Cloning and sequencing of VL and VH genes from a novel clone ZCH-7-2F9 of anti-hCD14 monoclonal antibody / 浙江大学学报·医学版

ABSTRACT

<p><b>OBJECTIVE</b>To acquire the genes of light chain variable region (VL) and heavy chain variable region (VH) of a novel clone ZCH-7-2F9(2F9) of anti-hCD14 for construction of anti-hCD14 single chain antibody(ScFv).</p><p><b>METHODS</b>From the mouse hybridoma cell line 2F9 and its fusion partner murine myeloma cell line NS-1, total RNA was prepared. The VL and VH genes were amplified by RT-PCR with family specific primer pairs, respectively. The PCR products were cloned into pGEM(sound recording copyright sign)-T Easy vectors, then transfected into DH5alpha and the positive recombinants were identified and purified. After sequencing with automatic DNA sequencer the sequences were analyzed online.</p><p><b>RESULTS</b>VL gene of the new clone of CD14 monoclonal antibody (McAb) 2F9 consisted of 321 bps encoding a peptide of 107 amino acid residues, and VH gene of the 2F9 antibody contained 360 bps encoding a peptide of 120 amino acid residues. According to IMMUNOGENETICS online analysis by IMGT/V-QUEST, the VL and VH genes belonged to mouse IGKV and mouse IGHV subgroups, respectively. On the position 23/88 of the light chain and 22/96 of the heavy chain genes there were cysteines, which play the key role in forming disulfo-bond in each chain. Both VL and VH chains had definitely 4 frame regions (FR) and 3 complementary determinant regions (CDR).</p><p><b>CONCLUSION</b>The treatment of CML consisting of myeloablative Allo-SCT combined with Gleevec before and after transplantation is an effective and safe method for CML.</p>