Screening And Identification Of Human ZnT8-specific ScFv From Type 1 Diabetes Phage Display Library

Type 1 diabetes(TID)is an autoimmunity disease resulting from the destruction of islet ? cells by components of the immune system.The autoimmune attack targets at a series autoantigens that are expressed in ? cells.The autoantigens have been identified in T1D including insulin,65-kDa isoform of glutamic acid decarboxylase(GAD),tyrosine phosphatase and Zinc transporter 8(ZnT8).Among them,ZnT8 was observed to be a major autoantigen in TID and a predictive marker in risk groups before onset of the disease.Wenzlau et al.found that individuals followed from birth to TID showed ZnT8 antibodies as early as 2 years of age and increasing levels and prevalence persisting to disease onset.Our study demonstrated that ZnT8 as the novel immunodominant epitopes are recognized in type 1 diabetes patients,suggesting that a strong inverse relationship between T1D and ZnT8.ZnT family play the role as the zinc ions diffusion facilitators which allow diffusion of zinc ions across biological membranes.ZnT and Zrt/Irt-like protein(ZIP)are the two main components that maintain the zinc homeostasis in the body.Among ZnT family,Chimienti et al.first showed that zinc transporter 8(ZnT8),coded by SLC30A8 gene,was the most expressed transporter in human pancreas and further identified that ZnT8 was exclusively expressed in secretory granules of islet ? cells.The overexpression of ZnT8 in islet cells can stimulate zinc accumulation and enhance glucose-stimulated insulin secretion compared with control cells.These findings show that ZnT8 is essential for both zinc accumulation and the proper storage,secretion,and the action of insulin.To our knowledge,the animal antibodies of ZnT8 have graduall shown important applications in scientific research,while humanized ZnT8 antibodies have not been identified.The detection of antibodies and CD8+T cells against ZnT8 is an important way for the prediction and diagnosis of TID.Antigen-specific therapy targets to ZnT8 maybe a potential way in preventing or arresting the progression of TID in the future.Humanized or human antibodies could have an important application value in the clinical diagnosis and theraphy of T1D.Our study aimed to construct a single chain Fv fragment(scFv)phage-display library from T1D and select ZnT8 specific scFv which could give new ideas and foundations to the development of new agents in diagnosis and therapies of T1D.Methods1.Expression of recombinant ZnT8 protein in prokaryotic expression systemThe ZnT8(N/C)gene and ZnT8(Arg325Trp)gene were amplified by PCR.Ligated the PCR products with plasmid pcold ? and transformed into E.Coli BL21 competent cells.After induced positive clones with IPTG,the precipitation cells were cracking by ultrasonic and then purified by affinity column.The recombinant ZnT8 proteins were identified by SDS-PAGE and Western blot.ELISA assay were used to identify the binding ability of ZnT8(N/C)protein to the commercial polyclonal of ZnT8 antibodies.2.Construction of scFv phage display library from T1DPeripheral blood mononuclear cells(PBMC)were isolated from newly diagnosed T1D volunteers' After extracted the total RNA,VH and VL genes were amplified through RT-PCR and scFvs were obtained by overlap-PCR.The scFvs were then ligated with phagemid vector pComb3XSS.Introduced the ligations into E.coli XL1-Blue by electroporation,then rescued and amplified the library using VCSM13 helper phages.A scFv phage display library was obtained after the supernatant precipitate with 4%PEG8000 and 3%NaCl.3.Selection of ZnT8 specific scFv from the libraryThe ZnT8(N/C)protein was coated onto a microtiter ELISA plate for bio-panning.After four rounds of bio-panning,bacterial clones were selected for phage ELISA.The positive scFv with higher OD values and right sequences were expressed and purified through HisTrap column.4.Identification of ZnT8 specific scFvScFvs were identified by affinity assays,Western Blot,ELISA,and immunohistochemical assays.Results1.The ZnT8(N/C)gene(?550bp)and ZnT8(Arg325Trp)gene(?650bp)were successfully amplified and ligated with the pcold ? vector respectively.The positive plasmids were confirmed by DNA sequencing.After induced with IPTQ recombinant proteins were expressed in the bacterias pellets.The purified ZnT8(N/C)protein and ZnT8(Arg325Trp)protein were analyzed by SDS-PAGE with 20 kD band and 25kD band respectively.The ELISA assay showed that the purified and denatured ZnT8(N/C).protein have the bioactivity to recognize polyclonal ZnT8 antibodies.The optimal dose of ZnT8(N/C)protein when screening scFv was 0.8?g/well.2.The scFv gene fragments(-750bp)were generated after RNA extraction,RT-PCR,and overlap PCR,and then ligated with the phagemid.After transformed into E.coli XL1-Blue and rescued by VCSM13,a phage display library containing 1.0×108 clones was constructed successfully.3.After four rounds of bio-panning,11 out of 160 clones were high positive against ZnT8(N/C)protein by phage-ELISA.After sequencing;7 unique scFvs were obtained.Proteins were purified with HisTrap column.The SDS-PAGE result showed a band at about 30kD of each scFv.4.Among the 7 scFvs,C22 and C27 showed higher OD value when detected by Phage ELISA.The affinity assays showed that both C27 and C22 scFv have a high affinity to ZnT8(N/C)protein,the kD(M)was 5.397×10-8 and 5.953×10-8 respectively.When detected with C27 and C22 scFv,ZnT8(N/C)protein showed a?20 kD protein band,which is the predicted molecular weight.The ELISA result showed that the binding capacity of serial diluted scFv to the ZnT8(Arg325Trp)decreased with the dilution ratio increased,which indicated the renatured scFvs could bind to ZnT8 in a concentration dependent manner.The Immunohistochemiscal assays showed that both the two scFvs are specific to human islet ?cells.Conclusion1.We constructed a T1D scFv phage-display library containing 1.0×108 clones successfully.It makes the foundations for the autoantibodies screening from type 1 diabetes.2.Seven scFv antibodies specific to human ZnT8 protein were selected from the library.Among them,C22 and C27showed high affinity and specificity to ZnT8 which expressed on islet ? cells.It will enable us to address the questions relating to the role of ZnT8 in T1D pathogenesis,and also provide the basis for developing sensitive diagnostics and therapeutics agents that could be used to treat or alleviate the disease.