Construction Of Naive Mouse ScFv Library And The Selection Of Pig IgG

Antibody,which is employed extensively in diagnosis and treatment of illness as well as laborator research plays an important role in the field of modern medical scince.The technology of Phage antibody library creats a whole new area for producing human monoclonal antibody. PCR was used to amplify from immunoglobulin all the variable fragments of heavy chain and light chain which then was recombined into appropriate phagemid vector,and finally be displayed on the surface of phage by fusion with phage protein. Then after panning,obtain the specific variable region gene without hybridoma or immunizing.In out study,we constructed a mouse phage antibody library by isolating lymphocytes from spleen of non-immunized mouse,then cloned the variable region of heavy chain and light chain which was then recombined into express vector.Recombinant phage-displayed antibody technique is currently the most popular method for the selecton of recombinant antibody fragments in vitro.This approach relies on a phage-display system in which fragments of antibodies are expressed as fusion proteins displayed on the phage surface.Using the polymerase chain reaction(PCR),immunoglobulin variable (V) region genes are first amplified from spleen cells.The VH(variable heavy)and VL(variable light)genes are then cloned into a phage vector and expressed as a fusion with a phage protein.Each recombinant phage genome contains the V genes that specify the antibody displayed on its surface.Since the displayed antibodies retain their antigen-binding capability, it is thus possible to enrich for recombinant phage expressing specific antibodies by affinity selection.With this approach,antibodies of defined specificity and affinity can be selected from a population.In this study, a phage-displayed antibody library against pig IgG was constructed using this new and leading biotechnique. Clones expressing antibodies against pig IgG were panned and screened from the library.Using RNA extraction method and the series methods of the Recombinant Phage Anibody System,the genes of VH and VL were gained from the spleen cells of non-immunized mice,which were about 360bp and 320bp respectively, and then were jointed together with a linker DNA(Gly4Ser)3 by splicing overlap extension PCR and a 750bp desired fragment(single chain Fv,ScFv) was generated.After digested with Sfil and Notl,the ScFv genes were ligated into the phagemid vector pCANTAB-5E and transformed into competent E?coli TGI cells.The recombinants were grown on the amp plate.Plasmid electrophoresis showed that there were insert fragments and the aimed fragments(750bp) were amplified using PCR.The results showed that we have successfully constructed a phage-displyed antibody library against pig IgG.Pig IgG was used for panning and screening ScFv antibody from the antibody library. Clones with good reactivity with pig IgG in ELISA selected soluble ScFv antibodies were produced and the expression conditions were optimized.Furthermore,the characteristics of pig IgG ScFvs were determined by ELISA.The products were purified by saturated animonlum sulfite and ion-exchange chromatography.This research is an explore and sample of molecular biology high technique research.One side,the complishment of this reseach inaugurate a new way on the research of gene engineering antibody,antibody preparation and the new type of biology production which is important theoretical meaning and practical appliance value.On the other hand,it provides a new method and technic flat for the selection of all kinds of antibodies.