| Prokaryotes and mammalian cells have a wide range of potential applications in the food industry.Food additives,enzymes,artificial milk and other new food or food raw materials based on synthetic biotechnology are constantly entering people’s field of vision.Studies have shown that adiponectin is closely related to the health status of the human body.It has been confirmed that adiponectin plays an important role in the regulation of insulin levels and cancer prevention.In addition,the content of adiponectin in serum can be used as an important biomarker for diabetes and cardiovascular disease.This study mainly focused on the expression of recombinant adiponectin protein.The core component of adiponectin is globular domain protein（gAd）.Taking gAd protein as the research object,the recombinant expression of gAd protein is realized based on prokaryotic expression and mammalian expression system respectively.On this basis,anti-gAd monoclonal antibodies are prepared based on hybridoma cell fusion technology,in order to provide key raw materials for the large-scale preparation of adiponectin and the development of adiponectin rapid immunoassay detection products.The main contents of this study are as follows:1)Based on the synthesis of the globular domain protein（gAd）gene of human adiponectin,the fusion protein expression vectors pET30a-gAd-NusA,pET30a-gAd-MBP,pET30a-gAd-GST,pET30a-gAd-SUMO,pET30a-gAd-MBP,pET30a-gAd-GST and pET30a-gAd-SUMO were constructed respectively.IPTG was used as an inducer to induce E.coli BL21 to express protein,the results showed that pET30a-gAd-NusA and pET30a-gAd-MBP expressed soluble fusion proteins,their expression yields were 10 mg/L and 14 mg/L,respectively.The purified fusion proteins were identified by SDS-PAGE and ELISA,respectively.The results showed that the purity of the two fusion proteins was greater than 90%,and they had specific binding to anti-human adiponectin（ADPN）antibody.2)This study also carried out the prokaryotic expression study of soluble gAd protein optimized based on induction temperature and IPTG concentration during inductionof product expression.The results showed that under the expression conditions of induction temperature of 12℃and IPTG concentration of 0.05 mmol/L,the pET30a-gAd expression vector achieved soluble expression in E.coli BL21,and its purified expression is 2 mg/L.The purified gAd protein was identified by SDS-PAGE and ELISA,the results showed that the soluble gAd protein existed in three forms of monomer,dimer and trimer,and all of them had specific binding with anti-human adiponectin（ADPN）antibody.3)In this study,the eukaryotic expression vector pCDNA3.1-gAd-EGFP of gAd was constructed.PEI reagent was used as transfection reagent,the transient transfection of gAd protein was carried out in Expi 293 cells.The results showed that the pCDNA3.1-gAd-EGFP expression vector was recombinantly expressed in Expi293cells,and its expression yield was 4μg/mL,The results of SDS-PAGE and Western blot showed that there were three expression products with different molecular weights,and all of them could specifically bind to the anti-human adiponectin monoclonal antibody.4)This study also carried out construction of CHO cell line with stabe expression of recombinant gAd-EGFP protein based on CHO-S cell.Adherent CHO-S cells were transfected with pCDNA3.1-gAd-EGFP as the expression vector.The optimal transfection conditions were as follows:cell density was 3×106 cells/mL,PEI dosage was 4μg/mL,plasmid dosage was 2μg/mL.After screening with G418-containing medium and three subcloning,three strains of cells stably expressing recombinant gAd-EGFP protein were obtained,and they were named 4D11,4G11 and 2F12 respectively.On this basis,the above three cell lines were successfully cultured in suspension.One of the cells（4D11）was amplified and cultured in suspension,and the yield of expressing recombinant gAd-EGFP protein was 6 mg/L.The results were the same as the gAd-EGFP recombinant protein obtained by transient transfection.5)In this study,gAd was used as the immunizing antigen to immunize three Balb/c female mice,preparation of hybridoma fusion cells.After three subcloning,eight hybridoma cell lines were finally obtained,which could specifically secrete anti-gAd antibodies,and they were named 7D10、3B7、9B10、3A7、9E2、3H9、2C8 and 7B9respectively.Preparation of monoclonal antibody ascites and determination of its titer.The results showed that the ascites titers of the 8 monoclonal antibodies prepared ranged from 1:128000-1:2560000.The affinity constants of 9B10 and 3A7 monoclonal antibodies detected by biofilm interferometry were 5.281×10-10 mol/L and 1.033×10-9mol/L,respectively.The antibody has high affinity and can be used in the manufacture of adiponectin immunoassay products. |
