【作者】 孙娜娜；

【导师】 王刚； 周丽君；

【作者基本信息】 第四军医大学 ， 皮肤病与性病学， 2009， 硕士

【摘要】 大疱性类天疱疮(bullous pemphigoid,BP)是一种常见的慢性自身免疫性水疱性皮肤病,临床表现为正常皮肤或红斑基础上的紧张性水疱、大疱,多见于老年人群,病情易反复发作。目前BP尚缺乏特异性治疗手段而主要依靠应用糖皮质激素和免疫抑制剂等方法,虽然可以暂时起到缓解病情的作用,但需要长期应用产生的副作用以及BP病情的反复发作是临床实际工作中最亟待解决的问题[1, 2]。研究发现BP的发病是由致病性自身抗体介导、补体等多种炎症成分参与而最终导致皮肤免疫损伤的复杂过程[3, 4]。主要病理改变是表皮下水疱并伴有中性粒细胞、嗜酸性细胞等炎细胞浸润。免疫学研究发现患者皮肤表皮真皮交界处的基底膜带(basement membrane zone, BMZ)有抗体(IgG、IgE等)和补体沉积,同时在患者血清中可以检测到特异性识别BMZ的自身抗体。BP180系真表皮交界处基底膜带半桥粒跨膜胶原蛋白,又称ⅩⅦ型胶原(COL17)或大疱性类天疱疮抗原2 (BPAg2),在维持表皮和真皮的正常粘附方面起着至关重要的作用。目前证实BP180是BP致病性自身抗体所识别的主要靶抗原,其细胞外区域特别是第16非胶原编码区(NC16A)是致病性自身抗体识别的主要靶表位区[3, 5, 6]。BP病理性自身抗体与自身抗原特异性结合并激活补体,肥大细胞脱颗粒,趋化中性粒细胞、嗜酸性细胞、单核细胞等炎细胞在局部浸润并释放中性粒细胞弹性蛋白酶(NE)、基质金属蛋白酶9 (MMP-9)等蛋白水解酶和多种炎症介质,造成明显的免疫炎症反应,导致在表皮真皮黏附中起重要作用的分子结构和功能受损,真表皮分离最终发生表皮下水疱。一项较新的研究已经在人源化动物模型体内证实了BP患者抗BP180-NC16A自身抗体的致病作用[7],更深层次地阐明了BP的发病机理。在此复杂过程中,病理性自身抗体与自身抗原结合进而激活补体是至关重要的环节。因此,我们提出从这一关键环节入手,制备人源性基因工程小分子抗体,利用其能特异性识别靶抗原同时又不具有Fc段因而不能激活补体的特性,封闭与BP发病相关的自身抗原表位,竞争性抑制自身抗体的结合,从而阻断自身抗体的致病作用,探讨更加有效和特异的治疗策略。本课题组在前期工作中,直接以大疱性类天疱疮患者的外周血淋巴细胞为基因来源,利用噬菌体抗体库技术筛选首次得到针对大疱性类天疱疮抗原BP180-NC16A的人源性基因工程抗体Fab片段P10[8]。本研究在此基础上构建scFv,为BP的基因工程抗体治疗提供新的候选抗体。目的:利用基因重组技术制备人源性抗大疱性类天疱疮抗原BP180的单链抗体(scFv),进行大量原核表达,获得纯化目的蛋白,并对其免疫学特性进行鉴定。为进一步研究BP的发病机制和靶向治疗奠定基础。方法:经PCR分别扩增抗BP180人源性Fab抗体P10轻链可变区(Vλ)和重链可变区(VH)基因,应用重叠延伸拼接法(SOE)通过连接肽Linker在体外直接完成轻重链可变区的组合形成scFv。所获得的scFv基因片段再与pGEM-T载体连接,经蓝白斑筛选,菌落PCR快速鉴定阳性克隆,进行SacⅠ和SpeⅠ双酶切鉴定后回收纯化目的产物。重组正确的scFv基因克隆入原核表达载体p3MH,电转化大肠杆菌XL1-Blue,经辅助噬菌体VCSM13感染表达噬菌体抗体,ELISA检测活性,双酶切鉴定及DNA序列测定。选取活性最强的噬菌体抗体质粒酶切去除载体上的基因Ⅲ,载体自连,获得人源性抗BP180-NC16A单链抗体的可溶性表达质粒。IPTG诱导进行scFv的大量可溶性表达,上清用50%饱和硫酸铵盐析沉淀,充分透析后利用亲和层析法分离纯化目的蛋白,SDS-PAGE鉴定纯度。ELISA和Western blot鉴定活性和特异性,以人皮肤冰冻切片为底物进行间接免疫荧光检测结合部位。结果:成功构建人源性抗BP180-NC16A单链抗体的表达质粒,双酶切和DNA测序完全正确,命名为pscFv10。噬菌体单链抗体经ELISA检测结合活性良好。载体自连后转化大肠杆菌,表达产物经SDS-PAGE鉴定可溶性目的蛋白的纯度在89%以上。ELISA和Western blot方法证实所获的单链抗体具备良好抗原结合活性和高度特异性。间接免疫荧光检测,在人皮肤的表皮真皮交界处形成线状荧光条带,表明该抗体特异性识别人皮肤基底膜带的BP180分子。结论:采用基因重组技术成功构建了人源性抗大疱性类天疱疮抗原BP180-NC16A的单链抗体,并获得了可溶性表达。通过原核表达及亲和层析法有效纯化目的蛋白,并进行体外生物学鉴定,证明所获单链抗体具备良好的抗原结合活性和特异性,为下一步靶向BP180分子免疫学发病机理的研究和开展可能的临床治疗应用奠定了基础。更多还原

【Abstract】 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.更多还原