Figure S1 - Humanization and Characterization of an Anti-Human TNF-α Murine Monoclonal Antibody

RT-PCR results showing the variable fragments and schematic representation of the strategy to assemble the heavy and light chains. (A) Amplification of the variable fragment cDNAs from the mouse hybridoma 357-101-4 secreting m357 IgG by RT-PCR with the Heavy Primers (Cat. No. 27-1586-01, GE Healthcare) and the Light Primer Mix (Cat. No. 27-1583-01, GE Healthcare), respectively. Lane M: Molecular weight marker. Lane 1: VH, ∼340 bp. Lane 2: VL, ∼325 bp. (B) Assembly of the cDNAs encoding the open reading frames of the heavy and light chains of the humanized 357 (h357) IgG1 by over-lapping PCR, respectively. The cDNA construct consisting the murine signal peptide (SP) derived from the cloning vector pSecTag2/Hygro, the VH region of h357, and the human IgG1 constant region (CH1, hinge, CH2 and CH3) derived from the cloning vector pFUSE-CHIg-hG1 were obtained by over-lapping PCR, followed by sub-cloning into the vector pSecTag2/Hygro at Nhe I and Not I sites. The cDNA construct consisting the above signal peptide, the VL region of h357, and the human kappa light chain constant region derived from the cloning vector pFUSE2-CLIg-hk were obtained by over-lapping PCR, followed by sub-cloning into the mammalian expression vectors pcDNA3.3-TOPO TA (Invitrogen, San Diego, CA). The locations of the primers and the restriction sites are shown in the diagram. SP, murine Ig kappa-chain V-J2-C signal peptide.
(EPS)

本数据集包含逆转录聚合酶链式反应（RT-PCR，Reverse Transcription Polymerase Chain Reaction）结果与重链、轻链组装策略的示意图。(A) 采用重链引物（货号：27-1586-01，通用电气医疗集团（GE Healthcare））与轻链引物混合物（货号：27-1583-01，通用电气医疗集团），从分泌m357 IgG（免疫球蛋白G，Immunoglobulin G）的小鼠杂交瘤细胞357-101-4中扩增可变片段互补DNA（cDNA，Complementary DNA）。泳道M：分子量标志物。泳道1：可变重链（VH，Variable Heavy chain），约340 bp（碱基对，Base Pair）。泳道2：可变轻链（VL，Variable Light chain），约325 bp。(B) 分别通过重叠聚合酶链式反应（Overlapping PCR）扩增编码人源化357（h357）IgG1重链与轻链开放阅读框的cDNA。通过重叠PCR获得包含以下元件的cDNA构建体：源自克隆载体pSecTag2/Hygro的小鼠信号肽（SP，Signal Peptide）、h357的VH区域，以及源自克隆载体pFUSE-CHIg-hG1的人IgG1恒定区（CH1、铰链区、CH2与CH3），随后通过Nhe I与Not I酶切位点将其亚克隆至载体pSecTag2/Hygro中。另一cDNA构建体包含上述信号肽、h357的VL区域，以及源自克隆载体pFUSE2-CLIg-hk的人κ轻链恒定区，经重叠PCR扩增后，亚克隆至哺乳动物表达载体pcDNA3.3-TOPO TA（美国 Invitrogen 公司，加利福尼亚州圣地亚哥）中。引物与限制性酶切位点的位置已在示意图中标注。SP：小鼠Ig κ链V-J2-C信号肽。(EPS)