肝癌标志物高尔基体蛋白73基因在大肠杆菌中的表达

Expression of the golgi protein 73 for the marker of hepatocellular carcinoma E.coli

目的克隆、表达和鉴定肝癌标志物高尔基体蛋A73（G1773）基因序列（1028～1327bp）。方法应用Biosun生物学软件分析GP73全序列，预测其抗原表位，将GP73蛋白基因克隆到表达载体PGEX-4T-2上，构建重组表达质粒PGEX-4T-2／GP73,转化大肠杆菌BL21，异丙基-β—D-硫代半乳糖吡喃糖苷（WFG）诱导表达，利用GST亲和层析柱对重组蛋白进行纯化．并用蛋白印迹分析技术（Western blot）和酶联免疫吸附测定方法（ELISA）检测其抗原性．并与商品化的GP73抗原进行比较。结果本研究纯化的GP73蛋白在大肠杆菌中获得高效表达，SDS～聚丙烯酰胺凝胶电泳（SDS—PAGE）显示其相对分子质量为37kD，与预计大小一致。Western blot和ELISA实验证实，纯化的GP73蛋白具有良好的抗原性。结论本研究成功克隆和表达了GP73序列．为制备抗体和肝癌诊断试剂的研发奠定了实验基础。

Objective To clone and express the recombinant golgi protein 73 （GP73） and make preparations for antibody preparation and detection. Methods The amino acid sequences of the GP73 were analyzed and aligned using the Biosun software. The gene of antigen epitope was cloned into PGEX-4T-2 vector to construct the recombinant plasmid PGEX-4T-2/GP73. Recombinant GP73 protein was expressed in E.coli and was purified by GST QZT 4FF. The reaction was evaluated by Western blot and ELISA. Results The recombinant capsid gene could be over experssed in E.coIi and had well antigenicity. Conclusion The capsid protein GP73 is successful cloned and expressed, which could be useful for developing antibody preparation and diagnose reagent for HCC.