肝细胞癌相关肿瘤抗原基因的筛选与分析

Screening and analysis of genes encoding hepatocellular carcinoma associated tumor antigens

目的筛选和分析肝细胞癌相关的肿瘤抗原。方法用肝细胞癌组织构建cDNA表达文库,通过重组cDNA表达文库血清学分析法,用自体患者和异体患者的血清对文库进行筛选。将所得阳性噬菌体克隆体内剪切获得其pBK-CMV噬菌粒。通过限制性核酸内切酶鉴定插入cDNA片段,并作测序和生物信息学分析。同时将阳性克隆中的LIMS1插入片段进行原核表达。结果自体血清筛选得到14种基因,异体筛选获得11种基因,两组中均筛选到kinectin,共有24种肝细胞癌相关的肿瘤抗原基因。其中有8种基因目前还不清楚其功能,其余16种基因根据确定的或者推断的功能可以分成8组。LIMS1原核重组蛋白表达成功。结论本研究为肝细胞癌的免疫治疗提供了候选基因,并为理解肝细胞癌发生和发展的机制提供了线索。

Objective To screen and clone the genes encoding hepatocellular carcinoma associated tumor antigens. Methods A hepatocellular carcinoma cDNA express library was constructed with ZAP vector and analyzed by serological analysis of recombinant cDNA expression library (SEREX) with sera from autologous and allogenous patients. Monoclonalized positive phage clones were converted into pBK-CMV phagemid forms by in vivo excision. The cDNA inserts were determined by restriction endonuclease digestion with EcoR I and Xho I. The cDNA inserts were sequenced and analyzed with bioinformatics. LIMS1 insert was cut from the clone HCL5-70 and constructed into pQE 31 express vector. The recombinant LIMS1 was expressed in M15 and analyzed with SDS-PAGE and Western blot. Results Fourteen genes were cloned from autologous screening and eleven genes were obtained with allogeneous analysis. One gene, kinectin, was identified in both autologous and allogeneous screening. Eight of the total twenty-four genes were unknown for their functions; the other sixteen genes can be classified into eight groups according to their established or putative function. Recombinant LIMS1 was expressed in M15. Conclusion The identification of hepatocellular carcinoma associated tumor antigens provides potential targets for immunotherapy of hepatocellular carcinoma patients and will help in the understanding of the carcinogenesis of hepatocellular carcinoma.