Cloning,Expression Of DOC-1 And Preparation And Application Of The Polyclonal Antibody Against DOC-1

Oral cancer is a common disease of a serious threat to human health, in recent years one of the important issues covered in the medical field. With the advance in molecular techniques, the study on the pathogenetic mechanism and the relationship among tumor, oncogene and tumor suppressor genes at molecular level has become one of the main developing directions in gene therapy on tumor. Mutation, deactivation and deregulated expression of oncogenes and tumour suppressor genes may be involved in the pathogenesis of oral cancer. Deleted in oral cancer-1 (DOC-1) is a novel putative oral tumor suppressor genes identified and isolated from the hamster oral cancer model in 1995 and p12 protein is a function of its encoded protein with tumor growth inhibition. As a new candidate tumor suppressor gene has been concerned by medical researchers. The DOC-1 gene is located on chromosome 12q24 and its full length cDNA is 1627bp. Translation of the DOC-1 cDNA predicts a protein of 115 amino acids residues with a molecular weight of 12.4kDa. DOC-1 induces apoptosis in malignant hamster oral keratinocytes. Recently, p12 has been show to be an S-phase regulator through two important cellular partners: CDK2 and DNA polymerase-a-primase. p12 is a CDK2-associated protein that negatively regulates its kinase activity, p12 suppresses tumor growth through reduction of the expression level of CDK2 activity, which to suppress the Rb activities. Ectopic expression of p12 in squamous carcinoma cells reversed the malignant phenotype of these cells, in part due an ability of p12 to bind to both DNA polymerase-a-primase and to cyclin-dependent kinase 2 (CDK2), thereby inhibiting their activities. In normal epithelial cells, transforming growth factor-P-1(TGF-P-1) induces p12 expression transcriptionally, which, in turn, mediates the growth inhibitory activity of TGF-β-1. In order to further study the role and regulation of DOC-1 and its protein in oral carcinogenesis process, it become the primary task of generate antibody at present.In order to address the function of DOC-1, we cloned the CDS of DOC-1 by RT-PCR strategy. Then we constructed the expression vector of this CDS and expressed protein in E.coli. The expressed protein was used as immunogen for making antibody. A rabbit polyclonal anti-p12D0C-1 protein serum was successfully obtained.Objective: To clone DOC-1 gene, construct prokaryotic expression vector of DOC-1 gene, express and purify its recombinant protein, prepared antibody against it for further study on function and modulation of doc-1 and its protein p12DOC-1 in Squamous Cell Carcinoma of the human oral cavity.Methods: l.The total RNA was extracted from the brain of human embryo. The segment of coding sequence of DOC-1 was amplified by RT-PCR and was further cloned into the vector(pMD18-T)and the prokaryotic expression vector(pGEX-4T-1) by turns, to produce the new construct pGEX-4T-1-D0C-1. Then the pGEX-4T-1-D0C-l identified by restriction enzymes digestion analysis and sequencing. 2. After sequenced, the pGEX-4T-1-D0C-1 was transformed into E.coli. GST-p12 recombinant protein was expressed under IPTG induction and purified by affinity column and SDS-PAGE. 3. The interest protein band was cut out as antigen, immunized the New Zealand rabbit to produce polyclonal antibodies. The specific antibody against p12DOC-1 was identified by Western blot. Detect titer of antiserum by means of Western blot. 4. We use the polyclonal antibody to detect the existence of p12^DCC-1 in the normal oral mucous membrane tissue and not in the oral cancer through the method of immunohistochemistry. Results: The segment of coding sequence of p12^DOC-1 was specifically amplified by RT-PCR from the brain of human embryo, and obtained approximately 348bp band as expected. DOC-1 was further cloned into the vector (pMD18-T) and the prokaryotic expression vector (pGEX-4T-1) by turns, and the sequence was confirmed. The constructed vector of pGEX-4T-1 -DOC-1 was transformed into E.coli BL21.After IPTG induction and affinity chromatography, a new protein band about Mr 38000 showed on SDS-PAGE and the high purity GST-p12 fusion protein was obtained. The interest protein as antigen, immunized the New Zealand rabbit to produce polyclonal antibodies. Western-blot analysis showed the antiserum could combine with prokaryotic expressed fusion protein. Immunohistochemistry suggests the polyclonal antibodies may react to p12 in the normal oral mucous membrane tissue.Conclusion: The prokaryotic expression vector pGEX-4T-1-doc-1 was constructed successfully, and the high purity GST-p12 fusion protein was obtained. GST-p12 antibody was generated in rabbit. Western blot analyses showed that the antibody had high titer and specifically recognized GST-p12 fusion protein in vitro, which will facilitate the study of the function and modulation of p12^DOC-1 protein in oral cancer.In a word, it provides important experiment instrument for further studying the function and regulation of p12 to have obtained the polyclonal antibody against p12, also lays a important experiment foundation for further study the function of p12 in oral carcinogenesis.