[Cloning of variable region and signal peptide genes of anti-CD20 monoclonal antibody by RLM-RACE]

Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2005 Jul;21(4):466-9.
[Article in Chinese]

Abstract

Aim: To seek a good method for simultaneous cloning V(L), V(H) and signal peptide genes of anti-CD20 monoclonal antibody (mAb) from hybridoma cells 1-28.

Methods: Total RNA was prepared from hybridoma cells 1-28 by Trizol. Two methods were used to clone the target genes. One was traditional rapid amplification of 5' cDNA end (T-5' RACE), and the other was RNA ligase-mediated rapid amplification of 5' cDNA end (RLM-RACE). The PCR products were then cloned into pGEM-T Easy vector, sequenced and analyzed with Blast method using Kabat and GenBank Databases.

Results: The target genes of L chain and H chain were both successfully cloned by RLM-RACE, while only V(L) gene and its signal peptide genes were obtained by T-5' RACE.

Conclusion: RLM-RACE is a good method for cloning antibody's V gene and signal peptide genes.

Publication types

  • English Abstract

MeSH terms

  • 5' Untranslated Regions / genetics
  • Amino Acid Sequence
  • Antibodies, Monoclonal / chemistry*
  • Antibodies, Monoclonal / genetics
  • Antibodies, Monoclonal / immunology*
  • Antigens, CD20 / immunology*
  • Base Sequence
  • Cloning, Molecular
  • Electrophoresis, Agar Gel
  • Humans
  • Immunoglobulin Variable Region / chemistry
  • Immunoglobulin Variable Region / genetics*
  • Molecular Sequence Data
  • Polymerase Chain Reaction / methods*
  • Protein Sorting Signals / genetics*
  • RNA / analysis
  • RNA / genetics
  • RNA / metabolism
  • RNA Ligase (ATP) / metabolism*

Substances

  • 5' Untranslated Regions
  • Antibodies, Monoclonal
  • Antigens, CD20
  • Immunoglobulin Variable Region
  • Protein Sorting Signals
  • RNA
  • RNA Ligase (ATP)