Cloning and sequencing of the light chain variable region from NS-1 myeloma

Abstract

The aim of this study was to clone the gene encoding the light chain variable region (V(L)) of the murine myeloma cell line P3/NS1/1-Ag4-1 (NS-1). Total RNA was prepared from the NS-1 cell line and its fusion hybridoma cell line 2F9. The V(L) gene was amplified using reverse transcription PCR (RT-PCR) with family-specific primer pairs. The PCR products were cloned into the pGEM(®)-T easy vector, transfected into E. coli DH5α cells and the positive recombinants were identified and purified with EcoRI digestion and agarose gel electrophoresis. The sequence was identified using an automatic DNA sequencer and analyzed online using the IMGT/V-QUEST program (version 3.2.21). The PCR product that was amplified with the P9 and P14 primers was approximately 392 bp in size. Following digestion with EcoRI, the band of interest was also detected in positive recombinants using agarose gel electrophoresis. The sequences from the NS-1 and 2F9 cells were identical. The whole sequence was 387 bp long, encoding 128 amino acids (AA), including a 60 bp leader sequence. There was a TAA stop codon at 385-387 bp and only one cysteine was found, at 112AA/128AA. Analysis using IMGT/V-QUEST determined the cloned V- and J-segments as murine IG(κ)KV3-12*01 and IG(κ)KJ2*01, respectively. Accordingly, the NS-1 V(L) gene belongs to the Ig(κ) gene family V3 subgroup. The NS-1 V(L) gene was successfully cloned and it is a pseudo-Igκ chain gene in NS-1 cells. This study may aid the sequencing of genes encoding monoclonal antibodies produced by mouse hybridomas raised with NS-1 myeloma.