| Aim: Prostate cancer is the most significant malignancy among men in the Western developed countries nowadays. Among many methods, prostate cancer diagnosis by prostate specific antigen (PSA) testing is so popular and widespread, but as the most useful tumor maker, PSA is not perfect, much of current studies were focused on finding various makers with enhanced sensitivity and speciality for Prostate cancer. A novel gene UROC28 was cloned and identified in 2000. A low level of expression was observed in normal tissues and BPH tissues, UROC28 were significantly up-regulated in prostate cancer with varying Gleason scores, and metastasis prostate cancer tissues. Western blot protocol was used to investigate whether UROC28 protein is present in serum samples from normal and prostate cancer individuals. The mean serum UROC28 protein level in individuals of prostate cancer is significantly higher than normal individuals, also, the mean URCO28 levels between normal versus prostate cancer were significantly different with a P < 0.0001. These preliminary results suggest that UROC28 may provide an alternative serum marker either alone or in combination with other markers such as PSA for a more accurate diagnosis of prostate cancer. Since it is really instructive research and required more and progressivestudies, we try to learn some preliminary function information about UROC28 in this study. Methods: the experiments includes seven steps1. Bioinformation analyses were used to analyses UROC28 gene and UROC28 protein.2. The UROC28 was cloned by PCR and then was inserted into plasmid to construct the expression vector of pRSET-UROC28 and expressed protein in E. Coli BL-21; further step to make sure higher expression experiment condition and then UROC28 protein purification was performed with Ni2+-NTA method.3. Immunize New Zealand White rabbits to produce polyclonal antibody (rabbit-anti UROC28). And polyclonal immune serum was purified.4. The pcDNA-UROC28 expression vectors was constructed and permanent transfected in to mammalian cell HEK293 and PC-3 cell line, and then the cell biological effect were observed during the overexpression process of UROC28.5. We constructed a shRNA expression vector pWH1-UROC28, andblocked the expression of UROC28 gene in prostate cancer cell line PC-3 by resorting RNA interference, and then the cell growth curve and cell cycle and morphological effect were observed.6. We constructed pEGFP-UROC28 green fluorescent protein fusion expression vector, and permanent transfected in to HEK293 and PC-3 cell line; Irnmnohistochemistry method was employed to detect the localization of UROC28. Indirect immunofliiroscence method and DAB substrate immunohistochemistry method were used to analyze and prove the subcellular distribution and expression of UROC28 antigen.7. Immnohistochemistry staining method with anti-UROC28 rabbit polyclonal antibody was performed on formalin-fixed sample sections of prostate cancer and BPH tissue by using SABC process. Result:1. The nucleic acid sequence and amino acid sequence were compared with genebank, and the amino acid sequence was analyzed by protein analysis software. Using genome blast, UROC28 was mapped to chromosome 6q23-24, and no similajr sequence else was found. The result showed that there was a complete open reading frame which was encoded by 135 amino acid; it is presumed that the molecular weight of this protein is 15.7 KD; PI is 9.77, and possibility of localization is: nuclear: 65.2%; mitochondrial: 30.4%; cytoskeletal: 4.3%; and two Transmembrane Regions were Predicted, one of those two at 114 acid has a high creditability with836 score.2. We constructed pRSET-UROC28 expression vector which was further identified by double-enzyme digestion and sequenced. The sequenced result was consistent with genebank reports. High expression of Fusion protein UROC28 was successfully induced by IPTG; SDS-PAGE showed that the molecular mass is 22 l03kDa, similar to the mass of theoretic fusion pro...
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