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


CONCLUSIONS
RLM-RACE is a good method for cloning antibody's V gene and signal peptide genes.
Department of Molecular Immunology, Institute of Basic Medical Sciences, Academy of Military Medical Sciences, Beijing 100850, China. wangyg03@yahoo.com.cn
Yu-gang
YG
Wang
Jian-nan
JN
Feng
Bei-fen
BF
Shen
D020121:5' Untranslated Regions / Q000235:genetics; D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000235:genetics / Q000276:immunology*; D018951:Antigens, CD20 / Q000276:immunology*; D001483:Base Sequence; D003001:Cloning, Molecular; D004587:Electrophoresis, Agar Gel; D006801:Humans; D007135:Immunoglobulin Variable Region / Q000737:chemistry / Q000235:genetics*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D021382:Protein Sorting Signals / Q000235:genetics*; D012313:RNA / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D011130:RNA Ligase (ATP) / Q000378:metabolism*
D020121:5' Untranslated Regions; D000911:Antibodies, Monoclonal; D018951:Antigens, CD20; D007135:Immunoglobulin Variable Region; D021382:Protein Sorting Signals; D012313:RNA; D011130:RNA Ligase (ATP)