| Tumor cells are transformed normal cells acquiring sequential mutations, which permanently damaged the genome. This damage leads to abnormal cell processes,including gene expression, signal transduction, and metabolism, which finally induce the malignant proliferation of tumor cells. However, CTLs have been reported that recognize epitopes of mutated proteins and aberrantly expressed protein. In some cases, patients bearing these CTLs, especially which recognized epitopes derived from mutated proteins, had better survival rate after immunological or other interventions which maintained the immune responses. These results indicated that tumor immunotherapy by vaccines derived from proper tumor antigens was practical in eradicating residue tumor cells by inducing and maintaining the anti-tumor immune responses in vivo. But regardless active immunotherapy or passive immunotherapy, a clinically successful vaccine should be derived from a tumor antigen, which is able to induce tumor specific immune responses.Although there has been a long search for tumor antigens, major progress has only recently been made in the molecular definition of human tumor antigens. nasopharyngeal carcinoma, one ofhuman common malignant tumors, cannot be easily diagnosed and has poor prognosis. Nasopharyngeal carcinogenesis is a multistage process involving interactions among multiple factors. The principal therapeutics is radiotherapy. Moiety patients prognosis is poor though improvement in therapeutic tool and method. Pathology found that massive leukomonocytes around nasopharyngeal carcinoma cell, display immunogenicity. So these expressed that immunologic system reacted in anti-carcinoma. In order to improve prognosis, it is crucial to screen and identify the tumor specific antigens for tumor diagnosis and immunotherapy.In this research, using xenogenic immune sera , tumor antigens were screened from a cDNA expression library of nasopharyngeal carcinoma by SEREX. To remive antibodies reactive with antigens related to the vector, sera were absorbed by E.coli lysates. Recombinant phage was amplified at a concentration of 2.5-3 X103 per 180mm plate, the library was screened by xenogenic immune sera in a 1:1500 dilution. A total 1.0 X 106recombinants were screened. After other there times screening, we got 18 positive clones, designated as NPC-1 to 18. Positive phage clones were converted to pBluescnpt phagemid forms by in vivo excision. Plasmid DNA was extracted and subjected to restriction enzyme analysis with EcoRI and Xhol, showed the inserts length were from 0.3kb to 3kb. All positive clones were sequenced using ABI Prism automated DNA sequencer. The positive nucleotide sequences of cDNA inserts were determined and analysed with DNASIS and BLAST software in EMBL and GenBank. These antigen genes included known genes, such as MAGE gene^ CREM gene> fi-2-microglobulin ^ Ribosomal Protein et al, and unknwn genes or ESTs in GenBank. mRNAexpression analysis of new ESTs in normal and malignant tissues or cell lines were evaluated by RT-PCR. The results showed that NPC-15 showed ubiquitous mRNA expression in all tissues. NPC-18 showed expression in tumors and cell lines but down-regulated in normal tissues of analyzed. NPC-14 was found to be expression in some tumors and cell lines but not expressed in normal tissues. At same time, we investigated 40 nasopharyngeal carcinoma patients and 40 health donors by Western Blotting and Secondary SEREX. Secondary SEREX results showed that auto-antibodies against NPC-14 NPC-15 and NPC-18 were detectable in some of tumor and normal samples, while positive rates of antibody against NPC-14 and NPC-18 in nasopharyngeal carcinoma patients were higher than those in healthy controls.In this study, we screened a cDNA expression library of nasopharyngeal carcinoma by SEREX, a new approach for tumor antigens screening, and got two antigens that may be valuable tumor markers in diagnosis of nasopharyngeal carcinoma, and to lay a foundation for future study of immunotherapy. |
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