Cloning And Function Research For The Novel Gene BRAP

Neuropeptide is causing more and more attentions for its role in airway hyperresponsiveness and in the occurrence of airway inflammation. Bombesin-like peptides (BLPs) are neuropeptides of broad biological functions. In mammals, the current members of BLPs have been identified as gastrin-releasing peptide (GRP) and neuromedin B (NMB). Correspondingly, in mammals,3 members in BLPs receptor family have been identified, namely GRP receptor, NMB receptor and bombesin receptor subtype 3 (BRS-3). The former two combined with GRP and NMB respectively with high affinity and induced the corresponding biological function. While, the native ligand of BRS-3 has not been identified, so it is still an orphan receptor. BRS-3 is a 7-time transmembrane G protein-coupled receptor consisting of 399 amino acids, and in most normal tissues, the expression of BRS-3 is low. Its biological significance, biological effect and gene expression regulation are not known well at present, while it expresses increasedly in stressed lung tissues and tumor tissues, suggesting that BRS-3 may be related to cell growth and injury repair.According to our preliminary studies, BRS-3 may play a role in the regulation of stress responses in lung and airway epithelia. And further on, we conducted a series of studies on the biological functions and regulation of gene expression of BRS-3, and the native ligand of BRS-3. With the bacterial two-hybrid technique, it is shown that BRS-3 can interact with a variety of proteins, and the functions of these proteins are involved in cell growth, differentiation, anti-apoptosis, cell cytoskeleton, tyrosine kinase, etc. Meanwhile, it was found that two new proteins can interact with BRS-3, while their biological significance is not known. Therefore, this subject focuses on the sequence characteristics, bioinformatics and its function for one novel gene (tentatively named as bombesin receptor activated protein BRAP,gene bank locus:NM-152734).Objective:Adopting the methods of cloning and predicting gene functions on the bioinformatics, we studied a new gene which interacts with BRS-3, and further on, observed the subcellular location of BRAP-coded protein and the function in the bronchial epithelial cell. These results constructed the basis for studies on the supramolecular assembly of BRAP and BRS-3 in the stress response in bronchial epithelial cell.Method:(1) Conduct bioinformatics analysis of BRAP by cloning analysis software on internet.(2) Use RT-PCR assay to amplify the new gene ORF, and ligate to pEGFP-C1 vector, build the recombinant plasmid pEGFP-C1/BRAP, transfect into Hela cells. Meanwhile, after transient transfection, we observe its expression protein by the laser confocal microscope, and analyze the expression of transfected protein by Western-blot.(3) Build the recombinant plasmid pcDNA3.1(+)/BRAP, transiently transfect into human bronchial epithelial cells (HBEs) and detect its impact on the cell cycle with flow cytometry. We further sort HBE cells which stably express pcDNA3.1(+)/BRAP, and observe their impacts on the wound repair ability of HBE. The wound repair ability was detected according to the small irregular injure area caused by mechanical injure on bronchial epithelial cell, and use the microscopic video analysis system to measure wound area every 4h. Then, we draw the linear regression equation of time and repair area. The repair index was used to assess the wound repair ability of transfected HBE cells corresponding to the linear slope of linear regression equation.Result:(1) BRAP cDNA contain a full length of 6721bp, encodes 354 amino acids, which is precisely located at 6p21.2. In addition, BRAP gene expresses highly in normal fetal brain tissue, fetal lung, fetal liver, human embryonic stem cells, brain tumor astrocytes, prostate cancer, lung cancer and other tissues, and there is a transmembrane region in it..(2) We build the recombinant plasmid pEGFP-C1/BRAP which consisted with the BRAP sequence through DNA sequencing. Its expression products are located in the membrane and cytoplasm, and its expression increased significantly in the transfected recombinant cells.(3) We build recombinant plasmid pcDNA3.1(+)/BRAP, which consisted with the BRAP sequence through DNA sequencing; the flow cytometry results demonstrated that the recombinant plasmid pcDNA3.1(+)/BRAP increase the S phase+ G2 phase of cell cycle by 25%.Conclusion:(1) Bioinformatics analysis suggested that BRAP has the signal molecular properties, and may be involved in the regulation of physical functions of cells.(2) The BRAP protein was located in the membrane and cytoplasm, suggesting that this protein may be a cytoplasmic protein and involved in cellular signal transduction.(3) BRAP promoted the cell cycle, and increased the wound repair ability of human bronchial epithelial cell.