摘要
目的构建抗CD25人鼠嵌合抗体基因真核表达质粒,并在293T细胞中进行瞬时表达。方法采用RT-PCR技术,设计针对信号肽的简并引物,钓取抗CD25杂交瘤细胞的重、轻链可变区基因,以质粒PAG4622为模板,钓取人抗体IgG1重、轻链恒定区基因,分别将重、轻链可变区基因与相应的恒定区基因进行拼接,将完整的重、轻链嵌合基因分别与真核表达载体pOptiVEC和pcDNA3.3连接,构建抗CD25人鼠嵌合抗体基因真核表达质粒,将其通过脂质体法共转染至293T细胞中进行表达。通过RT-PCR法检测嵌合抗体基因的转录水平,ELISA法检测嵌合抗体的表达量,流式细胞术(FCM)分析嵌合抗体的结合活性。结果抗CD25人鼠嵌合抗体基因真核表达质粒构建正确;RT-PCR显示嵌合抗体基因在293T细胞中成功转录;转染后48、72、96和120h,细胞培养上清中嵌合抗体的含量分别为7.47、11.72、8.02和18.28ng/ml;FCM检测其能特异性与IL-2Rα链结合。结论已成功构建了抗CD25人鼠嵌合抗体基因的真核表达质粒,并能在293T细胞中瞬时表达。
Objective To construct a eukaryotic expression vector for human-mouse chimeric antibody gene and express transiently in 293T cells. Methods The genes encoding variable regions of heavy and light chains of CD25 hybridoma cells were amplified by RT-PCR using the designed degenerate primers specific to signal peptide. The genes encoding constant regions of heavy and light chains of human IgG1 were amplified using plasmid PAG4622 as a template. The amplified genes encoding variable regions of heavy and light chains were spliced to the corresponding genes encoding constant regions. The obtained chimeric genes encoding heavy and light chains were inserted into eukaryotic expression vectors pOptiVEC and pcDNA3.3 respectively, and the constructed re- combinant plasmids were co-transfected to 293T cells for expression. The transcription level of chimeric antibody gene was determined by RT-PCR, the expression level of chimeric antibody by ELISA, and the binding activity of chimeric antibody by flow cytometry （FCM）. Results The eukaryotic expression vector for human-mouse chimeric antibody gene was constructed correctly. RT-PCR proved that the chimeric antibody gene was successfully transcribed in 293T cells. The chimeric antibody contents in culture supernatant of 293T cells 48, 72, 96 and 120 h after transfection were 7. 47, 11. 72, 8. 02 and 18. 28 ng / ml respectively. FCM showed specific binding of the chimeric antibody to IL-2Rα chain. Conclusion The eukaryotic expression vector for human-mouse chimeric antibody gene was successfully constructed and expressed transiently in 293T cells.
出处
《中国生物制品学杂志》
CAS
CSCD
2010年第2期113-117,共5页
Chinese Journal of Biologicals
关键词
CD25
嵌合抗体
瞬时表达
CD25
Chimeric antibody
Transient expression
作者简介
通讯作者：杨晓明，E-mail：xmyang@wibp．com．cn
