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Zinc transporter 8 polyclonal antibody and Functional Analysis

Author: LiuBaoYing
Tutor: ChenBing
School: Third Military Medical University
Course: Internal Medicine
Keywords: Zinc transporter 8 T2DM Prokaryotic expression Polyclonal antibody Pancreas Adipose tissue Exendin-4
CLC: R587.1
Type: PhD thesis
Year: 2010
Downloads: 48
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Abstract

I. Background Diabetes absolute lack of insulin secretion or peripheral tissue is characterized by reduced sensitivity to insulin. From the genetic and environmental risk factors in diabetes plays an important role. Since the 1934 discovery of zinc is a component of insulin crystals since the relationship between zinc and diabetes research concern. Many studies have tried to elucidate the role of zinc in diabetes, in order to further reveal the pathogenesis of diabetes and the development of new therapeutic drugs. Various tissues and organs in the body, the highest concentrations of zinc in pancreatic islets, and research has shown that islet zinc content reduces the incidence of diabetes is closely related. Previous studies show that there are two families involved in the regulation of intracellular zinc levels. One is ZnT (zinc transporter) family, encoded by the gene SLC30A it by making a cytosolic zinc into the cells or intracellular organelles and reduce the concentration of zinc. Another is the ZIP (zinc-binding protein) family SLC39A encoded by genes, it is the role of zinc into the extracellular or intracellular organelles in the transfer into the cytosol and zinc increase the zinc concentration in the cytoplasm. So far, it has been found 10 ZnT family members and 15 ZIP family members. Zinc transporter 8 (zinc transporter 8, ZnT8) is one of ZnT family members, it is considered the islet β cell-specific zinc transporter. Recent studies have shown that ZnT8 also expressed in adipose tissue, peripheral blood lymphocytes, thyroid and adrenal glands. Several studies have shown that ZnT8 single nucleotide polymorphisms (single nucleotide polymorphism, SNP) rs132266634 and type 2 diabetes (type 2 diabetes, T2DM) association. This SNP rs132266634 danger bases and intravenous glucose tolerance test in reduced insulin secretion association; and proinsulin to insulin conversion process barriers associated; associated with elevated fasting glucose. Therefore, ZnT8 can be used as genetic markers of T2DM. In addition, recent studies have found ZnT8 is type 1 diabetes autoantigen one. The SNPrs132266634 about ZnT8 associated with type 1 diabetes have been reported. Further study confirmed that the use of shRNA decreased islet β cells after expression ZnT8, β cells decreased insulin and glucose-stimulated insulin secretion (glucose-stimulated insulin secretion, GSIS) decreased. Furthermore ZnT8 knockout mice showed reduced plasma insulin and GSIS reduced, while overexpression ZnT8 of INS-1 (insulinoma cell line) cells increased GSIS. Previous studies have shown ZnT8 regulate insulin secretion may be a key factor, and may become the treatment of diabetes drug target. In addition to the pancreas, the fat tissue is another expression ZnT8 organization. Adipose tissue in which the expression of leptin with increasing zinc content, and vice versa. Leptin is a hormone secreted by fat cells, which the body regulating food intake and energy metabolism plays an important role. Glucagon-like peptide -1 (glucagon like peptide-1, GLP-1) with insulin secretion, inhibition of glucagon secretion, stimulate the pancreatic β cell proliferation and differentiation, inhibition of β cell apoptosis, inhibition of gastric emptying and so, in recent years become the treatment of diabetes drug research hotspot. Magnusson N and other studies have shown that GLP-1 can be increased INS-1 cells ZnT8 mRNA expression, but GLP-1 expression in vivo is still not clear how they affect ZnT8. Natural GLP-1 in vivo quickly dipeptidyl peptidase - Ⅳ (dipeptidylpeptidase Ⅳ, DPP-Ⅳ) degradation, its half-life is only a few minutes. Exendin-4 analogue is GLP-1, it is better stability in the body, with the GLP-1 receptor binding and play a hypoglycemic effect. Currently ZnT8 in T2DM pancreas and adipose tissue situation has not yet been reported. ZnT8 play in the pathogenesis of T2DM what role remains to be further studied. Second, the aim of the present study aims to investigate the role of ZnT8 in T2DM and its possible mechanism. Using molecular cloning techniques to build ZnT8 prokaryotic expression vector, for ZnT8 prokaryotic protein expression and purification. The purified protein to immunize New Zealand rabbits get ZnT8 polyclonal antibodies, and antibody purification. Select the db / db mice (T2DM mice) as the research object, from the mRNA and protein levels detected in the pancreas and adipose tissue ZnT8 expression. Exendin-4 also choose to intervene (db / db mice by intraperitoneal injection of Exendin-4), was observed after the intervention ZnT8 in the pancreas and adipose tissue changes. Third, the method 1. Based ZnT8 carboxyl terminus (ZnT8-C ,268-369 amino acids) gene sequence (GenBank NO: NM ₁ 73851.2) were designed with restriction sites BamHI and NotI primer to Chimienti kindly provided by Prof. p-ZnT8 vector as a template, PCR amplification ZnT8-C fragment and cloned into the prokaryotic expression vector pET32a, by sequencing correct, it will convert it into E. coli BL21 (DE3) pLysS, induced expression, Ni2 -NTA chromatography and identified with His antibody, obtain a purified recombinant protein ZnT8-C. The purified ZnT8-C recombinant protein emulsified with Freund's adjuvant, as a New Zealand rabbits were immunized to obtain rabbit antiserum was purified by saturated ammonium sulfate to obtain preliminary purified rabbit polyclonal antibody anti-ZnT8 through more indirect ELISA clonal antibody titer, Western blotting and immunohistochemistry preliminary testing its activity. (2) Appropriation db / db mice pancreas and adipose tissue total RNA and total protein, application real-time PCR and Western blotting techniques from technical level of nucleic acid and protein expression levels of detection ZnT8. Exendin-4 was observed after administration of intervention in db / db mice pancreas and adipose tissue ZnT8 expression. Fourth, Result 1. (1) successfully amplified ZnT8-C of the cDNA, by sequence alignment, and in GenBank coding ZnT8 carboxy-terminal cDNA sequence exactly. (2) The recombinant prokaryotic expression plasmid pET32a-ZnT8-C in E. coli BL21 (DE3) plysS obtain soluble expression, molecular weight of about 30kDa, by Ni2-NTA chromatography, Western blotting was identified as the desired ZnT8-C protein. (3) to obtain purified recombinant protein ZnT8-C, and used to immunize New Zealand rabbits, rabbit anti ZnT8 obtained polyclonal antibody. A recombinant protein antigen with indirect ELISA titer antiserum ZnT8 in l: 3200 - l: 6400. Western blot analysis: ZnT8 antiserum at a molecular weight of about 30kd prokaryotic expression of the corresponding protein bands showing specific binding, indicating that the polyclonal antibody has a good immunological activity. 2. (1) db / db mouse pancreatic tissue ZnT8 nucleic acid and protein expression compared to control mice heterozygous significantly reduced. db / db mice testes fat and adipose tissue in the center ZnT8 nucleic acid and protein expression compared with control mice was also significantly reduced. (2) Exendin-4 after administration of db / db mice ZnT8 pancreatic tissue with the nucleic acid and protein expression was significantly higher than the control group, and the adipose tissue ZnT8 nucleic acids and protein expression compared with the control group, no significant difference . V. CONCLUSIONS The main conclusions of this study: 1. Successfully prepared with good activity ZnT8 polyclonal antibodies. 2.db/db mouse pancreas and adipose tissue expression ZnT8 lower than the control group, suggesting ZnT8 may be involved in the pathogenesis of T2DM and the progression of the disease; Exendin-4 administration allows the db / db mice of pancreatic tissue ZnT8 upregulation, and its expression in adipose tissue ZnT8 no effect, suggesting ZnT8 role of GLP-1 may be drug targets.

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CLC: > Medicine, health > Internal Medicine > Endocrine diseases and metabolic diseases > Islet disease > Diabetes
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