| The bone morphogenetic proteins (BMPs) play a pivotal role in endochondral bone formation. Using differential display polymerase chain reaction, Francesca Gori,et.al have identified a novel gene, named BIG-3 (BMP-2-induced gene 3 kb), that is induced as a murine prechondroblastic cell line, MLB13MYC clone 17, acquires osteoblastic features in response to BMP-2 treatment. BIG-3 is a target gene on the BMP signaling pathway. Alkaline phosphatase activity of MC3T3 transfected with BIG-3 gene increase earlier than that of the cell transfected with empty vector, and, by 14 days, the cells transfected with BIG-3 have a 10-fold increase in AP activity relative to cells transfected with empty vector. Expression of BIG-3 increased mRNA levels encoding Cbfal, type I collagen, and osteocalcin and accelerated formation of mineralized nodules.This gene encodes a 328-amino acid protein with a molecular mass of -34 kDa. BIG-3 is a new member of a family of highly conserved proteins, the WD-40 repeat proteins, found in all eukaryotes but not in prokaryotes. The WD-40 family of proteins participates in a wide variety of cellular functions including signal transduction, mRNA processing, gene regulation, and the cell cycle.The fact that BIG-3 accelerates chondrocyte differentiation in vitro, combined with the observation that BIG-3 is expressed in the growth plateduring embryonic development, suggests that this novel protein is likely to play an in vivo regulatory role in the developing growth plate. BIG-3 expressed in osteoblast dramatically accelerates the program of osteoblastic differentiation. This suggests that BIG-3 protein plays a role in the regulation of endochondral bone formation.1. In this study, we first analysed the homologogy and consistency of human and murine BIG-3 gene whole coding sequence by computer software. The results showed that the homologogy of coding sequence was 99.9% and the consistency of coding sequence was 87.7%. These sequences were highly conserved during the evolution. Then we analysed the homologogy and consistency of amino acid sequence by computer software. The results showed that homologogy and consistency of amino acid sequence were both 100%.2. We searched the BIG-3 gene sequence in Genebank, designed the specific primers that is based on this sequence and synthesized the primers in Shanghai. The total RNA was isolated from new-born mice skull, the cDNA was gained through RT-PCR. The vector and the cDNA were digested with two restriction enzymes. By ligation and transform into competent cells, the positive clone was selected, the sequence was identified and was constructed the prokaryote expression vector. The results suggested that the sequence of the gene we obtained consisted with the BIG-3 gene sequence in Genebank.3. The BL21 transfected with recombinant plasmid pGEX-4T-2-BIG-3 treatment with IPTG showed an expression line on position of 60kDa, which consisted with our design. After treatment with IPTG for 1h, the bacilli was collected and subjectedto lysis by ultrasonic. The supernatant flowed through the Sepharose 4B pole, samples were taken to do SDS-PAGE electrophoresis. Then we got the purified GST-BIG-3 fusion protein.4. The rabbits were immunized with the GST-BIG-3 fusion protein purified by electrophoresis to produce the poly-cloned antibody. The effect and specificity of antibody were assessed by double immunodiffusion and western blot. The results showed that the titer of antibody we got was 1:128 and the specificity was high.5. The pMSCVpuro-BIG-3 plasmid was constructed, the PT67 cell was transfected with pMSCVpuro-BIG-3 plasmid to produce the recombinant BIG-3 retrovirus. The retrovirus was identified by PCR. The results consisted with our design.6. The human BMSCs were cultured in 12-core plate. After cells achieved 60% fusion, the cells were infected with recombinant BIG-3 retrovirus 200ul. 7 days later, using the DMEM with 5 V-g/ml puromycin to select the cells, then alkal... |
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