Prokaryotic Expression And Monoclonal Antibody Preparation Of EDAG-1-a Kind Of Transcription Regulatory Factor Interrelated Hematopoiesis

EDAG-1 is a novel human hematopoietic tissue-specific gene (China patent number 01118268.8 open number cn 1388130A). Previous studies showed that EDAG-1 involves in the regulation of apoptosis and hematopoietic development. Nowadays, the mechanisms of EDAG-1 regulating proliferation, differentiation and apoptosis of haematopoietic cells remain largely unknown. We prepare the monoclonal antibody of EDAG-1 for study the mechanisms. The monoclonal antibody of EDAG-1 is important tool for functional studies of EDAG-1.We cloned the EDAG-1 cDNA from human fetal liver cDNA library and inserted it into the pGEX-4T-3 vector. Then we succeed to set up prokaryotic expressed vector marked by GST, after we choose PGEX-4T-3 as our vector and then we culture at 37℃to better that expression system. And when the OD600 is 1.4-1.5 we add IPTG of 0.1mM to further induce it. After four hours we can get highly-efficient expression of protein. And the fusion protein takes a thirty percentage in the general protein of the bacteria and the expression protein exists in the bacteria in a soluble form, through the purification of the induced protein, we can get highly-pure protein of EDAG-1.We use the fusion protein GST-EDAG-1 from pro-nucleus expression to immunize the mouse BALB/C. Using spleen cell from immunized mouse and the SP2/0 myeloma cells of the mouse from the same class as the immunized mouse with PEG participating in this process of cell fusion. And then we do a preliminary selection among fused cells cultured in HAT base. And we do experiment of ELISA on hybridoma cells to judge whether the related anti-body exists. If that happens, we can get mono-clone hybridoma cells which can express EDAG-1 anti-body. From the results of our preliminary experiment, we found that the monoclonal anti–body we made has the trait of stability and low background. The monoclonal antibody we made can be used to detect the expressed product of leukemia cell and someday in the future it offer a tool for the determination and therapy for cancers such like leukemia but not the least. At the same time, it's very important for our future deeper study on some biological functions like the molecular mechanism of EDAG-1 regulating hematopoiesis.