Construction Of Human Naive Fab Library And Characterization Of Anti-RH(D) Fab Fragment Generated From The Library

In all blood-group systems, the D antigen is the most effective red blood cell surface antigen except the A, B antigens, its corresponding antibody is Rh(D) antibody. Rh(D) antibody has very vital clinical significance. For the blood transfusion patient, Rh(D) antibody can cause the acute hemolytic blood transfusion response; for the D masculine embryo, Rh(D) antibody which the parent substance produces, can enter embryo in vivo through the placenta, and initiate the newborn to dissolve homeopathy (HDN). So it's necessary to research Rh(D) antibody. However, because mouse's immune system cannot distinguish the independent D antigen, it's unable to develop the Rh(D) antibody by the traditional hybridism technology.Fab fragment genes were obtained by RT-PCR from peripheral lymphocytes of Rh(D-) patients with anti-Rh(D) antibodies. The obtained Fab genes and pComb3×ss vector were digested by Sfi I enzyme and ligated between each other. The recombinant genes were electro-transformation into competent E.coli XL1-blue. The transformed cells were infected with VCSM13 helper phage to yield recombinant Fab phage antibody. A human Fab phage antibody library with 7.4×10~6 capability was constructed successfully.The constructed library was panned by 5 rounds of"adhesion - amplification - elution", and the original antibody library gained enrichment in great degree. After the 5 rounds of panning, a positive clone against Rh(D) was screened from the phage antibody library, and was characterized by agglutination and western-blotting. The positive clone was sequenced, and after the alignment of the partial nucleoside acid sequences of Fd and Vκgene cloned in this experiment with that submitted to Kabat, the Fd gene cloned in this experiment is VH3-23, and the Vκgene cloned in this experiment isκappa.In this experiment, we successfully cloned Fab fragment genes, constructed a human Fab phage antibody library with 7.4×10~6 capability, and a positive clone against Rh(D) was screened from the phage antibody library. These successful results provide reliable foundation for manufacturing whole molecule IgG engineering antibody and hemolytic disease of newborn and hemolyticus transfusion reaction.