Xiang J H, Roder J, Pan Z G, Roifman C, Hozumi N
Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Department of Immunology and Medical Genetics, University of Toronto, Ontario, Canada.
Mol Immunol. 1991 Jan-Feb;28(1-2):141-8. doi: 10.1016/0161-5890(91)90097-4.
A mouse/human chimeric B72.3P-1-6 antibody was produced by construction of a novel expression vector mpSV2neo-EP2-V-Crl containing the same gene fragments as the expression vector mpSV2neo-EP1-V-Crl (Xiang J., Roder J. and Hozunni N., submitted to Molec. Immun., 1991) except the promoter (P2) fragment in which the translation start codon ATG is retained. The expression vector was transfected into a heavy chain loss mutant cell line, B72.3M1. The translation of the chimeric heavy chain may start at the exogenous start codon ATG within the P2 fragment, which is 27 base pairs upstream of the endogenous start codon ATG in B72.3 heavy chain V region cDNA fragment, leading to an alteration in leader sequence cleavage sites and the formation of chimeric heavy chain with an elongation in the FR1 region. Chimeric B72.3P-1-6 antibody retained binding specificity to TAG72 antigen, but showed an eight-fold decrease in binding affinity to TAG72 compared with chimeric B72.3-1-3 antibody. This suggests that residues in FRI contribute to the correct folding of the antibody binding region of the B72.3 antibody.
通过构建新型表达载体mpSV2neo-EP2-V-Crl产生了一种小鼠/人嵌合B72.3P-1-6抗体，该载体包含与表达载体mpSV2neo-EP1-V-Crl（Xiang J.、Roder J.和Hozunni N.，已提交给《分子免疫学》，1991年）相同的基因片段，但启动子（P2）片段中的翻译起始密码子ATG得以保留。将该表达载体转染到重链缺失突变细胞系B72.3M1中。嵌合重链的翻译可能起始于P2片段内的外源性起始密码子ATG，其位于B72.3重链V区cDNA片段内源性起始密码子ATG上游27个碱基对处，导致前导序列切割位点发生改变，并形成FR1区域延长的嵌合重链。嵌合B72.3P-1-6抗体保留了对TAG72抗原的结合特异性，但与嵌合B72.3-1-3抗体相比，其对TAG72的结合亲和力降低了八倍。这表明FR1中的残基有助于B72.3抗体结合区域的正确折叠。
