Construction of human recombinant antinuclear antibody Fab library on the surface of phage / 解放军医学杂志

ABSTRACT

Objective To construct a phage antibody Fab library of human antinuclear antibody. Methods Total RNA was extracted from peripheral blood lymphocytes of 4 patients suffering from autoallergic disease, with antinuclear IgG antibody titers in the serum higher than 1∶10 000 as estimated by indirect immunofluorescence technique. Taking oligo-dT as the primers, human immunoglobulin light chains ?/? genes as well as heavy chains Fd genes were reversely transcribed to cDNA by PCR. The ?/? light chains were initially cloned into pComb3Hss vector to construct a human recombinant light chain library, and the heavy chain genes were subsequently inserted into the corresponding sites of ?/?-pComb3Hss plasmid to generate a recombinant ?/?-Fd-pComb3Hss plasmid. The plasmid was then transformed into E. coli XL1-Blue, which was subsequently infected by the helper phage M13KO7. A random recombinant antibody library was expressed on the surface of filamentous phage. Results Human immunoglobulin ?/? light chain and heavy chain Fd genes (660bp) were amplified successfully. The light chain library with the capacity size of 2?104, and the human recombinant Fab antibody library with the capacity size of 4?104 were also successfully obtained. Finally, the phage antibody library of the Fab fragment of human antinuclear antibody, containing 2?109 CFU/ml phage, was constructed. Conclusion A phage antibody library of Fab fragment of human antinuclear antibody is constructed successfully. This research lays a foundation for the preparation for phage library of Fab fragment of human antinuclear antibody, and also paves the way for the advanced assessment for its effect on the immuno-targeting therapy of tumors.