Construction And Identification Human TRAb Fab Fragment Phage Library

Background and objectiveTSH RECEPTOR(TSH-R)autoantibodies(TRAbs)in patients with Graves' disease(GD)are functionally heterogeneous;they include mainly thyroid-stimulating antibodies(TSAbs)and occasionally also thyroid-blocking antibodies(TSBAbs)and neutral TRbs.Phage surface display technique,which has become an increacingly important tool in biotechnology,provides a new way for antibody development.In this project we constructed a human TRAb Fab fragment phage display library,pro vide a platform for human antibdy preparation and a basis to diagnosis technology and new therapy for autoimmune thyroid disease.Method1)Total RNA was isolated from peripheral blood cells of a woman with hyperthyroidism.2)human immunoglobulin ?/? light chain and heavy chains Fd genes were amplified by polymerase chain reaction.(PCR).3)The ?/? light chain were first cloned into pComb3Hss vector to construct a human recombination light chain library.The heavy chain genes were subsequently inserted into the corresponding sites of ?/?-pComb3Hss plasmid to generate a combinatorial ?/?-Fd-pComb3Hss plasmid.4)phage display:The plasmid transformed into E.coli.XLl-Blue,then E.colli.XL1-Blue was infected by helper phage M13K07.A random combinatorial library was expressed on the surface of filamentous phage.6)The reconstructed humanized TRAb Fab library was enriched by five rounds panning.plasmid DNA isolated from positive clone was cut off g? gene.After ligated itself,the recombinant plasmid transformed E.coli.XL1-Blue,then XL1-Blue was induced by IPTG to product soluble human TRAb Fab.7)Preparation of affinity chromatography column for the purification of human TRAb Fab fragment.8)Purification of human TRAb Fab fragment by prepared chromatography column.9)Identification of purified human TRAb Fab fragment by western blotting.10)The DNA fragment from the positive clones were sequenced.Results1)With designed oligo-(dT)nucleotide primers,cDNA library was gained successfully by reverse transcription technology from total RNA of peripheral blood cells.Human immunoglobulin ? light chain and heavy chain Fd genes were obtained by PCR successfully.2)We gained a phage antibody library by phage display technology based on combine antibody library successfully.3)after the five round panning,unique clones were isolated from the human IgG Fab fragment library.It was proved the recombinant plasmid had been cut off g?genes and ligated by itself through electrophoresis after digested by XhoI.4)The result of indirect ELISA indicated all of the clone of Fab had specific to bind with TSHR-N.5)We gained purified human TRAb Fab fragment by prepared affinity chromatography column.The result of western blotting showed molecular weight of the protein is 45KD and it can be captures by anti-human IgG Fab.6)These sequence analysis showed all of the heavy chain Fd gene and light chain gene we received are derived from human.ConclusionA human TRAb Fab fragment library was constructed by phage display technology.Through solid phase absorption of antigen screen method.one unique clone was isolated from human IgG Fab fragment library.We finished the expression and preliminary purification of soluble Fab fragment.The result of western blotting and ELISA indicated the prepared protein was human IgG Fab and has antibody activity against TSHR-N.