High Expression Of Human Differentiation Inhibitor Id-2 And Id-3 In E.coli And Preparation Of Its Polyclonal Antibody

Inhibitor of differentiation (Id) is also called DNA binding inhibitor, it belongs to the helix-loop-helix protein family. Id can down-regulate the activity of basic helix-loop-helix protein, thus inhibit the cell differentiation, prompt cell proliferation and take some reponsbilities in regulation of cell cycle. At present the known mamlian Id have 4 subtypes, they are Id-1 to Id-4. Among them Id-2 usually plays an important role in control of cell differentiation and proliferation. It can down-regulate the transcription factor of liver cells, and inhibit liver stellate cell differentiation, it can also suppress the some of the liver cells transcription factor. The expression of Id-2 is significant lowered in the progress of aging. Some studies showed that over-expression of Id-2 in osteoclast, breast epithelial cells, fiberblast cells etc can effectively detain the cell aging process or make the cell become immortal. Id-2 gene also shares the chacteristic of oncogene, thus the Id-2 protein plays an role in some kinds of cancer. The very early expression of Id-3 was first found in mouse fiberlast cells. Id-3 take some responsibility in the process of T and B cell growth, mucle cell differention, vascular smooth muscle cells proliferation and the growth of bone tissues. Thus the Id molecule has became an very important taget in both life science research and drug development.In this study we colned human Id-2 and Id-3 in pGEX-6P-1 prokaryotic expression vector. The construct of both Id-2 and Id-3 GST fusion protein were verified by PCR, sequencing and endonuclease digestion.The following IPTG induced expression of Id-2 and Id-3 in E.coli were confirmed by western blot, ELISA and bi-direction agar diffusion assay. The polyclonal antibodies of Id-2 and Id-3 were purified from the serum of immunized rabbits.The results of our study provide a feasible way to detect of Id-2 and Id-3 protein in different tissues, also it is expected to be useful in analysis of Id-2 and Id-3 structure and epitope indentification for futher studies.