Construction and expression of single-chain Fv antibody against human bladder carcinoma.
We designed two sets of oligonucleotide primers to amplify the immunoglobulin heavy- and light-chain variable-region genes from genomic DNA by polymerase chain reaction (PCR). The genomic DNA was extracted from hybridoma BDI-1 cells, which secreted a monoclonal antibody (mAb) against human bladder carcinoma. The primers contained special restriction sites that allowed the variable-region genes to be easily cloned for sequencing and expression. The recombinants were sequenced by Sanger's method. It was proved that the full lengths of the VH and VK genes were 366 and 324 bp, respectively. Compared with other published sequences, the VH gene was a member of mouse heavy-chain VH subgroup II and originated from the rearrangement of VH, Dsp2.2 and JH4. The VK gene was VK subgroup IV and from VK and JK4. The VH and VK genes was inserted expression vector pWAI80. By inducement, the ScFv antibodies were expressed and secreted from Escherichia coli. Binding activities against the bladder carcinoma cells were detected. We suggest that ScFv antibody recognized the antigen specifically.
Yu
L Z
LZ
Institute of Urology BMU, Beijing Medical University, P.R. of China.
Xiao
S
S
Huang
H L
HL
Gu
Z
Z
Gu
F L
FL
Guo
Y L
YL
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000235:genetics*; D000912:Antibodies, Neoplasm / Q000096:biosynthesis* / Q000235:genetics*; D000918:Antibody Specificity; D000951:Antigens, Neoplasm; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005803:Genes, Immunoglobulin; D006801:Humans; D006825:Hybridomas / Q000276:immunology; D007135:Immunoglobulin Variable Region / Q000096:biosynthesis* / Q000235:genetics*; D051379:Mice; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D015202:Protein Engineering; D001749:Urinary Bladder Neoplasms / Q000276:immunology*
D000911:Antibodies, Monoclonal; D000912:Antibodies, Neoplasm; D000951:Antigens, Neoplasm; D017931:DNA Primers; D007135:Immunoglobulin Variable Region