Cloning Sequencing And Expression Of The Variable Regions Of Two Human Monoclonal Antibody Against Hemorrhagic Fever Virus

Monoclonal antibodies have been available for more than twenty years. Despite their great utility in the laboratary and in diagnostic testing, they have yet to make a significant impact as a clinical tools. Because most of the monoclonal antibodies are murine antibodies, they cause the response of a human anti-mouse antibody(HAMA) in patients. It is difficult to produce human monoclonal antibodies andmost of the available human monoclonal antibodies currently are of the IgM isotype, which limited their usefulness. Genetic engineering offers a new approach to overcoming the limitations of monoclonal antibodies.Human-human hybridoma cell line 87-2 secretes anti-HFRSV human monoclonal antibody. Total RNA was extracted from the cells and was reverse transcribed to the first strand cDNA using oligo-d(T) as a primer. A set of oligonucleotide primers were designed to amplify the variable regions of human immunoglobulin heavy and light chain from the cDNA by polymerase chain reaction. The amplified VH and VL fragments were ligated to the phage M13 vector DNA. The recombinants were sequenced. The sequences of the ampified VH and VL fragments were confirmed as the human immunoglobulin variable genes by comparing with those of the published genes in EBML gene bank. There are framework(FR) and complementarity determining (CDR) regions in the VH and VL genes, the VH consists of 405 base pairs encoding 135 amino acids. The VL consists of 321 base pairs encoding 107 amino acids.