人组织中大肠癌负相关基因ST13的蛋白表达研究

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ST13蛋白的生物学特性。方法将ST13 ORF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ST13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10^4-10^5，并具有对ST13蛋白的高度特异性。Western—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蛋白系表观分子量50KD的细胞浆可溶性分子，可表达于大肠等多种上皮组织。ST13蛋白在大肠癌组织呈低表达，在多种正常和肿瘤组织中的表达程度不一。cDNA同源性及蛋白质特性比较表明ST13与Hip(hsp70-interacting protein)为同一蛋白质，提示ST13／Hip经Hsp70等分子伴侣途径而在大肠癌发生发展中起作用。

Characterization of ST13 Protein Expression in Human Colorectal Cancer Tissues

Objective: To characterize the expression of ST13 protein in human tissues for investigation of the function of colorectal cancer related gene ST13.Methods: ST13 ORF was cloned and over-expressed in E.coli. The recombinant ST13 protein was purified by affinity chromatography. ST13 monoclonal antibodies were generated and affinity purified with the recombinant protein. Immunoblot and immunohistochemical staining were employed to analyze ST13 protein expression in human tissues. Results:The expression and purification of the recombinant ST13 protein were confirmed by SDS-PAGE. The protein yield reached about 2.5 mg/L of induced bacterial culture with a purity of 91.3%. Three strains of hybridoma were obtained with antibody titers from 10⁴ to 10⁵ in ascites fluids and with high specificity for ST13 protein. Immunoblot showed that the apparent Mr of ST13 protein in SW480 cells and human tissues estimated by SDS-PAGE mobility was approximately 50000, which was about 10000 larger than the 41324 calculated, but the glycosylation of the protein was excluded. Computer modeling revealed the protein to be a hydrophilic molecule. Immunohistochemical staining showed that ST13 protein was evenly distributed in cytoplasm and expressed in colon, stomach, liver, and other epithelial cells. Differences in the staining intensity of the protein were observed between normal and cancer tissues as well as among different normal or carcinoma tissues. Conclusion: ST13 protein is a cytoplasmic molecule with an apparent Mr of 50000. The protein is expressed in colorectal and other epithelial tissues. The expression level of the protein is down-regulated in colorectal cancer and varies among different normal and/or carcinoma tissues. Comparison of cDNA sequences and protein characteristics indicates that ST13 protein and hsp70-interacting protein (Hip) are same proteins, raising the possibility that ST13 protein is involved in the development of colorectal cancer through Hsp70 molecular chaperone machinery.