Construction Of Swine Phage Single-chain Antibody Variable Fragment (scFv) Library And Selection Of Antibody To RSpaA

In recent years Single-chain antibody variable fragment (scFv) in the target to drugs, gene therapy, and residue detection and detection aspect has been widely studied and applied. The phage antibody library display system has became one of the main technical platform in the field of recombinant antibody nowdays. This research constructs the swine phage single-chain antibody library, by bio-panning obtained with scFv to bind specifically to the rSpaA. And for the screening of the scFv were expressed in prokaryotic expression, which provides a new way for recombinant antibody preparation of anti-rSpaA, for swine erysipelas disease detection and comprehensive prevention.The main experiment of this project was as follows:1. Total RNA was extracted from spleen lymphocytes and synthesized first stand cDNA by RT-PCR then used it as template to amplify fragments consisting of a variable heavy chain and a variable light chain with PCR and join with their linkers. The scFv and phagemid vector pComb3Xss were digested by T4 DNA Ligase.The recombinant vector electric transformed into XL1-blue.Thus we constructed the swine scFv bacterial gene library and the library contains 2.5 x 106 clones/mL.2. After the transformated cells has been infected by the helper phage M13KO7,the phage display scFv library was constructed.After 5 rounds biopanning enrichment and screening with rSpaA.Finally,the phage scFv enriched library titer was 2.4x 106 pfu/mL,and the library had been enriched for 12 times.3.52 strains of rSpaA-specific phage antibody was selected from bio-panning enrichment,44 was positive detected by PCR.These positive strains were screened by phage-ELISA, and 31 clones were positive.Three strains were selected for sequencing analysis and homology modeling analysis was carried out by online database.4. Two stains recombinant scFv protein were constructed into pET-28a and then transformed into E.coli Rosetta (DE3),The SDS-PAGE analysis showed that 2 recombinant scFv protein were expressed successfully, but mostly in the form of inclusion bodies.