Expression And Purification Of Globular Domain Of Human Adiponectin In E.coli And Preparation Of Its Monoclonal Antibody

With the rapid development of science and technology，the level of living wasimproved rapidly. Especially after World War Two, the composition of our diet haschanged largely, and the sharply increasing consumption of meat and fat was biggestchange. Something followed, the incidence of diabetes, Cardiovascular and tumoralso increased quickly. Now China has been one of countries with fat populationfastest growth, the second large diabetes population, and the first in the incidence oftype2diabetes mellitus. The research indicated that the level of adiponectin in bloodwas connected closely with these diseases, and globular adiponectin was the mainstructure of its function. So, it could provide gridance for clinical diagnosis of thesediseases and clinical medication though detection of adiponectin. In the research,getting fusion protein of human gadiponectin with biological activity by genetic engineering, andmonoclonal antibody of human gadiponectin was prepared with it, and paving the way ofestablishing the detection of human adiponectin.According to the human adiponectin gene sequence NM₀04797.2in NCBI, analysis thecodon and optimizing them, then chmical synthesis the gene sequence, and to contruct expressionvector pET41a-gAd by disgestion. In E.coli, fusion protein of human gadiponectin was expressedthrough optimization of inducer concentration, induction tempreture, and induction time. The bestcondition was37℃，0.1mmol/L IPTG and induction for6hours. And it was purified withNi-affinity chromatography, to renature fusion protein of human gadiponectin by inclusionrefolding technology and to obtain bio-activity human gadiponectin. Purified human gadiponectinwas immunized BABL/C mice with, and subcutaneous injecting several sites with proper dosage.Mouse with higher immunizing potence was Selected, and performing cell fusion technique withits splend cells and myeloma cells. Then indirect ELISA was performed for screening positivecells of monoclonal antibody against human gadiponectin with HAT culture, cloning screenedpositive cells by limiting dilution, and obtaining cell strain that secreted monoclonal antibodyagainst human gadiponectin. Amplification culture the cell was performed and collecting them viacentrifugation, then inoculating each mice against5×105hybridoma cells in the abdominal cavityfor induction ascites, collecting induced ascites, and performing with ammonium sulfate precipitation and DEAE-affinity chromatography to purify ascites, then analysis the purifizationthrough gray level of SDS-PAGE, to gain monoclonal antibody against human gadiponectin and toidentify its characteristics by indirect ELISA.Plasmid pET41-gAd was constructed, and high level expression fusion proteinof humangadiponectin with biological activity, which relative molecular weight is about45kD, and itspurify was90%, approximately. One hybridoma cell strain that secreting monoclonal antibodyagainst human gadiponectin was obtained, and the purifization of the antibody was91%, and thesubtype was IgG1.