抗人肝癌基因工程嵌合抗体的构建及在Sp2/0细胞的稳定表达  

Construction of an engineering chimeric antibody against human hepatoma and stable expression in Sp2/0 cell

作　　者：邢金良[1] 杨向民[1] 宋斐[1] 姚西英[1] 陈志南[1] 

机构地区：[1]第四军医大学基础部细胞工程研究中心,西安710032

出　　处：《中华微生物学和免疫学杂志》2004年第6期480-483,共4页Chinese Journal of Microbiology and Immunology

基　　金：国家自然科学基金课题 (No .3 0 2 0 0 3 3 0 )

摘　　要：目的　构建抗人肝癌嵌合抗体 ,并在Sp2 0细胞中进行稳定高效表达。方法　分别将HAb18轻、重链可变区基因克隆入真核表达载体pDHL构建重组载体pDHL chHAb18。酶切及测序鉴定后 ,转染Sp2 0细胞 ,经甲氨蝶蛉 (MTX)筛选获得抗性克隆。采用有限稀释法单克隆化后持续MTX加压培养。通过ELISA筛选高效表达的单克隆细胞株。表达产物纯化后 ,进行SDS PAGE和Westernblot检测 ,同时采用ELISA和免疫荧光法检测其抗原结合特异性。结果　成功构建了重组嵌合IgG抗体chHAb18的真核表达载体pDHL chHAb18。转化Sp2 0细胞后 ,初步筛选得到了高效表达chHAb18的细胞株。经MTX加压培养 ,1株细胞的chHAb18表达量达到 10mg L。SDS PAGE和Westernblot检测结果表明 :目的蛋白分子质量约为 16 0× 10 3,还原后为 2条带 ,分子质量约为 5 2× 10 3和 2 7× 10 3。上述蛋白条带均与羊抗人IgG抗体特异结合。结论　成功构建了抗人肝癌基因工程嵌合抗体chHAb18,并在骨髓瘤细胞中获得高效表达。为其进一步的应用研究奠定了基础。Objective To construct a chemeric antibody against human hepatoma which stably and highly express in Sp2/0 cell. Methods The V H and V L genes of McAb HAb18 were separately cloned into eukaryotic expression vector pDHL to construct a recombinant vector pDHL/chHAb18. The vector pDHL/chHAb18 was transfected into Sp2/0 cells and positive clones were obtained by MTX screening. The Sp2/0 cells containing pDHL/chHAb18 were monoclonized by limited dilution method and continuously cultured with an increased concentration of MTX. Moreover, monoclonal cell strains with high expression of chHAb18 were screened by ELISA. The antigen binding specificity was identified by ELISA and immunofluorescense staining. Results A eukaryotic expression vector pDHL/chHAb18 of recombinant chimeric antibody chHAb18 was successfully constructed. The cell strains highly expressing chHAb18 were obtained by screeing. Among them, the antibody expression level of one strain cell is up to 10mg/L after continuously culturing with increased MTX concentration. The molecular weight of protein products was approximately 160kD under unreduction condition, and it became two new bands after treatment with reduction reagent, with the molecular weight of 52kD and 27kD, and that all bands can bind with goat anti-human IgG. Conclusion An engineering chimeric IgG antibody against human hepatoma was successfully constructed and highly expressed in Sp2/0 cell, which lay a solid foundation for application of chHAb18 in the treatment of human hepatoma.

关 键 词：抗人肝癌基因工程 抗体 sp2/0细胞 基因表达 生物学活性 

分 类 号：R735.7[医药卫生—肿瘤]