人IgG Fc基因克隆及人源抗ImsAg全分子抗体在CHO细胞中表达  

Cloning of Human IgG Fc cDNA and Expression of Whole Human Anti-HBsAg Antibody in CHO Cells

作　　者：童贻刚[1] 徐静[1] 刘国奇[1] 张永国[1] 陈万荣[1] 刘树玲[1] 王海涛[1] 

机构地区：[1]军事医学科学院微生物流行病研究所,北京100071

出　　处：《中国生物学文摘》2006年第1期30-34,共5页Chinese Biological Abstracts

基　　金：国家863基金资助项目.No.Z18-01

摘　　要：利用mRNA提取试剂盒直接从健康人外周血白细胞中提取mRNA，逆转录为cDNA，合成引物扩增人抗体分子IgG1亚型Fc基因，克隆到PGEMT-Vector中并测序。合成引物扩增人抗体分子轻、重链信号肽序列，分别克隆并测序。将轻链信号肽和轻链（kappa）VL-CL基因进行重组形成轻链全分子基因，再将其克隆到哺乳细胞表达载体pcDNA3．1中。将重链信号肽、重链（gamma）V H-CH1和Fc基因进行重组形成重链全分子基因，再将其克隆到哺乳细胞表达载体pCI-DHFR1中。将轻、重链全分子表达载体同时转染中国仓鼠卵细胞（CHO细胞），以药物G418和氨甲喋呤（MTX）筛选细胞克隆，检测细胞克隆的培养上清中抗HBsAg抗体活性，结果筛到数株较高表达的克隆．当MTX药物浓度增加到1×10^-7mol／L时，平均每10^6个细胞培养上清抗HBsAg最高表达虽可达8μg/d。回收细胞培养上清，以Protein L亲和层析柱纯化重组抗体。该抗体在非还原SDS-PAGE中表现为一条高分子量（〉100kD）的蛋白质带。在还原SDS-PAGE中表现为一条约50kD的带和一条约25kD的带，Western印迹分析结果显示．该蛋白质的全分子和重链分子可以和羊抗人Fc抗血清发生特异性免疫反应。Messenger RNA was extracted from human peripheral lymphocytes and first strand cDNA was prepared by reverse-transeiption. The cDNA of Fc fragment of human IgG1 was then obtained by PCR and was cloned into the p GEM T-vector. The DNA sequences encoding signal peptides of both light and heavy chains were synthesized and cloned, respectively. For construction of the light chain expression plasmid, the light chain signal sequence was linked with the light chain variable and constant regions （ VL-CL ） which had been cloned previously by screening of phage display libraries with HBsAg. The resulting full-length light chain sequence was then inserted into pcDNA3.1, a mammalian expression vector. For construction of the heavy chain expression plasmid, the heavy chain signal sequence, the variable region, the first constant region （ VH-CH1, cloned previously by screening of phage display libraries with HBsAg） and Fc fragment sequence were ligated to form a fulllength heavy chain ORF, which was then cloned into another mammalian cxprcssion vector, pCFDHFR1. CHO（ dhfr ） cells were cotransfected with the above light and heavy chain expression plasmids, and cell clones expressing human anti-HBsAg antibodies were selected by G418 and methotrexate （MTX）. The recombinant human antibodies were purified with protein L affinity chromatography from the cell culture medium. As human serum IgG, the recombinant IgGexhibited only one band with a molecular weight of more than 100 kD in nonreducing SDS-PAGE; In reducing SDS-PAGE, however, it turned out to be two bands of approximately 50 kD and 25 kD, respectively. Western blot analysis demonstrated that the whole IgG in the norrreducing SDSPAGE, and the heavy chain in the reducing SDS-PAGE both reacted with goat anti-human Fc antiserum.

关 键 词：抗体工程 全分子抗体 哺乳细胞 基因表达 

分 类 号：Q78[生物学—分子生物学] Q785