| Oral carcinogenesis is a complex, multistep process, in this process the oncogene and tumor suppressor gene have got the same important function. Cell hybrid experiments show it is necessary that tumor suppressor gene lose for oral carcinogenesis. 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, p12 protein is a tumor growth suppressor coded by DOC-1. As a new candidate tumor suppress gene, up to the present its some functions and mechanisms have been studied. In short, p12 suppresses tumor growth by direct inhibition of DNA-pp or/with by negatively regulating the CDK2-mediated phosphorylation of DNA-pp, also probably by negatively regulating the active pool of CDK2, which to suppress the Rb activities; ectopic p12 in malignant hamster oral keratinocytes induces apoptosis, and in SW48 increased p12 is associated with significantly increased expression of proapoptosis components of the caspase cascade (caspase7,caspase9) .But still having a series problem to solve, for instance, how to induce apoptosis; why to be deleted in oral cancer; which phase to be deleted in oral cancer and so on. For further study on function and modulation of doc-1 and its protein pl2, at present it becomes the initial mission to generate antibody against p12. Objective: To predict and to design antigenic epitopes sequence inamino acid sequence of human p12; to generate and to identify the polyclonal antibody against p12-MAPs; to examine if there are speciality of histology and site in the loss of pl2 protein expression in the oral cancer, to evaluate the feasibility of this antibody in early diagnoses of oral squamous carcinoma. Methods:1. By bioinformatics technology and protein-nucleic acid analysis software, including GODEKEYE, EMBOSS, COILS 2.1, ANTHEPROT V5.0 etal, epitopes of p12 were analysed according to amino acide sequence (aa No. BAA22937) of human pl2. With its main function and general principle for designing antigenic determinant, the appropriate antigenic peptide sequence was choosed and designed.2. The p12-MAPs was synthesized using the solid synthetic peptide approach.3. According to the routine Immunization, the New Zealand rabbit was immunized by p12-MAPs to generate antibody against this antigenic peptide. The titer and the specificity of the polyclonal antibody was detected by ELISA and western blot.4. By immunohistochemical method and immunocytochemical method, p12 expression were examined in some cell lines and in the oral squamous carcinoma specimens including oral squamous carcinoma and premalignant lesions et al.5. Using tissue microarray , p12 expression characteristics were examined in multiple diseased tissue of oral cavity, soft tissue tumor, malignant tumor in different site, human fetus multiple organ tissues by immunohistochemical method.Result:1. The designed antigenic peptide sequence of pl2 is the ECLAETERNARS.2. 11mg of 8 branch p12- MAPS (ECLAETERNARS) was synthesized, its purity examined by HPLC: 89.43%.3. The PolyAb against p12-MAPs has been obtained, western blot suggests the polyAb may react to p12 in human placenta tissue and HaCaT cell line. ELISA suggests its titer was 1 -.51200.4. Cell Staining: p12 expression is positive in HaCaT cell, but is negative in SGC7901, Hela,Tca-8113 and 293 .5. P12 staining rate in the oral squamous carcinoma is obviously lower than premalignant lesions of oral cavity (p<0.05).6. P12 staning rate was reduced or absent not only in different site squamous carcinoma but also in the other tumor origined from different tissue.Conclusion:1. It is feasible to predict and design antigenic epitopes sequence in amino acid sequence by bioinformatics technology and protein-nucleic acid analysis software. Epitope sequence, ECLAETERNARS, can be used to generate antibody against p12.2. It is feasible to generate antibody against p12 by... |
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