 3h. ResultsFirst, SDS-PAGE analysis revealed that most protein with an expected molecular weight of 25 KD exists in precipitate in inclusion body. It's also be proved by Western blot with His tag monoclonal antibody.Second,12% SDS-PAGE and Western Blot analysis as to fractions containing pure target protein showed that the fusion protein was purificated.Third,12% SDS-PAGE and Western Blot analysis as to protein after enterokinase digestion showed that the His tag is removed successfully by enterokinase.Fourth, data obtained from the mice experiments showed that compared with the normal control group and the diabetes control group injected with normal saline, the treatment group injected with fusion protein was found blood glucose significantly lowering at 2h,2.5h,3h.ConclusionsFirst, most fusion protein presents in inclusion body.Second, the fusion protein is purificated and refolded.Third, the His tag is successfully removed by enterokinase.Fourth, data obtained from the mice experiments showed the hypoglycemic activity of gAd-GLP-1-A fusion protein.