To Fish The Human UROC-28 Binding Protein By The Yeast Two Hybrid System

Prostate cancer is a malignant tumor menacing men's lives and it's morbidity is increasing in recent years. The diagnosis and treatment of Prostate cancer have attracted significant attentions and wide studies. UROC28 expressed highly in prostate> bladder and mammary cancer and the expression is increased accompanying with the Gleason score's increasing of prostate cancer. UROC28 is highly expressed in the serum of prostate cancer patients when compared with that of BPH patient or health. It is predicted that UROC28 may play a key role in the tumorgenesis of prostate. UROC28 may be a potentially special marker for prostate cancer just like PSA. It may offer some significant help to the early-stage diagnosis> prognosis and gene therapy of prostate cancer.The interactions between proteins exist in each cell of the life and they control lots of activities in cells. These interactions are the basis of life and each activitiy of life is carried out by suchinteractions. Studying the roles of such proteins is helpful for us to discover some basic problems about life.UROC28 is a novel gene, and studies of it is very few. To make some original studies on the function and mechanism of UROC28, we carried out some experiments. First we isolated the total RNA from human bladder cancer tissue and prostate cancer cell line PC-3, then we designed two primers according to the gene sequence of UROC28. The UROC28 cDNA gene was got by RT-PCR amplification. Then we cloned the cDNA gene into pUC19 and pGBKT? vectors respectively. The results were confirmed by sequencing the products. At the same time the cDNA gene were cloned into GST fusion expression vector pGEX-4Tl. By SDS-PAGE and Western blot, we confirmed that the expressed fusion protein existed in inclusion body and with a molecular weight of 42KD. This protein will be used as an antigen to produce the antibody and then offer a basic tool for further studies on it's localization in different tissues, function and characterization.To fish the novel proteins interacting with UROC28, we screened foetus liver and brain cDNA library by yeast two hybrid system. Firstly we transformed the Bait vector into yeast strain AH-109. The self-activation of bait protein was excluded by phenotype selection and p-galactosidase assay. By screening the library we at last got 38 candidated genes which encode proteins that can interact with UROC28. Further analysis of these candidated genes can give some clues for the further studies of UROC28.