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: V_H, ∼340 bp. Lane 2: V_L, ∼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 V_H 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 V_L 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)