Hoogenboom H R, Raus J C, Volckaert G
Dr. L. Willems Instituut, Limburgs Universitair Centrum, Diepenbeek, Belgium.
J Immunol. 1990 Apr 15;144(8):3211-7.
The cloning, construction and expression of chimeric Ig genes, encoding a mAb directed against the human transferrin receptor, is described. From a mouse hybridoma cell line, secreting an antitransferrin receptor antibody, mRNA was prepared and converted into cDNA using Ig-specific oligonucleotides. H and L chain encoding cDNA fragments were isolated and sequenced. Chimeric genes were constructed by linking the murine V region cDNA fragments to human C region exons. After sequential transfection of nonproducing mouse hybridoma cells with the expression vectors containing the chimeric H and L chain genes, antibody secreting transfectomas were obtained. ELISA and immunoblot analysis clearly demonstrate the secretion of human kappa- and gamma-1 chain. Flow microfluorimetry analysis of the chimeric antibody shows that the Ag-binding capacity has been retained. The chimeric antibody most likely will be less immunogenic then the original mouse antibody when used in human cancer therapy.
本文描述了编码针对人转铁蛋白受体的单克隆抗体的嵌合Ig基因的克隆、构建及表达。从分泌抗转铁蛋白受体抗体的小鼠杂交瘤细胞系中制备mRNA，并使用Ig特异性寡核苷酸将其转化为cDNA。分离并测序编码重链和轻链的cDNA片段。通过将鼠源V区cDNA片段与人C区外显子连接来构建嵌合基因。用含有嵌合重链和轻链基因的表达载体对不产生抗体的小鼠杂交瘤细胞进行连续转染后，获得了分泌抗体的转染瘤细胞。ELISA和免疫印迹分析清楚地证明了人κ链和γ-1链的分泌。对嵌合抗体的流式微荧光分析表明其保留了抗原结合能力。当用于人类癌症治疗时，嵌合抗体的免疫原性很可能低于原始小鼠抗体。
