| Adiponectin is a secreted plasma protein expressed exclusively in differentiated adipocytes. It is highly correlated with insulin resistance, arteriosclerosis and so on. To test serum adiponectin concentration is one of means for elucidating its biological roles and functional regulation. It is of significant for predicting the occurrence and development of some diseases. On the other hand, the globular domain is the major domain for adiponectin activity. Comparing with the inclusion body, the soluble proteins are likely to display the correct conformation of adiponectin, which is the structure basis of its biological activities. So the soluble expression of adiponectin and gadiponectin may contribute to the research of their biological function and the associated molecular mechanism. At the same time, using the expressed proteins, monoclonal and polyclonal antibodies against various epitopes in adiponectin for ELISA can be prepared. At present, there are two major methods of assaying serum adiponectin concentration in abroad. One is ELISA and the other is RIA. Most of the ELISA methods use biotin-avidin system, so their procedures are complex and time-consuming. No similar kits are available in domestic market. Objectives: To develop an ELISA method for the quantitative detection of human serum adiponectins in aged individuals with or without type2 diabetes mellitus. And further to clone and express adiponectin and gadiponectin solubly in E.coli expression system and purify active proteins to prepare polyclonal antibodies that might be used for further optimizing the established ELISA method in the future. Methods: Recombinant human adiponectins were expressed in E. coli and the inclusion bodies were purified by affinity column and used for preparing polyclonal and monoclonal antibodies against adiponectin by immunizing rabbits and mice, respectively. The purified monoclonal antibodies were used as coating antibody while the purified polyclonal anti-human adiponectin antibodies werelabeled by horseradish peroxidase and serve as the detection antibody. Standard curve was made and the concentrations of adiponectin in the sera of type2 diabetes families were detected with a newly prepared serum or plasma sample diluent and the correlation of adiponectin concentration with type2 diabetes was analyzed. For further optimization of the established ELISA method, new antibodies are needed. Then the DNA coding for the full-length adiponectin as well as gadiponectin were amplified from pQE30-adiponectin plasmid respectively by PCR and were subcloned into pGEX-4T-2 vector to construct recombinant expression plasmids. After being transformed into competent host E.coli BL21, the soluble expression of fusion proteins were induce by IPTG at 25 °C. The soluble proteins were purified by GSTrap affinity column. And polyclonal antibodies were prepared by immunizing rabbits. The specificity of the polyclonal antibody prepared was examined by Western-blot analysis. The biological functions of purified adiponectin as well as gadiponection were tested using liver cancer cell line HepG2 and mononuclear blood cells from healthy donors as target cells.Results: The sensitivity of the established ELISA method was 0.5ug/ml. Both intra- and inter-assay imprecision values were<8%. The dilution curves of plasma showed good linearity, and the recovery was 88%-108%. The newly prepared serum or plasma sample diluent, which is in the procession for applying a patent, is essential for detecting the serum adiponectin. There was good correlation between serum adiponedtin concentrations by the ELISA and a commercially available Linco ELISA Kit(r=0.71). Serum adiponectin concentrations were lower in type2 diabetic patients compared with healthy controls (4.04±2.39 vs. 5.87±2.96ug/ml; t=3.12 P=0.002), and this difference was significant after controlling for age, gender and body mass index. Plasma adiponectin showed a significant correlation with triglyceride ( TG; r=-0.367, P=0.009)in normal subject and was negatively associated with body mass index(BMI; r=-0.413, P=0.023)in normal women. The assay results of sera from Endocrinopathy laboratory of 301 Hospital also showed that serum adiponectin concentrations were significant lower in type2 diabetic patients compared with healthy controls (4.26 + 2.65 vs 7.98±4.39ng/mlt=4.38, P<0.0001).The DNAs coding for 710bp adiponectin and 430bp gadiponectin were amplified from pQE30-adiponectin plasmids by PCR, respectively. And the... |
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