The Screening And Study On The Human Antibody Against Human Interferon Human Interferon α1b(huIFN-α1b)

Systemic lupus erythematosus is a autoimmune disease and a serious threat to human health. Studies revealed that the elevated levels of human interferon alpha(huIFN-α) in the patients’ serum might be a causal role in human SLE, and the antibody against huIFN-a might be an effective drug to control this rheumatism.In this studying, using phage display technology, human antibodies against human interferon alb were screened from a human synthetic antibody library, the research include three parts, as the following:1. the screening、expression and identification of human recombinant scFv antibody against human interferon alb (huIFN-alb)human recombinant scFv antibody against human interferon α(huIFN-α) were screened form a human synthetic antibody library with the library size of2×109against purified huIFN-alb using high throughput technology(HTS).3000individual clones were randomly picked, and the binding property of these scFv phage antibody were identified by phage-ELISA, and20%of the3000clones could bind specifically with huIFN-alb. Sequence analysis of CDR3of86positive clones revealed that the length and sequence of CDR3were distinct, and9different antibody genes were identified by sequence analysis, and7antibody genes were derived from VH3and VL3germline family,2antibody genes were derived from VH3and VL1germline family.5antibodies witch could specificly bind huIFN-alb but not with unrelated antigen huIFN-α2b and huIFN-γ were obtained by phage-ELISA assay. After digested by SfiI and NotI, The5scFv antibody genes were cloned into expression vector pET22b. Expression of scFv antibody protein was induce by IPTG, and expressed antibody protein were secreted into periplasmic space of bacteria.4mg purified scFv antibody protein was obtained from600ml bacterium in liquid medium.3scFv antibody of5with specific binding to huIFN-α1b but not to huIFN-α2b and huIFN-γ were gained by ELISA and Western blot assay.To obtain IgG antibody against huIFN-alb, the light chain and heavy chain of positive scFv antibody genes were cloned into IgG expression cassette vector (pAc-K-CH3) by digested with SacⅠ and HindⅢ、XhoⅠ and NdeⅠ. The recombinant plasmid and baculovirus DNA were cotransfected into insect cell sf9, and antibody genes expressed in recombinant baculovirus/insert system. The flurescence of infected sf9insect cells detected with FITC conjugated anti-human Fc antibodies demonstrated that human IgG antibody were expressed well. The recombinant human IgG molecules were purified by protein-A affinity chromatography. Heavy and light chains of the expected sizes were observed with SDS-PAGE, and the result showed that the IgG antibody against huIFN-α1b produced in insect cell sf9could correctly assemble and secreted into the culture medium. To identify the binding properties of recombinant IgG antibody to huIFN-alb,ELISA and Western blot were performed, and the result demonstrate that the3IgG antibodies could specifically bind to huIFN-α1b but not to huIFN-α2b and huIFN-γ2. the epitopes、affinity and function properties of recombinant IgG moleculeCompetitive ELISA were adopted to determine whether the binding sites of3antibodies to huIFN-alb were identical or not. The result demonstrated that recombinant IgG AIFNal IgG1molecule compete with3scFv antibodies for the binding sites to huIFN-alb, and this may result from the binding sites of the3scFv to huIFN-alb contain some overlap with each other. Utilizing competitive ELISA, the binding sites of3human IgG antibodies molecule and a mouse IgG antibodies molecule4G10which could bind huIFN-alb and neutralize the anti-virus property of huIFN-alb were identified. The result demonstrated that mouse IgG antibodies molecule4G10compete with3human IgG antibodies molecule for the binding sites to huIFN-alb, and this may result from the binding sites of the3human IgG antibodies to huIFN-alb contain some overlap with4G10and this gave some hint about the3human IgG antibodies for neutralizing property. Making use of BIAcore3000, the affinity of three human IgG antibodies to huIFN-alb were evaluated, and the affinity of AIFNal IgGl to huIFN-alb was74.7×10-9, and the affinity of AIFNal IgG2to huIFN-alb was5.44×10-9, and the affinity of AIFNa3IgGl to huIFN-alb was11.4×10-9. Two gene ISG15and IFIT-1with expression being down regulated by huIFN-alb were selected for the function assay of antibodies by Real-time PCR, and the result demonstrate that in the4hour the expression of ISG15and IFIT-1induced by huIFN-alb could both being blocked by human IgG antibodies AIFNal IgGl. In another function assay, WISH-VSV system were utilized to determine whether the3human IgG antibodies could block the anti-virus function of huIFN-alb or not, and the result showed that the3IgG antibodies (AIFNa1IgG1、AIFNal IgG2and AIFNal IgG3) could not neutralize the anti-virus function of huIFN-alb in the concentration of200ug/ml, however, the mouse IgG antibodies molecule4G10could neutralize the anti-virus function of huIFN-alb in the concentration of100ug/ml, and this may indicate that the human antibodies (AIFNal IgG1、AIFNal IgG2and AIFNal IgG3) were not directed to the epitope related with anti-virus function of huIFN-α1b.3. the research on the antigen-antibody interactionIn order to study the antigen-antibody interaction, the huIFN-alb protein were expressed in E.coli strain. The lymphocytes from health person were isolated. The total RNA was extracted from the human isolated lymphocytes and the cDNAs was synthesized using primer oligo-dT. After the huIFN-alb gene was amplified by PCR with cDNAs as template, the huIFN-alb gene were cloned into the vector pET30a by digesting with NdeⅠ and XhoⅠ. The recombinant plasmid were transformed into E.coli, and expression of huIFN-alb protein could be induced by IPTG and be used for the epitope mapping by Western blot test. Through site directed mutation and delete mutation in the amino acids sites of26-35, this loopAB region(26-35) of huIFN-α1b was important for the antigen-antibody interaction, and especially the amino acids sites27、33、34、35. Based on the epitope mapping and function assay, we further studied the antigen-antibody interaction by molecule modeling and docking. Using crystal structure of huIFN-α2b from PDB database as template, the3D structure of huIFN-alb was constructed(the amino acid sequence similarity between huIFN-α2b and huIFN-alb was83%), and using crystal structure of2A9M from PDB database as template, the3D structure of scFv antibody (AIFNalscFvl) was constructed(the amino acid sequence similarity between2A9M and AIFNal scFvl was77%). The obtained structures of scFv antibody and huIFN-alb were further optimized by chamm course, and the structures with lowest energy were finally got. In the Discovery Studio(DS) software system installed on server computer, using Z-dock program, the3D molecules of AIFNalscFv1and huIFN-alb was docked. Based on the parameter of RMSD、 E_Rdock、epitope mapping assay on the docked result, the reasonable modeling structure were obtained, and after analyzing of docked antigen-antibody complex structure, the function of antibody could be explained.