[Construction, expression and identification of a single chain antibody variable (scFv) against human CD25 molecule].

AIM: To construct and express a single chain fragment variable (scFv) fragment against human CD25 molecule and identify its bioactivity. METHODS: V(H) and V(L) genes of anti-murine CD25 monoclonal antibody were cloned by RT-PCR from hybridoma cell WuTac secreting anti-CD25 mAb. scFv gene was spliced by sequence overlap extending (SOE) PCR, and then it was ligated into pMD18T vector to be identified by endonuclease digestion, PCR and sequencing. scFv gene was cloned into pBAD/gIIIA expression vector and transformed into TOP10 E.coli. After positive clones were induced by L-arabinose for 4 hours, the purity of protein was detected by SDS-PAGE and its bioactivity was identified by competitive inhibition ELISA test. RESULTS: scFv genes of V(L)-(GGGGSGGGGSSGGGS)-V(H) was constructed successfully. The V(H) chain consisted of 351 bp and encoded 117 amino acids, which belonged to heavy chain subgroup III (C) of mouse immunoglobulin variable region. The V(L) chain consisted of 318 bp and encoded 106 amino acids, which belonged to light chain subgroup IV of mouse immunoglobulin variable region. The scFv antibody expressed by TOP10 fused with 6xHis and C-myc tag protein and the relative molecular mass of fusion protein was about 31,000. Competitive inhibition ELISA test indicated the scFv antibody had specific activity. CONCLUSION: The expressed product of the single-chain antibody shows some specific binding capacity, which provides a basis for the clinical application of anti-CD25 single-chain antibody.

Molecular cloning and sequence analysis of heavy- and light-chain variable region genes of anti-CD71 monoclonal antibody.

OBJECTIVE: To clone heavy-chain and light-chain variable region (VH and VL) gene of mouse-anti-human CD71 monoclonal antibody (mAb). METHOD: One-step method was used to extract total RNA, and a set of oligonucleotide primers were designed to amplify the cDNAs with reverse transcriptase-polymerase chain reaction (RT-PCR), and the resultant products were respectively cloned into PMD18-T vector and their sequences analyzed. RESULTS: The PCR product obtained with the oligonucleotide primers for the variable region of mouse immunoglobulin heavy chain was about 350 bp and that with oligonucleotide primers for the light chain was about 320 bp, and their DNA sequences were determined. CONCLUSION: The length of the cloned heavy chain variable region was 348 bp, belonging to mouse heavy-chain subgroup II(A); the light-chain variable region was 336 bp that belongs to mouse kappa light-chain subgroup II.