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目的原核表达大肠癌负相关基因ST13，制备ST13蛋白单克隆抗体，研究人大肠等组织中表达的STl3蛋白的生物学特性。方法将ST13ORF克隆至GST融合蛋白表达质粒，原核表达并以Glutathione纯化GST-ST13蛋白，以凝血酶切得到ST13蛋白。以GST-ST13融合蛋白免疫小鼠，以ST13蛋白筛选杂交瘤细胞株，按杂交瘤技术制备STl3蛋白单克隆抗体，并以ST13蛋白亲和层析纯化单抗。以该单抗作Western-blot及免疫组化检测临床标本。结果蛋白表达和纯化以SDS-PAGE证实。融合蛋白表达量占大肠杆菌总蛋白20％，每升培养物回收融合蛋白2．5mg，纯度为91．3％。建立了3个ST13蛋白单抗的杂交瘤细胞株，腹水中单抗的ELISA效价达104～105，并具有对STl3蛋白的高度特异性。Westem-blot证实组织细胞中天然ST13蛋白的SDS-PAGE表观分子量约50KD，比其计算分子量大约10KD，但无糖基化修饰。免疫组化表明该蛋白均匀分布于SW480细胞及大肠上皮细胞浆，计算机分析系亲水性分子。免疫组化还表明该蛋白可表达于大肠、胃及肝等多种组织上皮，但在各种正常组织和肿瘤组织之间，正常组织之间，以及肿瘤组织之间的的着色强度不一。结论原核表达了ST13蛋白，制备了相应的单抗，建立了检测组织标本中ST13蛋白的方法。研究表明人组．织ST13蛋白系表观分?
Objective To express prokaryotic expression plasmid of ST13 in colorectal cancer and to prepare the monoclonal antibody of ST13 protein to study the biological characteristics of ST13 protein expressed in human colon and other tissues. Methods ST13 ORF was cloned into the GST fusion protein expression plasmid. The prokaryotic expression of GST-ST13 protein was purified by Glutathione, and the ST13 protein was obtained by thrombin digestion. The mice were immunized with the GST-ST13 fusion protein, the hybridoma cell lines were screened with ST13 protein, and monoclonal antibodies against ST13 protein were prepared by hybridoma technology. The monoclonal antibodies were purified by ST13 protein affinity chromatography. The monoclonal antibody was used for Western-blot and immunohistochemical detection of clinical specimens. Results Protein expression and purification was confirmed by SDS-PAGE. The fusion protein expression amount accounted for 20% of total E. coli protein, fusion protein 2.5mg per liter of culture recovery, purity of 91.3%. Three hybridoma cell lines with ST13 monoclonal antibody were established. The ELISA titer of McAbs in ascites was 104-105 and highly specific for ST13 protein. Westem-blot confirmed that the apparent molecular weight of SDS-PAGE of native ST13 protein in tissue cells was about 50KD, which is about 10KD more than its calculated molecular weight, but no glycosylation. Immunohistochemistry showed that the protein was evenly distributed in SW480 cells and large intestine epithelial cytoplasm, computer analysis of hydrophilic molecules. Immunohistochemistry also showed that the protein can be expressed in the large intestine, stomach and liver epithelium, but in a variety of normal and tumor tissue, normal tissue, and between the tumor tissue of different color strength. Conclusion The prokaryotic expression of ST13 protein, prepared the corresponding monoclonal antibody, established a method for detection of ST13 protein in tissue samples. Research shows people groups. Strain ST13 protein expression of the Department of points?