人类肿瘤相关蛋白hyrdC的原核表达、抗体制备及其在胃癌中的初步检测

Human tumor-related protein hyrdC: prokaryotic expression,antibody preparation and its preliminary detection in gastric cancer

目的:制备原核表达的hyrdC纯化蛋白及相应单克隆抗体,并以该抗体初步检测胃癌中hyrdC蛋白的表达。方法:用RT-PCR方法获取人hyrdC基因序列,插入原核表达载体pET32a构建重组质粒,纯化得到TRX-hyrdC融合蛋白,将其作为抗原免疫BALB/c小鼠,按常规方法制备抗hyrdC单克隆抗体,以ELISA、Western印迹法筛选及鉴定;采用免疫组织化学法(SP法)比较胃癌组织及瘤旁正常组织中hyrdC抗体水平的差异。结果:测序结果显示克隆的hyrdC ORF与GenBank中登录的序列一致。所获TRX-hyrdC融合蛋白相对分子质量与理论计算值相符,获得2株分泌抗hyrdC单克隆抗体的细胞株;Western印迹法显示该抗体能特异性地结合人hyrdC蛋白;SP法测定显示,hyrdC蛋白在胃癌组织中表达明显高于正常胃组织(P<0.001)。结论:成功制备了抗hyrdC单克隆抗体;SP法测定胃癌组织与瘤旁正常组织中hyrdC抗体水平有显著差异。

Objective： To prepare pure prokaryotically expressed protein of hyrdC and the monoclonal antibody against hyrdC, and to detect the hyrdC protein in gastric cancer with the prepared monoclonal antibody. Methods： The target DNA sequence of hyrdC was obtained by RT-PCR and subsequently inserted into prokaryotic expression vector pET32a. The TRXhyrdC fusion protein was purified and was used to immunize 6-week-old BALB/c female mice to prepare monoclonal antibodies. The supernants of hybridoma cells were screened by enzyme-linked immunoabsorbent assay（ELISA） and identified by Western blot. The prepared monoclonal antibody was used to detect the expression of hyrdC protein in gastric cancer and adjacent normal tissues by immunohistochemical method （SP）. Results： The sequence of hyrdC ORF was conformed to the reported in GenBank. Two lines of hybrids secreting monoclonal antibodies against hyrdC were established,both belonging to IgG2a subtype. Western blot showed that the prepared monoclonal antibodies specifically combined with human hyrdC protein. Immunohistochemistry （SP） results showed that the expression of hyrdC protein in gastric cancer was significantly higher than that in normal gastric mucous membrane （P〈0. 001）. Conclusion： The monoclonal antibody against hyrdC has been successfully obtained. There are significant differences in the expression of hyrdC between the gastric tumor tissues and normal tissues.