Construction, Expression And Purification Of Human Anti-BP180 Single-chain Fv Antibody

Bullous pemphigoid(BP) is the most common and chronic autoimmune blistering skin disease characterized clinically by tense bullae that may develop on normal or erythematous skin, which is seen predominantly in the elderly. Up to recently, BP is usually treated with systemic glucocorticosteroids, frequently in combination with other immunosuppressants. Although the condition can be temporarily relieved, the severe side effects of glucocorticosteroids and the frequent recurrence of the disease remain to be the most serious problems[1, 2].It has been confirmed that the pathogenesis of BP is a complicated course initiated by the binding of autoantibodies, and mediated by complements and other inflammatory factors[3,.4]. The major histologic feature of BP is subepidermal blisters with variable degrees of dermal inflammation. The immunopathological tsets reveal linear deposition of C3, IgG, and/or IgE along the basement membrane zone. A majority of the patients with BP demonstrate circulating autoantibodies reactive with autoantigens located in the region of the basement membrane zone.BP180, also referred to as type XVII collagen or bullous pemphigoid antigen 2 (BPAg2), is a transmembrane protein that is associated with hemidesmosome and is thought to harbor the pathogenic autoantigen responsible for the initiation of BP. The extracellular domain especially the 16th non-collagenous A domain (NC16A) is the main pathogenic target epitope for BP autoantibodies[3, 5, 6].The pathogenic autoantibodies of BP bind to dermal-epidermal junction (DEJ) components and activate the complement system that mediates a series of inflammatory events including dermal mast cell degranulation and generation of neutrophils, eosinophils, monocyte rich infiltrates and proteinases and inflammatory mediators released, result in the loss of cell-matrix adhesion structure, and finally, subepidermal blister formation. Newly developed humanized BP animal model further elucidated the pathogenic evects of the autoantibody in vivo[7]. It was confirmed that the activation of the complement cascade via the classical pathway is required for the development of the disease. It is therefore rational to develop new therapeutic strategies by preventing complement activation via blocking autoantibody binding to the corresponding pathogenic autoantigen using targeted scFv antibody fragments.In our previous studies, phage antibody library was constructed using the the lymphocytes from BP patients and a Fab monoclonal antibody (clone P10), which specifically bind to BP180-NC16A, was isolated[8]. The purpose of the present study is to generate anti-BP180-NC16A scFv.Objective: To construct and express anti-BP180-NC16A scFv by genetically engineering technology, and to identify the binding activity and specificity of the expressed scFv.Methods: Variable region genes of VH and Vλwere cloned from Fab fragment against BP180-NC16A separately, linked with a linker sequence by SOE PCR method, and then ligated with the vetor pGEMT. After rapid screening and identification, the correct ligasion of VH-linker-Vλwas inserted into the prokaryotic expression vector p3MH, and transformed to E.coli by electroporation. The recombinant was identified by restriction double-enzyme digestion and sequence analysis. After that, soluble scFv express vector was obtained by removing the gene III fragment in the phagemid vector. Anti-BP180-NC16A scFv was expressed, and was purified using Ni affinity chromatography method. The reactivity and specificity of the expressed scFv with rhNC16A and other control antigens were tested by ELISA and Western blotting. In order to evaluate the binding of anti-BP180-NC16A scFv to the naive BP180 molecule, indirect immunofluorescence (IIF) was performed on the frozen section of normal human skin.Results: Anti-BP180-NC16A scFv was successfully constructed. DNA sequence analysis showed that there was no any mutation in Vλand VH genes of the constructed scFv. The soluble anti-BP180-NC16A scFv was successfully expressed and was purified by Ni affinity chromatography. SDS-PAGE analysis showed that the purity of scFv products was higher than 89%. By ELISA and Western blot characterization, the isolated anti-BP180-NC16A scFv was found to have a good antigenic specificity as well as excellent binding activity with the NC16A domain of human BP180. Indirect immunofluorescence (IIF) staining showed linear binding of the scFv along the dermal-epidermal junction (DEJ).Conclusion: Our success in generating the anti-BP180-NC16A scFv makes it possible to create a novel specific therapy for BP. In addition, it would be also useful in furthering the studies on the pathogenesis of the disease.