| Backgrounds: The esophageal carcinoma is one of common malignant tumors of digestive system. The incidence rate in malignant tumors is situated in sixth in the world. 70 percent of esophageal carcinomas were happened in our country and the incidence rate is situated in fourth. It can not be diagnosed in early stage. When the symptoms appear, most of the cancer cells have been diffused and metastasesed. The survival rate in five years of esophageal carcinoma hovered at 10 percent all the time. The outcome of esophageal carcinoma is far from satisfaction to the advanced stage with metastasesed and recurred patients by the traditional treatment such as surgery, radiotherapy and chemotherapy. If diagnosed and treated in the early stage, however, the survival rate will be greatly improved. Therefore, the best approach to make the patients with esophageal carcinoma live longer is to diagnose and treat in the early stage. At present, tumor immunology becomes new studying pattern and focus. Identifying human esophageal squamous carcinoma-specific/associated antigens by combining tumor immunology theory and molecule biology technology will lay foundation for diagnosing esophageal carcinoma in the early stage and developing vaccines. It can also provide a lot of molecule information for approaching molecule mechanism of tumor genesis and growth.Objectives: In this trial, we screened cDNA expression library of human esophageal squamous carcinoma to find new tumor antigens by using SEREX.Methods: The SEREX(serological identification of antigens by recombinant cDNA expression libraries)is a convenient and effective method to identify tumor antigens by screening of the cDNA expression libraries derived from human tumors with autologous or allogeneic sera antibody of patients with esophageal carcinoma. Firstly, the sensitive E.coli XL-1 Blue and E.coli XL-1 Blue lysate were prepared and the sera of the patients with esophageal carcinoma were pre-absorbed by the extracted E.coli XL-1 Blue lysate. Secondly, incubate the recombinant phage and transfer the plagues to nitrocellulose. Thirdly, immunoreaction was conducted on the NC membranes to identify the positive phage clones. The pre-absorbed serum of the patients is the first hand antibody and the HRP-conjugated goat anti-human IgG is the secondary antibody. Fourthly, the positive phage clones were screened secondly and thirdly with same procedures, in order to exclude false positive clones. At last, the cDNA of the exact positive clones were amplified by PCR and ligated to pUCm-T vector. Then, the nucleotide sequences of cDNA inserts were determined by bio-tech corporation. Sequence alignments were performed with BLAST software on GenBank databases.Results: Three rounds of serological screening of the cDNA expression library of human esophageal squamous carcinoma yielded 3 positive monoclones. The size of cDNA inserts were in the rang of about 750-1500bp. The nucleotide sequence of the first positive clone had a 98% homology with Homo sapiens ribosomal protein S13(RPS13). The nucleotide sequence of the second positive clone had a 99% homology with Homo sapiens connective tissue growth factor(CTGF) and the third had a 98% homology with Homo sapiens cAMP responsive element binding protein 1(CREB1).Conclusions: In this trial, we identified 3 different antigens by SEREX relating to cell metabolism, differentiation and transduction. They are highly expressed in some kinds of tumors and can induce serological immunoreactions of esophageal carcinoma. It suggests that they may play important roles in the incidence and the development of esophageal carcinoma. The next step, we will further study of the expression of these genes in normal tissues, esophageal carcinomas and different tumors. It will make contribution to supply candidate markers for diagnosis, vaccine development and pathogenesis research of esophageal carcinoma. |
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