| Cell apoptosis is an important process of basic life which is one of the the basic way to maintain the number of dynamic equilibrium of body cells. Cell apoptosis plays an important role in embryonic development, cell proliferation, clearance of aging and disease related cell, the occurrence and development of tumor, autoimmune diseases, etc. Apoptosis is a kind of precise process regulated by gene which can be roughly divided into the following processes, apoptosis signaling contact of cells, interactions of apoptosis regulating protein, the activation of the proteolytic enzymes, protein kinase activation cascade. The initiation of apoptosis a signaling system which can be divided into two pathways: the biochemical pathway of membrane receptor signaling about apoptosis, and the release of cytochrome C and the activation of Caspases.At present, the known mammalian genes of apoptosis related protein contains dozens of species.According to the different functions, these genes can be mainly divided into three categories: apoptosis inhibiting genes, apoptosis promoting genes and bidirectional regulation genes. These genes mainly includes:(1) The Bcl-2 family, the family contains a variety of inhibiting apoptosis related genes which expressed in hematopoietic cells, epithelial cells, lymphocytes, nerve cells and various tumor cells, the cells were mainly distributed in the membrane of mitochondria, endoplasmic reticulum and nuclear envelope and so on;(2) The Fas family, Fas protein belongs to the tumor necrosis factor receptor members family,and its ligand Fas L is a member of TNF family gene. The combination of Fas and its ligand FasL induced cell apoptosis;(3) The p53 family, p53 gene encoding the P53 protein had a positive effect on cell apoptosis. P53 is a DNA binding protein, a transcriptional activation. The mechanism of P53 induced apoptosis in different cells may be different whereas the P53 dependent apoptosis may ultimately leads to the activation and release of cytochrome C and Caspases.To screen host proteins interacting with apoptosis promoting protein Bad,tandem affinity purification(TAP) was utilized.The full-length bad gene was amplified from He La cells by RT-PCR,and then the fragment was inserted into pcDNA3.0-Flag-HA plasmid to construct pcDNA3.0-FLAG-HA-Bad eukaryotic expression vector which can induce the expression of fusion protein with FLAG and HA tags linked to the C terminal. Then, TAP method was utilized to screen out host protein which interacts with Bad as a bait protein followed by liquid chromatography/mass spectrometry(LC/MS) performing to identify Bad-interacting host proteins. Gelsolin was selected for further study among the identified proteins.Co-immunoprecipitation(Co-IP) was applied to confirm the interaction between Bad and Gelsolin after gelsolin gene cloning and transfection. This study established and improved the TAP method on interacting-protein identification, and screen host protein bond to Bad. The further study will focus on the interaction effect of these two kinds of proteins on cell apoptosis, and further focus on the growth of tumor cells inhibition. This study is also helpful to find cancer drug targets and analysis of drug treatment of anti tumor effect in the future. |
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