Construct A Large Of Phage Antibody Library

Molecular cloning of antibody repertoire in combinatorial phage display vectors has turned out to be a powerful technique for obtaining human monoclonal antibody genes and probing the antibody immunoresponse against a given antigen.In this study,we construct a phagemid and a large size of naive antibody library has been constructed with phage display library technology and Cre-Loxp recombinant system. Peripheral blood cells from 120 normal adultts were collected for lymphocytes issolation and total RNA preparation,cDNA was synthesized using random oligo-dT primers and reverse transcriptase following standard protocols,antibody V genes were amplified with specific primers,Light chain genes were mixed according the natural ratio of antibody distribution then inserted into the vector PDF-D-SacB first to obtain a light chain library was constructed by repeated electrotransfer,the insertion ratio of light chain was 100% among random picked clones.A primary library was constructed after heavy chain insertion according the natural ratio.Primary library infected BS1365 which can secrete Cre enzyme inducing the genes to shuffle at 30℃at MOI>100, the recombinant phage antibody library infected E.coli Transl-Blue at MOI<100 and establish working phage antibody library.A library containing 1.0×1011 clones was harvested.Ten clones was randomly tested.Ten out of the ten clones (100%)has indicated the insertion of heavy chain and light chain.The main. results are as follows:In this studis,all the lengths of amplified VH and VL subclass genes were consistent with the theoretical values.A large size of naive antibody library has been constructed with phage display library technology and Cre-Loxp recombinant system. Both the cloning efficacies of VH and VL genes were 100%.The capacith and titer of primary phage antibody library were 7.2×1012 and 6.0×1013,and those of working phage antibody library were 1.0×1011 and not less than 1.0×1013 respectinely.