【摘要】 制备OVA12蛋白和OVA12单抗,探讨其在临床肿瘤辅助诊断中的应用前景。将重组质粒pET32a-OVA12转化至BL21(DE3)中,经诱导表达、亲和层析纯化和超滤获得纯化了OVA12蛋白;应用杂交瘤技术制备并通过亲和层析技术纯化得到OVA12单抗;合成OVA12抗原多肽片段,分析单抗识别的抗原表位;建立ELISA法,用纯化的OVA12蛋白及其抗原多肽分析肿瘤患者血清中抗OVA12抗体的含量;用单抗经免疫组化法检测临床不同类型肿瘤组织中OVA12蛋白的表达差异。结果显示,获得了纯化的OVA12蛋白和两株OVA12单抗;检测得知,肿瘤患者血清中的抗OVA12抗体水平显著高于健康人血清;OVA12蛋白在肿瘤组织中的表达也高于癌旁组织及正常组织,提示OVA12蛋白和抗OVA12抗体可作为一个新的肿瘤标志物应用于临床肿瘤的辅助诊断。更多还原

【Abstract】 This study aims to explore the application prospect of OVA12,a new tumor-associated antigen,in clinical tumor assisted diagnosis and as a molecular target for cancer.The recombinant plasmid pET32a-OVA12 was transformed into E.coli BL21(DE3)for prokaryotic expression.OVA12 fusion protein was purified by nickel affinity chromatography and ultra filtration.Two strains of anti-OVA12 monoclonal antibody were obtained by hybridoma technique and affinity chromatography.Antigen polypeptides were synthetized to identify the antigen epitope.By establishing an ELISA method,we determined the expression level of anti-OVA12 antibody in serums of different kinds of tumor patients with OVA12 and the designed polypeptides.And we determined the expression level of OVA12 in different kinds of tumor tissues with the two strains of monoclonal antibodies by immunohistochemistry.The results showed that we have obtained purified OVA12 and its monoclonal antibodies.The expression level of anti-OVA12 antibody in the serum of tumor patients tested was significantly higher than that in serum of healthy people.The result of antigen peptides screening sera showed the same trend.And OVA12 expression in tumor tissues was relatively higher than non-tumor tissues,indicating that OVA12 may act as a new tumor marker which might be applied to clinical auxiliary diagnosis for tumor.更多还原