用定点整合系统表达抗基孔肯亚病毒人-鼠嵌合抗体的研究  

Expression of human-mouse chimeric antibody directed against Chikungunya virus with site-specific intergration system

作　　者：李建民[1] 陈薇[1] 贾秀杰[2] 安小平[1] 李冰[1] 樊英茹[1] 童贻刚[1] 

机构地区：[1]军事医学科学院微生物流行病研究所应用分子生物学研究室,北京100071 [2]吉林市昌邑区卫生防疫站,吉林吉林132000

出　　处：《细胞与分子免疫学杂志》2005年第3期312-315,318,共5页Chinese Journal of Cellular and Molecular Immunology

基　　金：解放军总后勤部"十五"指令性课题资助(No.18D501d)

摘　　要：目的:构建含有FRT序列的CHO/dhfr-工程细胞株,并在此细胞株中用定点整合系统表达抗基孔肯亚病毒人鼠嵌合抗体。方法:首先克隆FRT序列与HBsAg的融合基因FRT-HBsAg,然后将FRT-HBsAg亚克隆到pCI neo的MCS中,构建表达载体pCI FRT-HBsAg。以此载体转染CHO/dhfr-细胞,用1g/L的G418筛选抗性克隆。检测抗性克隆HBsAg的表达,筛选表达量高的克隆作为宿主细胞株,命名为CHO/dhfr-FRT+。将表达质粒pA FRTHFLF和pOG44以1∶9的质量比混合,取2μg转染CHO/dhfr-FRT+。转染48h后,换选择培养基(撤掉H、T)用96孔板进行克隆化,2wk后对长成的单克隆进行检测。筛选正确整合到CHO/dhfr-FRT+细胞中FRT位点的克隆,并不断提高MTX的浓度,进一步筛选表达量更高的克隆。在MTX的浓度为2×10-7mol/L时,获得稳定表达细胞株,其最后的表达量为5mg/L。对此细胞株扩大培养后,收集上清并纯化,以纯化的嵌合抗体进行SDS-PAGE及Westernblot和抗原结合活性的检测。结果:构建了高转录活性位点整合FRT序列的CHO/dhfr-细胞株,并在此细胞株中表达抗基孔肯亚病毒人鼠嵌合抗体,表达量为5mg/L。结论:构建了含有FRT序列的CHO/dhfr-工程细胞株,并在此细胞株中用定点整合系统表达抗基孔肯亚病毒人鼠嵌合抗体,为用此系统表达抗体分子和其他蛋白分子奠定了基础?AIM: To obtain CHO/dhfr^- cells line with integrated FRT sequence in the chromosome transcription active site and to express human-mouse chimeric antibody directed against Chikungunya Virus by using the cell line. METHODS: The fusion gene of FRT and HBsAg was constructed by PCR and cloned into the MCS of pCI-neo to construct pCI-FRT-HBsAg. The pCI-FRT-HBsAg was transfected into CHO/dhfr^- cells and cell clones with high expression of HBsAg were screened by detecting the amount of HBsAg with ELISA. A CHO cell clone with the highest expression was chosen and named as CHO/dhfr^- FRT^+. pAFRT HFLF, a expression plasmid of chimeric antibody with RFT sequence was transfected into CHO/dhfr^- FRT^+ cells and cell clones with high expression of the chimeric antibody were screened by increasing concentration of MTX. A CHO cell clone with high expression of the chimeric antibody was cultured in large scale and supernatant was collected from which the chimeric antibody was purified. The purified chimeric antibody was analyzed by SDS-PAGE, Western blot and IFA. RESULTS: A CHO/dhfr^- cells line with integrated FRT sequence in the chromosome transcription active site was obtained successfully. A cell clone with yield of 5 mg/L of chimeric antibody was obtained, as compared with routine CHO cell expression system with a yield of (2 mg/L). CONCLUSION: A cell line with integrated FRT sequence in the chromosome transcription active site was obtained and with it human-mouse chimeric antibody directed against Chikungunya virus was expressed, it lays a solid foundation which can be used for expressing antibodies and other proteins.

关 键 词：定点整合 FRT CHO/dhfr^-细胞 基孔肯亚病毒 嵌合抗体 

分 类 号：R392.11[医药卫生—免疫学]