肿瘤相关抗原基因HCA520重组蛋白的表达纯化及其肝癌患者血清中相应抗体的分析  

Expression of a Tumor Related Antigen Gene HCA 520 Encoded Protein and Analysis of Its Serological Reactivity to the Sera of HCC Patients

作　　者：韩克军[1] 杨美香[1] 王歈[1] 李燕[1] 陈慰峰[1] 

机构地区：[1]北京大学医学部免疫学系,北京100083

出　　处：《中国生物化学与分子生物学报》2003年第1期31-35,共5页Chinese Journal of Biochemistry and Molecular Biology

基　　金：国家"973"基金资助项目 (No .G19990 5 3 90 4);北京市自然科学基金资助项目 (No .70 0 10 0 2 )~~

摘　　要：HCA5 2 0是用SEREX(serologicalidentificationofrecombinantcDNAexpressingcloning)方法 ,即用肝癌病人血清从肝癌组织cDNA表达文库中筛选得到的肝癌相关性抗原编码基因 .利用RT PCR技术检测了HCA5 2 0mRNA在各种组织中的分布情况 ,构建了GST融合表达载体并且用亲和层析的方法纯化表达的融合蛋白 .最后用Western印迹检测了重组蛋白的免疫反应性 ,用斑点印迹检测了肝癌病人血清中HCA5 2 0的天然抗体的存在情况 .结果表明 ,HCA5 2 0在各种组织中呈丰度差异分布 ,构建好的pGEX 4T 3重组载体经IPTG诱导后高效表达GST HCA5 2 0融合蛋白 ,其分子量约 4 9kD .经GST Agarose亲和层析 ,重组蛋白得到高度纯化 .Western印迹证实 ,纯化蛋白为目的重组蛋白 ,重组蛋白具有与天然蛋白相同或相似的免疫反应性 .斑点印迹分析表明 ,2 0份肝癌病人血清中有 1份HCA5 2 0抗体阳性 ,而 4份正常人均为阴性 .HCA5 2 0的足量提供 ,可用以研究其在致癌中的作用 ,并可以免疫动物制备抗体 .它作为抗原 ,分析其抗体在不同肿瘤病人中的表达情况 。To analyze tissue expression profile of HCA 520, a novel tumor related antigen encoding gene was cloned and its recombinant protein was prepared for investigating immunoreactivity toward antibody in patient serum. Tissue distribution of HCA 520 mRNA was detected by RT PCR. Its recombinant protein was prepared as GST HCA520 fusion protein in E.coli expression system and purified by GST agarose affinity resin. Immunoreactivity was analysed using Western blot and dot blot. In dot blot, serum samples obtained from hepatocellular carcinoma were used as the natural source of antibody. HCA 520 mRNA was detected in the majority of 16 normal tissues tested, especially in colon, small intestine, prostate and kidney. GST HCA520 fusion protein was successfully expressed and purified in E.coli . The molecular weight of purified protein was approximately 49 kD. The GST in fusion protein did not change the immunoreactivity of HCA520 protein. However, anti GST antibody was often raised in many HCC patients. The availability of HCA520 recombinant protein makes it possible to study its role in tumorgenesis and to analysis its potential in cancer diagnosis.

关 键 词：HCA520 肿瘤相关性抗原 重组融合蛋白 免疫反应性 

分 类 号：Q786[生物学—分子生物学]