Construction and primary identification of a naive Fab fragment phage library / 中国免疫学杂志

ABSTRACT

Objective:To construct a human Fab fragment phage display library and provide a platform for human antibody preparation.Methods:Peripheral blood lymphocytes were collected from healthy donor.The heavy chain Fd fragment and light chain of human immunoglobulin's genes were amplified by RT-PCR,and then cloned into phagemid pComb3XSS to generate human phage antibody library.Cutting with endonucleases such as SacⅠ,XbaⅠ,XhoⅠand SpeⅠto identify the insertion of the light chain or heavy chain Fd genes.IL-2 and digoxin as the antigen was used to scan the phage antibody library.Results:A phage antibody library of Fab had 8.4?107 members and it's recombinant rate was 70%.Through DNA sequencing of one positive clone,it was showed that its heavy chain belonged to IgG subvariety and its light chain to ? family.Conclusion:The success of constructing a nave human phage antibody library proves the useful of phage display system in human antibody preparation,and it can be used to select,purify and express of amylin Fab antibody.