抗人肝癌单克隆抗体嵌合轻链在巴氏毕赤酵母的高效分泌表达  

Secretory expression of chimeric light chain of HAb 18 against human hepatoma in Pichia pastoris

作　　者：董红霖[1] 邢金良[2] 杨向民[2] 姚西英[2] 李郁[2] 窦科峰[1] 陈志南[2] 

机构地区：[1]第四军医大学西京医院肝胆外科 [2]第四军医大学细胞工程研究中心,陕西西安710033

出　　处：《第四军医大学学报》2005年第8期734-736,共3页Journal of the Fourth Military Medical University

基　　金：国家 863计划重点课题基金项目(2001AA2l5101)

摘　　要：目的: 通过整合,用巴氏毕赤酵母表达抗人肝癌mAbHAb18的cFab的嵌合轻链.方法: 将抗人肝癌mAbcFab/HAb18原核表达载体pET32a/cFab中的cL亚克隆到酵母表达载体pPIC9K,构建成重组质粒pPIC9K/cL测序鉴定.重组质粒pPIC9K/cL整合到酵母菌GS115的染色体上,经G418筛选得到高拷贝转化子及Mut表型鉴定后,用含5mL/L甲醇的培养基诱导表达.结果: 该系统成功表达了抗人肝癌mAbHAb18cFab的嵌合轻链,表达水平为22mg/L;WesternBlotting证实,表达产物具有良好的HAb18GE结合活性和特异性.结论: 嵌合轻链/HAb18在巴氏毕赤酵母获得成功表达,为高效分泌表达cFab抗体奠定了基础.AIM: To express secretively chimeric light chain of HAb18 against human hepatoma in Pichia pastoris.METHODS: Genes of cL chain fragment of cFab antibody HAb18 from the plasmids pET32a/Fab were subcloned into vectors pPIC9K.After confirmed by DNA sequence analysis,the recombinant plasmid pPIC9K/cL was transducted into the genome of GS115 Pichia pastoris using integrating technology.Mut +multiple inserted transformants were screened by G418 ○R and induced by 5 mL/L methanol to express.RESULTS: After 4 days of methanol induction,high-yield secretory expression (22 mg/L)of the cL fragments was achieved.Western Blotting assay proved that the expressed protein had specific binding activity with HAb18GE antigen.CONCLUSION: The successful expression of cL of HAb18 in Pichia pastoris lays a solid foundation for the secretory expression of cFab HAb18.

关 键 词：肝肿瘤 抗体 单克隆 嵌合轻链 毕赤酵母 分泌表达 

分 类 号：R256.4[医药卫生—中医内科学]