Predicting, Prokaryotic Expression And Identification Of A Highly Immunogenic Fragment Of Human Zinc Transporter 8

BackgroundType 1 diabetes(T1DM) is a complex, multifactorial disease caused by the selective destruction of insulin-producingβcells in the islets of Langerhans. The presence of circulating islet cell autoantibodies distinguishes T1D from other diabetic syndromes and determination of autoantigen genes and proteins is instrumental in understanding T1DM as a clinical entity and in investigating the pathogenesis of the disease. Prospective studies in humans have established that the appearance of multiple autoantibodies, such as islet cytoplasmic autoanti-bodies (ICA), glutamate decarboxylase (GADA), antibodies to insulin (IAA) and protein tyrosine phosphatase antibody (IA-2A), are strongly associated with the development of type 1 diabetes. The combined measurement of autoantibodies to IAA, GADA and ICA, although highly predictive of T1DM susceptibility, fall short of being able to determine individual risk in the general population, and the search for new molecular targets is an important goal.Zinc transporter 8 is an important member of a newly discovered of the zinc transporter family members, located in pancreaticβcells a small amount ofαcell, as the islet-specific zinc transporter protein. ZnT8 is postulated as important for providing Zn to allow for the proper maturation, storage, and secretion of insulin. Recently by radioimmunoprecipitation assays against new-onset T1DM and prediabetic sera. ZnT8 was identified as a novel and most potential autoantigen candidate. It provides an important additional and independent predictive marker for T1DM. In fact ZnT8 shows high specicities and sensitivities that complement current T1DM autoantibody assays and add to the predictive value of their measurement.ObjectiveThis research through bioinformatics tools, uses DNAstar software to forecast consolidatedly ZnT8 protein secondary structure, hydrophilicity, flexibility, surface accessibility and antigenicity of a consolidated forecast screens with a strong immunogenicity of the N-terminal fragment ZnT8 (N) and from the forecast results, use of genetic engineering techniques to build pET32a-ZnT8 (N) prokaryotic expression vector, which expressed in E. coli to achieve. All this explore its clinical value of diagnosis of diabetes mellitus for further in-depth study of its features and make the foundation of the clinical diagnosis of the role of the foundation.Methods1. Use bioinformatics software to predict the structure of ZnT8, from which the strong immunogenicity of the N-terminal fragment ZnT8 (N).2. According to ZnT8 gene coding sequence in the GenBank, design the upstream and downstream primers with NcoI and XhoⅠrestriction sites design, in order to pZnT-8-EGFP plasmid as a template for PCR amplification.3. The construction and identification of expression vector pET32a-ZnT8 (N).4. After the correct recombinant plasmid pET32a-ZnT8 (N), select restriction enzyme digestion and empty vector pET32a was transformed into E. coli IPTG induced for expression.5. ZnT8 (N) fusion protein in Western blot of identification.Results1. Predicted by DNAstar software, ZnT8 before the first N-terminal 140 amino acids were found rich inα-helix, hydrophilic, flexibility and surface accessibility were high, most of membrane area and antigenic analysis was significantly higher than in other fragments, indicating that the fragment (including 140 amino acids, ZnT8) antigens and strong, so select the Cloning and Expression of the fragments.2. The pET32a-ZnT8 (N) prokaryotic expression vector was constructed successfully.3. Through SDS-PAGE analysis showed that the purpose of bands observed with the expected molecular weight size of the match.4. Through Western-blot analyzed,the fusion protein expression level approximately 45% of the total bacterial protein.ConclusionScreening the source of human pancreatic islet cells of ZnT8 strong immunogenic fragments, and the success of prokaryotic expression system for further studies ZnT8 nature such as the foundation.