Prokaryotic Expression Of Adiponectin Gene Without Signal Peptide And Enzyme Linked Immunosorbant Assay Method Research

In recent years, the incidence of obesity, typeâ…¡Diabetes Mellitus and Atherosclerosis became more, and adiponectin (ADPN) in plasma had a negative correlation with the happening of diseases. Therefore, it could be reflected these diseases through detection human plasma ADPN. In this study, obtaining human adiponectin without signal peptide recombinant protein through genetic engineering, and preparation of standard substance, human ADPN ELISA quantitative detection method was established preliminary.According to ADPN sequence (NM₀04797.2) published in NCBI, a pair of primers was designed with Primer5.0. Extracting total RNA from human adipose tissue, RT-PCR was performed to get 678bp human ADPN without signal peptide gene fragments. The target gene and vector pET-41a were digested separately by double enzyme, then performing agarose gel electrophoresis, and purifying target fragments and vectors to ligate. The ligative product was transformed into DH5, inoculated on LB solid medium and picking single bacterial colony, and extracted plasmids after the colonies cultured overnight. Then the plasmid was digested by double enzymes to identify, and the positive recombinant plasmids were named pET-41a/ΔSADPN. The recombinant plasmid was transformed into E.coli BL21(DE3), expressing by IPTG induction. The SDS-PAGE showed that the expressed recombinant protein was soluble with better immunoreactivity.After optimizing induction conditions, the best inductive conditions was 25℃, 0.03mmol/L IPTG, and induction expression 8h. For fusion proteins containing histidine tag, target protein can be purified with nickel ions affinity chromatography, and the purified fusion protein was detected with concentration 2.1 mg/mL.The anti-ADPN monoclonal antibody was used as the coating antibody, and modified sodium periodate method was performed to conjugate anti-ADPN polyclonal antibodies with horseradish peroxidase (HRP-peroxidase), as enzyme labeled antibodies. The best coating concentration Anti-ADPN monoclonal antibodies was 1.0μg/mL by chessboard titration, and the best dilution times of HRP-peroxidase was 1:4000. The recombinant protein ofΔSADPN severed as standard product to make standard curve, the linear range from 1ng to 30ng. Compared with other similar kits in the clinical test results, there was no significant with t test, P>0.05, difference.The recombinantΔSADPN protein was obtained with immunoreactivity through genetic engineering methods, preparing of ADPN standard product, and preliminary establishing ADPN double antibody sandwich detection model, and laid the foundation for researching and developing ADPN quantitative detection kit.