摘要
目的 在CHO细胞中表达基孔肯亚病毒人 鼠嵌合抗体 ,并对其活性进行分析。方法以质粒pcDNA5 /FRT为骨架 ,应用pCI dhfr1(本室构建并保存 )的dhfr基因和hCMV启动子及SV4 0polyA ,构建了含有共扩增基因的哺乳动物双启动子表达载体 ,RT PCR克隆具有中和活性鼠源单克隆抗体的轻重链可变区基因 ,与人IgG1轻重链恒定区基因融合后亚克隆到表达载体pA的多克隆位点 ,构建了表达质粒pA HL ,脂质体转染CHO(dhfr )细胞 ,撤掉胸腺嘧啶和次黄嘌呤并不断提高MTX的浓度 ,筛选表达量高的克隆 ,扩大培养、收集上清 ,并纯化 ,纯化的嵌合抗体进行SDS PAGE、Western印迹、抗原结合活性和微量细胞中和实验。结果 在MTX的浓度为 2× 10 -7mol/L时 ,获得稳定表达细胞株 ,表达量为 5mg/L。结论 成功地在CHO(dhfr )细胞表达了具有中和活性的基孔肯亚病毒人 鼠嵌合抗体 ,为制备抗基孔肯亚病的被动免疫制剂奠定了良好的基础。
Objective To express human-mouse chimeric antibody against Chikungunya virus and to analyze its biological activity. Methods A new mammalian bipromoter expression vector was constructed with dihydrofolate reductase (dhfr) gene as the selection and coamplication marker. We cloned the light and heavy chain variable region gene of the anti-CHIK mouse monoclonal antibody CHIK-IF1 by RT-PCR and cloning human IgG1 constant region gene respectively, then cloning human-mouse chimeric antibody gene by fusion PCR, and then inserting human-mouse chimeric antibody gene into MCS of pA, constructing a expressing plasmid pA-HL. Plasmid pA-HL was transfected into CHO(dhfr-) engineering cells and high production cell clones were screened by enhancing MTX concentration. Collecting medium and purifying chimeric antibody with affinity chromatogram, purified chimeric antibody was used to SDS-PAGE, Western blot. Results A stable and high production cell line was acquired at MTX concentration 2×10 -7mol/L. Conclusion The human-mouse chimeric antibodies were successfully expressed in CHO cells. [
出处
《中华微生物学和免疫学杂志》
CAS
CSCD
北大核心
2004年第10期785-789,共5页
Chinese Journal of Microbiology and Immunology
基金
总后十五指令课题 ( 18 D5 0 1d)
