Construction Of Single Chain Antibody Library Against Porcine Adipocyte Membrane Protein

In this research we use the subtractive immunization technique. Liver cells were selected as immune tolerance antigen. The cyclophosphamide was injected into mice through intraperitoneal immunization at 15min, 24h and 48h after mice were immunized with liver cells. Blood was collected from eyeball on the 10th day. The antibody titre was measured by cell-ELISA. The antibody litre of CY100mg/kg dosage group was almost identical to the unimmunized mice. The result showed the dosage of CY100mg/kg could make the mouse produce the effective immune tolerance. Mice, having low antibody titre, were immunized with porcine adipocytes. After mice were immunized three times, the antibody titre was measured. The total RNA was isolated from the mice having high antibody titre.The RNA was extracted from the spleen of immunized mouse. The mRNA was purified by Oligo (dT)-cellulose and it was used as template to synthesize the first strand cDNA. Using mouse specific VH and VL primers, VH and VL genes were amplified from the cDNA and spliced as single chain variable fragment (scFv) by the linker sequence encoding (Gly4Ser)3 .The scFv genes were cloned into phagemid vector pCANTABSE and E.coli TGI competent cells were transformed. The clones selected on SOBAG plates were superinfected by M13KO7 helper phage. The scFv phage library was established.In a word, we take some method in researching single chain antibody by constructing scFv phage library against porcine adipocyte plasma membrane protein, which is very important to study immunization against adipocyte plasma membranes. The construction of phage library offers a platform for screening and expression of antibody in the future.