| The human ndrg2 (N-myc downstream regulated gene 2) gene was first discovered and cloned from cDNA library of normal human whole brain in our lab in 1999. This gene locates on human chromosome 14q11.2, including 16 exons and 15 introns, and its full mRNA is 2,024 nt in length, encoding NDRG2 protein whose Mr is 4.0×104.The first member of ndrg gene family found was ndrg1, which was isolated as a new gene upregulated in N-myc mutant mouse embryos and it was repressed by N-myc and c-myc. Ndrg gene family consists of 4 members: ndrg1, ndrg2,ndrg3 and ndrg4, which has high similarity in nuclear acid or amino acid sequences, but their expression levels are distinct in spatio-temporal distribution and periods of cells and tissues development. The precise molecular and cellular functions of members of this family are still unknow so far. The expression of NDRG protein family is regulated by lots of intracellular and extracelluar factors. NDRG1 protein expression is upregulated by homocystein,tunicamycin and P53. In addition, the expression is also activated when differentiation is induced by chemicals such as retinoic acid or when cells are under oxidative stress conditions caused by hypoxia,nickel and other reductive agents. On the other hand, the expression is repressed by the proto-oncogenes N-myc and c-myc, and in some cases the expression is also repressed by testosterone or dihydrotestosterone (androgen) at mRNA level. Infering from all the references that can be obtained, the functions of NDRG family may be involved in cellular differentiation events,maintaining the balance of cell redox potential (in oxidative stress condition or biological conversion process), or counteracting cell malignant transformation, and so on. Therefore, checking the protein interacted with NDRG family, then identifying the roles of NDRG family and investigating the possible sigaling pathway which are involved in will be very significant. Therefore ,this study were undertaken to address the above issues.The main results were as follows: 1.Construction of Prokaryotic Expression vector of ndrg family: To construct the recombinant vector pGEX-4T-1-ndrg1, pGEX-4T-1-ndrg2, pGEX-4T-1-ndrg4b,we digest the ndrg1,ndrg2,ndrg4b cDNA from recombinant vector pMD18-T-ndrg1, pMD18-T-ndrg2, pMD18-T-ndrg4b which were already sequenced and transfer into vector pGEX-4T-1. 2.Prokaryotic Expression of ndrg family: Recombinant protein of pGEX-4T-1-ndrg1, pGEX-4T-1-ndrg2, pGEX-4T-1-ndrg4b were expressed by inducedwith IPTG. We found that GST-NDRG1 is soluble ,while GST-NDRG2,GST-NDRG4b is insoluble. 3. Purification and western of recombinant protein: After the fusion protein was purified by Glutathione Sepharose 4B, two bands at Mr 6.6×104 were obtained, And western blot showed that that both of them were GST-NDRG1. 4. Selection the high expression cells: Three types of cells such as 7721, A549, HHCC were cultivated. NDRG1 was found to be highly expressed in HHCC by western. 5. Identifying the proteins interacted with NDRG1: Incubating purified protein GST-NDRG1 with HHCC, and at the same time GST-NDRG1 was incubated with lysis as contrast. After that, we find at least a new strip at Mr 4.3×104,pH6.0through 2D Gel Electrophoresis. It must be the interact protein with NDRG1. |
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