Cloning and sequence analysis of light variable region gene of anti-human retinoblastoma monoclonal antibody.
OBJECTIVE
To clone the variable region gene of light chain of monoclonal antibody against human retinoblastoma and to analyze the characterization of its nucleotide sequence as well as amino acid sequence.


METHODS
Total RNA was extracted from 3C6 hybridoma cells secreting specific monoclonal antibody (McAb) against human retinoblastoma (RB), then transcripted reversely into cDNA with olig-dT primers. The variable region of the light chain (VL) gene fragments was amplified using polymerase chain reaction(PCR) and further cloned into pGEM -T Easy vector. Then, 3C6 VL cDNA was sequenced by Sanger's method. Homologous analysis was done by NCBI BLAST.


RESULTS
The complete nucleotide sequence of 3C6 VL cDNA consisted of 321 bp encoding 107 amino acid residues, containing four workframe regions (FRs) and three complementarity-determining regions(CDRs) as well as the typical structure of two cys residues. The sequence is most homological to a member of the Vk9 gene family, and its chain utilizes the Jkl gene segment.


CONCLUSIONS
The light chain variable region gene of the McAb against human RB was amplified successfully, which belongs to the Vk9 gene family and utilizes Vk-Jkl gene rearrangement. This study lays a good basis for constructing a recombinant antibody and for making a new targeted therapeutic agents against retinoblastoma.
Zhong
Xiufeng
X
Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou 510060, China.
Li
Yongping
Y
Huang
Shuqi
S
Ning
Bo
B
Zhang
Chunyan
C
Zheng
Jianliang
J
Feng
Guanguang
G
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000235:genetics; D000912:Antibodies, Neoplasm / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular; D005784:Gene Amplification; D005803:Genes, Immunoglobulin / Q000235:genetics; D006825:Hybridomas / Q000378:metabolism; D007147:Immunoglobulin Light Chains / Q000096:biosynthesis / Q000235:genetics*; D007135:Immunoglobulin Variable Region / Q000096:biosynthesis / Q000235:genetics*; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D019572:Retinal Neoplasms / Q000235:genetics / Q000276:immunology; D012175:Retinoblastoma / Q000235:genetics / Q000276:immunology; D017421:Sequence Analysis
D000911:Antibodies, Monoclonal; D000912:Antibodies, Neoplasm; D007147:Immunoglobulin Light Chains; D007135:Immunoglobulin Variable Region