CN104922670A - Application of fused immune protein to preparation of medicine for treating multiple sclerosis - Google Patents

Application of fused immune protein to preparation of medicine for treating multiple sclerosis Download PDF

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CN104922670A
CN104922670A CN 201510223676 CN201510223676A CN104922670A CN 104922670 A CN104922670 A CN 104922670A CN 201510223676 CN201510223676 CN 201510223676 CN 201510223676 A CN201510223676 A CN 201510223676A CN 104922670 A CN104922670 A CN 104922670A
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eae
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sequence
brain
treatment
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CN 201510223676
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张蕲
周翔鱼
祝道成
周雄
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武汉奥斯梅得生物医药有限公司
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The invention discloses application of a fused immune protein to preparation of medicine for treating multiple sclerosis and belongs to the field of biological engineering. The fused immune protein has specific effect of relieving chronic EVE (experimental autoimmune encephalomyelitis) mouse clinical impairment. In a treatment course, hormone-like drug withdrawal rebound phenomenon does not appear. The fused immune protein can be used for preparing the medicine for treating multiple sclerosis independently or can be used for preparing the medicine for treating multiple sclerosis with other medicines.

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融合免疫蛋白在制备治疗多发性硬化症药物中的应用 Application of the fusion immunoglobulin in the manufacture of a medicament for the treatment of multiple sclerosis

技术领域 FIELD

[0001] 本发明涉及一种融合免疫球蛋白在制备治疗多发性硬化症药物中的应用,属于生物工程领域。 [0001] The present invention relates to a fusion immunoglobulin use in the manufacture of a medicament for the treatment of multiple sclerosis, which belongs to the field of biological engineering.

背景技术 Background technique

[0002] 多发性硬化症(MS)是一类临床表现和病理改变复杂多样,病程及预后千差万别, 其病程中常有缓解复发的神经系统损害症状。 [0002] Multiple sclerosis (MS) is a class of clinical and pathological changes complex and diverse, and prognosis of course vary, which is often the course of remission of recurrent neurologic symptoms. MS是以中枢神经系统白质散在/多灶性髓鞘脱失为主要病理变化,可伴有不同程度轴索损伤的炎性自身免疫性疾病。 MS is a central nervous system white matter scattered / multifocal demyelination major pathological changes, may be associated with varying degrees of axonal injury inflammatory autoimmune disease. 脱髓鞘病灶于脑白质小静脉和侧脑室周围常见,伴随淋巴细胞和单核巨噬细胞浸润,免疫荧光检查可见IgG 鞘内沉积,提示有免疫激活现象。 Demyelinating lesions in the white matter surrounding the lateral ventricles and venules common, lymphocytes and monocytes accompanied by infiltration of macrophages, intrathecal IgG immunofluorescence examination showed deposition, suggesting immune activation phenomenon. 根据少突胶质细胞保留的程度、丢失的髓鞘蛋白、炎症细胞浸润类型、免疫球蛋白或补体沉积情况可将髓鞘脱失或少突胶质细胞损伤模式分成四种类型:(I) T细胞/巨噬细胞介导的髓鞘脱失;(2)抗体/补体介导的髓鞘脱失;(3)少突胶质细胞营养不良并伴有少突胶质细胞凋亡;(4)原发性少突胶质细胞变性,特征与病毒感染或中毒所致的少突胶质细胞损害类似。 The degree of retention of oligodendrocytes, the myelin protein loss, inflammatory cell infiltration type, an immunoglobulin or complement deposition may be where demyelination or oligodendrocytes damage pattern into four types: (I) T / macrophage cell-mediated demyelination; (2) the antibody / complement-mediated demyelination; (3) oligodendrocytes and malnutrition with oligodendrocyte apoptosis; ( 4) primary oligodendrocyte degeneration, with features similar to a viral infection or poisoning caused by damage to oligodendrocytes. 由此可见,MS/EAE(EAE指实验性变态反应性脑脊髓炎)的免疫病理过程包括两个方面,一是周围免疫系统的激活,二是免疫反应在中枢神经系统的持续存在并被放大。 Thus, MS / EAE (EAE Experimental allergic encephalomyelitis means) immunopathological process includes two aspects, one surrounding the activation of the immune system, and second, the immune response and amplified in the central nervous system persists . 传统观点认为,中枢神经系统以髓鞘脱失为主的损伤主要是由强致炎细胞因子γ干扰素(IFN-γ)介导,IFN-Y通过诱导Thl型细胞(辅助性T细胞) 分化引发针对髓鞘的免疫应答;然而在进行IFN-γ或其受体基因敲除动物研宄时,却发现EAE仍能发生,甚至实验动物更易患病,说明MS/EAE可能是多种机制共同作用引起的自身免疫性疾病;其中可能涉及固有免疫、特异性免疫应答和免疫应答调节机制等诸多方面。 Conventional wisdom holds that demyelination of the central nervous system damage is mainly dominated by a strong proinflammatory cytokine gamma] interferon (IFN-γ) mediated, IFN-Y by inducing the differentiation of Thl-type cells (helper T cells) initiate an immune response against myelin; however during IFN-γ or its receptor knockout animal study based, but found that EAE can still occur, even more susceptible experimental animals, indicating that MS / EAE common variety of mechanisms may be autoimmune diseases caused by action; may involve innate immunity, specific immune response and immune response mechanisms, and many other aspects. 有大量研宄结果认为,EAE模型发病机制中,Thl/Th2型细胞反应失衡,Treg细胞(调节性T 细胞)分化受阻或功能减弱是最重要的两个因素。 A large study based on the results that, EAE pathogenesis models, Thl / Th2 type cellular response imbalance, Treg cells (regulatory T cells) differentiation is blocked or weakened two most important factors. IL_17(白介素)在EAE发病中起致炎作用,疾病进展过程中Thl7细胞的迀移至中枢神经系统是病变部位具有高度选择性的基础。 IL_17 (interleukin) plays in the pathogenesis of EAE proinflammatory role during disease progression Gan Thl7 cells in the central nervous system is moved to the lesion having a highly selective basis. 比较认可的MS免疫应答反应过程可能包括:特异性自身反应性T淋巴细胞、B淋巴细胞在外周首先被激活,在多种炎症介质的趋化作用下,经异常开放的血-脑屏障迀移、定位于中枢神经系统存在髓鞘的部位并引起免疫应答。 MS recognition process is relatively immune response may include: specific autoreactive T lymphocytes, B lymphocytes outer periphery is first activated, in a variety of inflammatory mediators of chemotaxis, the abnormal opening of the blood - brain barrier Gan shift , located in the site of presence of central nervous system myelin and elicit an immune response. 自身反应性T淋巴细胞经中枢自身髓鞘抗原成分反复不断地刺激,通过特异性细胞毒性作用或趋化诱导单核巨噬细胞和小胶质细胞等其他免疫细胞激活并释放多种炎性细胞因子,共同对中枢神经系统髓鞘进行直接攻击损害。 Autoreactive T lymphocytes by antigen component central myelin itself over and over again stimulated by specific cytotoxic effects or other immune cells induced by chemokine monocyte macrophages and activated microglial cells and the release of more inflammatory cells factor, the common central nervous system myelin direct attack damage. 其中自身反应性CD4+Thl淋巴细胞及其释放的细胞因子被认为起主导作用,同时B细胞来源的抗髓鞘成分抗体经由补体激活途径,趋化各种炎性细胞,并刺激针对髓鞘的调理作用而产生免疫损伤。 Wherein autoreactive lymphocytes and CD4 + Thl cytokine release is thought to play a dominant role, while the component against myelin-derived antibody activates B cells via complement pathway, various inflammatory cell chemotaxis and for stimulating myelination opsonization and immune damage. 之后由于髓鞘自身抗原持续存在,以上免疫应答过程迀延往复,髓鞘反复出现损伤和再生修复,临床症状随之出现特征性的缓解-复发进程。 After due myelin self-antigens persist, more immune response Gan Yan and forth, myelin recurring injury and regeneration, clinical symptoms have eased characteristic appearance - relapse process.

[0003] 实验性变态反应性脑脊髓炎(EAE),是发生于各种敏感实验动物中枢神经系统的炎性变态反应性疾病。 [0003] Experimental allergic encephalomyelitis (EAE), occurs in various inflammatory allergic diseases of the central nervous system susceptible animals. 诱导EAE模型抗原一般为组成髓鞘的成分蛋白,可通过注射特定的全髓鞘成分蛋白或其表达强抗原性表位的某一肽段加完全弗氏佐剂(CFA)主动免疫;也可以通过经髓鞘蛋白或其肽段致敏的CD4+T细胞过继免疫实验动物诱导。 Antigen-induced EAE model is generally a component of myelin proteins, by injection of specific components of whole myelin protein or an antigenic epitope strong expression of a peptide fragment with complete Freund's adjuvant (CFA) active immunization; may be by myelin protein or peptide sensitized CD4 + T cells in adoptive immunotherapy by inducing in experimental animals. 其中髓鞘成分蛋白或其肽段作为抗原,CFA中的所含的结核杆菌,是模型诱导的一个重要因素,可直接影响模型的发病率,甚至发病形式。 Wherein component myelin protein or peptide as an antigen contained in CFA Mycobacterium tuberculosis, is an important factor-induced model, the model can directly affect the incidence of, or even in the form of incidence. CFA还可以诱导ThO细胞向Thl细胞分化而辅助免疫诱导,没有CFA的辅助作用,一般不能被诱导出EAE模型。 CFA also induce ThO cells into Thl cells induced by adjuvant immunotherapy, no secondary effect of CFA, generally can not be induced EAE model. 另外,百日咳毒素可以提高正常血-脑屏障的通透性,有利于活化的自身反应性淋巴细胞向中枢神经系统迀移、浸润,引起炎症反应扩散。 Further, the Pertussis toxin can increase the normal blood - brain barrier permeability, activation of autoreactive lymphocytes facilitate the shift of the central nervous system Gan, infiltration, causing inflammation diffusion. 诱导发病过程中,树突状细胞为主的抗原提呈细胞(APC)被活化并将髓鞘蛋白抗原提呈给初始T淋巴细胞,特异性的自身反应性T淋巴细胞因此在外周被激活,穿过通透性增加的血脑屏障迀移至中枢神经系统(CNS)。 Induced pathogenesis, dendritic cell-based antigen presenting cells (APC) is activated and antigen presenting myelin protein is thus activated to the original outer peripheral T lymphocytes, specific autoreactive T lymphocytes, through increased permeability of the blood-brain barrier Gan moved to the central nervous system (CNS). 进入CNS后,T淋巴细胞通过释放炎性介质和细胞因子等途径招募炎性细胞至CNS,由于CNS中存在同源髓鞘蛋白抗原而被本地APC再活化, 增殖并释放炎性介质,并浸润递呈主组织相容性复合基因MHC II-抗原肽复合物的APC,导致炎症发生,进而出现脱髓鞘及轴突受损。 After entering the CNS, T lymphocyte pathway by releasing inflammatory mediators like cytokines and recruitment of inflammatory cells to the CNS, due to the presence of the homologous protein myelin antigens in the CNS is another local APC activation, proliferation, and release of inflammatory mediators, and invasion presenting the main histocompatibility complex gene MHC II- peptide complex of APC, leading to inflammation, demyelination and axonal damage occurs in turn. EAE模型动物大多数表现为加重-自然缓解单相病程,MBP68-86肽段在诱导Lewis大鼠EAE模型时一般发病率很高,而且疾病严重程度较均一,变异很小;但是该模型主要表现为急性炎性反应反应,中枢神经系统血管周围的炎细胞以中性粒细胞和单核细胞为主,少有淋巴细胞浸润;缺乏缓解-复发的临床特征,也没有髓鞘脱失和再生修复的病理学变化。 EAE animal model showed most aggravating - natural relief for single-phase course, MBP68-86 peptide induced EAE in the Lewis rat model is generally a high incidence and severity of the disease more uniform, variation is small; but this model is mainly the reaction, perivascular cells in the central nervous system inflammatory neutrophils and monocytes mainly acute inflammatory response, few lymphocytes; lack of ease - recurrent clinical features, nor demyelination and regeneration changes in pathology. PLP139-151肽段可诱导S几小鼠复发-缓解型EAE, MOG35-55肽段可在C57BL/6小鼠诱导出慢性非缓解型EAE,所见病理改变与与人类MS极为相似。 PLP139-151 peptide can induce several mouse S relapsing - remitting EAE, the MOG35-55 peptide can induce chronic non-remitting EAE in C57BL / 6 mice, pathological changes seen in humans with MS are very similar. 一般而言,血清各种抗髓鞘成分或非成分抗体不能诱导出EAE模型,但利用致敏T淋巴细胞被动传递则能成功,提示EAE/MS病变发生的本质可能属于细胞免疫机制。 In general, a variety of serum antibodies against myelin components or ingredient not induce EAE model, but the use of passive sensitized T lymphocytes is successfully passed, suggesting the nature of EAE / MS lesions may belong cellular immune mechanisms. 现在认为其他各种抗髓鞘成分自身抗体在EAE中不具有致病性,只有抗少突胶质细胞糖蛋白(MOG) 抗体可能与疾病的发生有关。 Now that various other autoantibodies against myelin components having no pathogenic in EAE, only anti-oligodendrocyte glycoprotein (MOG) antibodies may be related to the disease. MOG是一种26-28KDa跨膜糖蛋白,表达在髓鞘膜和少突胶质细胞外表面,其胞外区有三个可供识别的抗原表位,M0G35-55肽段是MOG胞外区致脑炎抗原表位之一,由于MOG选择性表达于成熟少突胶质细胞髓鞘外表面,因此更易受到免疫细胞直接攻击。 26-28KDa MOG is a transmembrane glycoprotein, myelin membrane and is expressed in the outer surface of the oligodendrocytes, which extracellular domain epitope identified for three, M0G35-55 peptide fragment is the extracellular domain of MOG one epitope induced encephalitis, since MOG selectively expressed in mature oligodendrocytes, myelin outer surface of glial cells, immune cells and therefore more vulnerable to attack directly. 近年来MOG作为EAE模型的诱导抗原已被广泛应用于动物实验研宄中,文献报道MOG诱导C57BL/6小鼠的EAE模型表现为两种形式,慢性进展型和慢性非缓解型,在病理和临床表现上最接近于人类MS。 In recent years, as the MOG EAE model induced by antigens it has been widely applied to the study based on animal experiments, reported in the literature MOG-induced C57BL / 6 mice EAE model in two forms, chronic-progressive and chronic non-remitting, pathological and closest to the clinical manifestations of human MS. 还有研宄结果证明MOG是髓鞘组成蛋白中唯一的既能引起T细胞反应又能引起髓鞘抗体产生的自身抗原;这也间接显示出MOG在诱导EAE模型建立及病理特点中的优势。 There is a Subsidiary proved MOG myelin protein composition caused both a unique T cell responses can cause myelin autoantigen antibodies; which indirectly shows the advantages of MOG-induced EAE model and the establishment of pathological features. C57BL/6小鼠是一种常用实验小鼠,国内较易获得和饲养,且遗传背景清楚。 C57BL / 6 mice is a common laboratory mouse, domestic and breeding easy to obtain, and genetic background is clear. M0G35-55肽段诱导C57BL/6小鼠的慢性EAE模型比急性EAE模型在临床、生化、免疫及病理等诸多方面与MS具有更相似的特征,尽管M0G35-55肽段诱导的EAE模型还不能完全真实地表现出人类MS全部病理特点,但仍是当前公认的研宄MS的发病机制和治疗策略的理想动物模型之一。 Chronic EAE model induced by peptide M0G35-55 C57BL / 6 mice having more than acute EAE model in many respects similar features with MS clinical, biochemical, immunological and pathology, although M0G35-55 peptide induced EAE model can not be completely true demonstration of all the pathological features of human MS, but is still recognized as one of the current study based on an ideal model of MS pathogenesis and treatment strategies.

[0004] 目前MS的治疗主要是缓解症状,尚没有治愈的方法,临床上用于治疗MS的一线药物有甲泼尼龙(糖皮质激素)、β干扰素(IFN-β)等。 [0004] The current primary treatment of MS is to relieve symptoms, no cure method, the first-line drugs for the treatment of MS are methylprednisolone (glucocorticoids) Clinically, interferon beta] (IFN-β) and the like. 甲泼尼龙是目前治疗MS急性期的首选药物,疗效较为确切,它能够抑制免疫细胞DNA的合成和有丝分裂,促进血脑屏障的恢复、改善轴索传导、诱导T淋巴细胞凋亡、减少炎性细胞因子的释放、对免疫系统的多个环节均有强力抑制作用。 Methylprednisolone drug of choice in acute treatment of MS, efficacy more precise, it is possible to inhibit the synthesis of DNA and mitosis immune cells, promoting the recovery of the blood-brain barrier, improve axonal conduction, induction of apoptosis of T lymphocytes, reduce the inflammatory release of cytokines, inhibition of the plurality of links have a strong immune system. 但是甲泼尼龙具有广泛而严重的毒副作用,患者的耐受性差异较大, 难以长期使用,停药后的反弹现象也经常出现。 But methylprednisolone has extensive and serious side effects, patients tolerated the difference is large, difficult long-term use, the rebound phenomenon after stopping too often. 而IFN-β则因价格昂贵限制其广泛使用。 IFN-β due to the high price limit its widespread use. 这些限制促使研宄者一直努力寻找一种更加有效、特异性更高并且毒副作用低的药物替代或联合治疗MS。 These limitations led study based on who has been trying to find a more effective, greater specificity and low toxicity of drug substitution or combination therapy MS.

[0005] 近年来的动物实验和临床研宄都显示,B淋巴细胞可能参与MS/EAE发生和发展的诸多环节。 [0005] In recent years, animal experiments and clinical research have shown traitor, B lymphocytes may be involved in MS / EAE development and progression of many links. 近年来多种单克隆抗体投入临床治疗并取得确切效果,这些现象也佐证了体液免疫的作用可能被人们所忽视。 In recent years, a variety of monoclonal antibodies into clinical treatment and achieved the exact effect, these phenomena also corroborated the role of humoral immunity may be overlooked. 有些研宄认为,MS患者不同的临床类型和对药物治疗反应的高异质性可能与不同的免疫应答机制参与有关。 Some study based on the view that the different types of MS patients and clinical response to drug treatment of high heterogeneity may be related to different mechanisms involved in the immune response. 因此,B淋巴细胞及其产生的多种抗体在MS/EAE病程中所起的作用越来越受到人们关注。 Thus, B lymphocytes, and a variety of antibodies produced in the MS / EAE role in the course of more and more attention. 在所有髓鞘蛋白成分中,少突胶质细胞糖蛋白(MOG)是唯一能够引起T细胞免疫和产生自身抗体的自身抗原。 In all of the protein components of myelin, oligodendrocyte glycoprotein (MOG) is the only capable of eliciting T cell immunity and the production of autoantibodies autoantigen. 可以通过活化自身杀伤细胞(NK细胞)和巨噬细胞的Fc受体发挥抗体依赖的细胞介导的细胞毒作用,或者形成膜免疫攻击复合物。 It can exert itself by activating killer cells (NK cells), macrophage Fc receptor, and cytotoxicity antibody dependent cell-mediated or immune membrane attack complex formation. MS/EAE中B淋巴细胞活化可能通过三条途径:旁路活化、分子模拟和表位扩展。 MS / EAE by B lymphocyte activation in three possible ways: pathway activation, molecular modeling, and epitope spreading. MS患者确定有体液免疫学异常,即在体内可以检测到多种自身抗体,脑脊液中也存在寡克隆带,只是还不了解寡克隆带的特异性靶抗原。 MS patients determine humoral immune abnormalities that can be detected in vivo in a variety of autoantibodies in CSF oligoclonal bands are also present, but do not know the target antigen specificity of oligoclonal bands. Owens等对MS患者脑脊液的研宄发现寡克隆带对已知髓鞘组成成分蛋白如M0G,髓鞘碱性蛋白(MBP),蛋白脂蛋白(PLP)等并不具有靶向性,这提示MS患者体内可能存在B淋巴细胞的表位扩展现象。 Owens et study based on MS patient CSF oligoclonal bands found known myelin protein components such as M0G, myelin basic protein (MBP), proteolipid protein (PLP) and the like do not have targeting, suggesting MS epitope spreading may be present in the patient B lymphocytes phenomenon. 有研宄发现,EAE 中产生的抗体所针对的抗原表位与T淋巴细胞结合的抗原表位在很大程度上是重合的,表明T淋巴细胞和B淋巴细胞可以通过结合相同的自身抗原而发生协同作用,这种TB淋巴细胞之间的相互作用可能是EAE的关键发病机制之一。 There are a Subsidiary found that EAE epitope antibodies generated against the T-lymphocyte epitope bound largely overlapping, suggesting that T and B lymphocytes can bind to the same autoantigen and synergistic effect occurs, interaction between the lymphocytes may be one such key TB pathogenesis of EAE. 以上事实都说明,抑制B淋巴细胞活化产生抗体及其抗原提呈功能,封闭自身抗体的活性是MS/EAE治疗可供选择的方法之一。 Facts described above, inhibition of B lymphocyte activation and antigen-presenting function to produce antibodies, autoantibodies blocking activity is one of the methods MS / EAE therapeutic alternative. 此外,抑制APC抗原提呈后的免疫应答,下调APC致炎细胞因子的表达也是治疗MS/EAE的有效备选方案之一。 Furthermore, suppression of the immune response following antigen presentation in APC, APC downregulation of proinflammatory cytokines are also an effective treatment alternatives MS / EAE's.

[0006] 本发明人通过总结前期部分理论成果提出如下假设:抗原提呈细胞(APC)对抗原识别、处理加工、提呈和自身活化是慢性EAE的早期免疫事件和发病的前提条件。 [0006] The present inventors hypothesize early theoretical results summarized Part: antigen-presenting cells (APC) for antigen recognition, processing and processing, and presenting themselves early immune activation is a prerequisite events and morbidity of chronic EAE. 固有免疫期抗原不能被有效清除或APC对这类抗原反应过于强烈,适应性免疫应答才随之发生。 Innate immunity of the antigen or APC can not be effectively cleared over-reaction to such antigens, the adaptive immune response only ensue. APC对淋巴细胞持续抗原提呈和自身活化最终引起细胞免疫应答在中枢神经系统的发生和放大而损伤髓鞘。 Lymphocytes in antigen presentation on APC and eventually causing their activation of a cellular immune response in the central nervous system, damage to myelin and enlarged. APC通过主要组织相容性复合物(MHC)分子和共刺激信号分子共同作用激活自身反应性T、B淋巴细胞是致病的中心环节和重要驱动因素,在EAE致病过程中可能起决定性作用。 APC activation of autoreactive T through interaction (MHC) molecules and costimulatory molecules of the major histocompatibility complex, B lymphocytes is a central and important pathogenic drivers, may play a decisive role in the pathogenesis of EAE . 外周自身免疫耐受机制终止、免疫反应的负反馈调节作用减弱或障碍,ThO 细胞选择性向Thl细胞的优势分化等多种因素共同作用,最终使中枢神经系统产生脱髓鞘病变。 An outer peripheral immune tolerance mechanisms terminated, a negative feedback regulation of the immune response attenuate or disorder, ThO cell selective advantage to differentiate into Thl cell interaction and other factors, and finally to central nervous system demyelinating disease. APC除了抗原提呈作用外,活化后产生的多种促炎性细胞因子对EAE发生也起到一定作用。 APC antigen presentation in addition, produced more after activation of pro-inflammatory cytokines also play a role in the occurrence of EAE. 增强免疫抑制性调节机制发挥,阻断APC的抗原摄取、加工、提呈,抑制自身反应性T、B淋巴细胞活化,减少APC促炎细胞因子分泌,促进ThO细胞向Th2细胞而非Thl细胞分化等都是潜在的有效治疗靶点。 Enhanced immunosuppressive regulatory mechanisms play, blocking the APC antigen uptake, processing, presentation, inhibition of autoreactive T, B lymphocyte activation, reduction APC proinflammatory cytokine secretion, promoting ThO cells into Thl cells but not Th2 cells are all potentially effective therapeutic targets.

[0007] 融合免疫球蛋白(mGE)(专利号:CN200410006498. 9),其组成成份全部来源于人源性免疫球蛋白,该蛋白作为一种药物进入人体,没有任何异体蛋白的免疫源性。 [0007] Fusion immunoglobulins (MGE) (Patent No:. CN200410006498 9), all of which constituent derived from a human endogenous immunoglobulin, the protein as a drug into the body, without any of the immunogenic foreign protein. mGE是一种具有剂量依赖效应,无明显不良反应的融合免疫球蛋白。 mGE is a dose-dependent effect of having no significant adverse reactions in the fusion immunoglobulin. mGE为约140KD的二聚体,将IgG 的Fc段与IgE的Fc段用15个氨基酸融合而成,可与抗ε -及γ-链特异性抗体相互作用。 is a dimer of about 140KD mGE the Fc region Fc fragment of IgG and IgE by 15 amino acids fused together, with the anti-ε - γ- chain specific antibody interactions. mGE的C区(Hinge)灵活,可转动,可将Fc γ RII (IgG受体)及Fc ε RI (亲和性IgE受体) 结合,从而诱发细胞内的抑制信号,阻止细胞释放各种生物活性物质。 mGE C region (Hinge) a flexible, rotatable, can be incorporated Fc γ RII (IgG receptor) and Fc ε RI (affinity IgE receptor), thereby inhibiting intracellular signaling induced, prevent the release of a variety of biological cells active substances. Fc ε RI通过IgE与多价抗原结合后使相邻两个Fc ε RI受体发生桥联,激活免疫受体酪氨酸活化基序(ITAM),进而启动下游信号通路并最终引起细胞的活化效应。 After IgE Fc ε RI by the multivalent antigen binding two adjacent Fc ε RI receptor bridging occurs, activation of the immune tyrosine-based activation motif (the ITAM), and thus start of downstream signaling pathways ultimately leads to the activation of the cells effect. Fc ε RI信号通路受IgG的抑制性受体FcyRIIb负反馈调节,Fe YRIIb是唯一胞内段含有免疫受体酪氨酸抑制基序(ΙΊΊΜ)的Fc 受体,可以招募下游信号转导分子去磷酸化而抑制信号转导。 Fc ε RI receptor signaling pathway by inhibiting the negative feedback regulation FcyRIIb IgG, Fe YRIIb unique within an intracellular segment contains an immunoreceptor tyrosine-based inhibitory motif (ΙΊΊΜ) an Fc receptor, can recruit the signal transduction molecule to the downstream phosphorylation inhibits signal transduction. Fc γ RIIb在生理条件下可与Fe ε RI发生聚合,并且磷酸化的Fe γ RIIb需要与Fe ε RI共聚集,通过SH2结构域的5-磷酸肌醇(SHIP)则可诱导产生抑制信号。 Fc γ RIIb can polymerize with Fe ε RI under physiological conditions, and phosphorylated Fe γ RIIb needs Fe ε RI and co-aggregation, can be induced by suppressing the signal inositol-5-phosphate (SHIP) SH2 domains. 该融合免疫球蛋白能够使Fey RIIb和Fe ε RI发生聚集,激活Fe ε RI相关酪氨酸激酶(Lyn)的抑制信号,从而导致Fe γ RIIb的ΙΊΊΜ的酪氨酸迅速磷酸化而抑制肥大细胞和嗜碱性粒细胞的脱颗粒反应(Degranulation)及B细胞分泌抗体向IgE的类别转换,其中,其作用原理见图17所示。 The fusion immunoglobulin is possible to Fey RIIb aggregation occurred and Fe ε RI activation Fe ε RI-related tyrosine kinase (Lyn) suppression signal, thereby causing the Fe γ RIIb ΙΊΊΜ rapid tyrosine phosphorylation and inhibition of mast cells and degranulation of basophils (degranulation) and antibody-secreting B cells to IgE class switching, wherein, its mechanism shown in Figure 17.

[0008] APC是调节免疫反应方向和产生免疫耐受所必需的调节器,在维持免疫耐受中起到关键作用。 [0008] APC is to modulate the immune response and immune tolerance directions necessary regulators, play a key role in the maintenance of immune tolerance. APC提呈抗原并激活T淋巴细胞时需要双信号刺激:Τ淋巴细胞表面抗原受体(TCR)与MHC-抗原肽复合物结合提供第一信号,APC表达的共刺激分子Β7-1/Β7-2与T细胞相应受体CD80/CD86/等结合提供活化的第二信号。 APC antigen presentation and activation requires two signals. Stimulation of T lymphocytes: Τ lymphocyte surface antigen receptor (TCR) binding MHC- peptide complex to provide a first signal, the APC co-stimulatory molecule expression Β7-1 / Β7- 2 corresponding to T cell receptors CD80 / CD86 / second binding other activation signal. 共刺激分子不仅只提供T细胞增殖的第二信号,同时也调控免疫反应强度,防止T进入无反应状态或细胞过度激活。 Costimulatory molecules not only provides a second signal of T cell proliferation, but also the regulation of the immune response strength to prevent entering unresponsive T cell or overactivation. 树突状细胞(DC)作为起主要作用的APC,有研宄者将其根据功能不同分为DCl和DC2两种类型,其中DCl可产生大量IL-12、TNF-a和少量IL-6,诱导ThO细胞向Thl细胞优势分化;而DC2可分泌大量IL-6和少量IL-12,诱导ThO细胞向Th2细胞优势分化。 APC dendritic cells (DC) play a major role as with a Subsidiary will be divided into two types DCl and DC2, DCl which can produce a large number of different functions in accordance with IL-12, TNF-a, and a small amount of IL-6, ThO cells into Thl inducing cells advantage; DC2 can secrete large amounts of IL-6 and a small amount of IL-12, induction of cell differentiation to Th2 cells ThO advantage. DC2可通过诱导Th2型反应而抑制Thl型应答的产生和强度,Th2型反应产生的IL-IO能影响DC的成熟,减少其MHCII类分子、共刺激分子和粘附分子等的表达,并能诱导Treg细胞分化,增强机体的免疫耐受。 DC2 can be suppressed and the strength of Thl type response inducing Th2 type response, IL-IO Th2-type reaction can affect DC maturation, reduce the MHCII molecules, costimulatory molecules and adhesion molecules, and can induce Treg cell differentiation, enhancement of immune tolerance. 研宄还发现,EAE动物中枢神经系统中小胶质细胞表达MHCII类分子,且应用INF-γ 诱导MHCII类分子表达能加重EAE病情,而应用IL-4、IL-10及TGF- β可抑制MHCII类分子表达并有助于EAE恢复,这与本实验前期所得出的APC抗原提呈及炎性细胞因子分泌是参与EAE的重要机制这个结论一致。 A Subsidiary found, central nervous system EAE animals microglial cells express MHCII molecules, induction of INF-γ and application of MHCII molecules expressed EAE aggravate the disease, and the application IL-4, IL-10 and TGF- β suppressed MHCII class molecule expression and help restore EAE, which is early in this experiment the results of APC antigen presentation and inflammatory cytokine secretion is an important mechanism involved in EAE conclusion of this agreement. 基于对MS/EAE发病机制多样化观点的认同,即多种类型免疫应答、多种免疫细胞参与和不同作用机制共同导致疾病发生,因此我们把治疗的靶点设为免疫细胞的Fe γ RIIb,通过增强其表达或功能而缓解MS/EAE的损伤症状。 Based on the recognition of MS / EAE pathogenesis of diverse views, that multiple types of immune responses, immune cells involved in a variety of different mechanisms of action and common cause disease, so we set the target for the treatment of Fe γ RIIb immune cells, by enhancing the expression or function and relieve symptoms of injury MS / EAE's. 本发明的目的是提供融合免疫球蛋白在制备治疗多发性硬化症药物中的应用,证实通过这一途径, 融合免疫球蛋白具有明确的缓解慢性EAE小鼠临床功能损害的作用,治疗过程中不会出现激素样停药反弹现象。 Object of the present invention is to provide an application in the manufacture of a fusion immunoglobulin in the treatment of multiple sclerosis drug, confirmed by this approach, the fusion immunoglobulin has a clear clinical remission of chronic EAE mice by dysfunction, the treatment process does not there will be hormone-like withdrawal rebound phenomenon.

发明内容 SUMMARY

[0009] 本发明公开了融合免疫球蛋白在制备治疗多发性硬化症药物中的应用,以充分的药理实验证明该融合免疫球蛋白可用于制备治疗多发性硬化症药物,且治疗过程中不会出现停药反弹现象。 [0009] The present invention discloses the use of a fusion immunoglobulin in the manufacture of a medicament for the treatment of multiple sclerosis, a sufficient pharmacological experiments prove that the fusion immunoglobulin can be used for the preparation of drugs to treat multiple sclerosis, and the treatment process will not withdrawal rebound phenomenon occurs.

[0010] 具体的说,该融合免疫球蛋白可以通过抑制APC的MHC分子表达,抑制其致炎细胞因子释放而缓解慢性EAE的神经功能损伤症状。 [0010] Specifically, the fusion immunoglobulin can be expressed by the inhibition of APC MHC molecules, which inhibit the proinflammatory cytokines releasing cells alleviated symptoms of chronic neurological impairment of EAE. 主要表现在以下几个方面: Mainly in the following areas:

[0011] (1)融合免疫球蛋白可以降低慢性EAE小鼠脑内补体C3和C5a、MHCI、MHCII类分子、B7-l、Fas、IL-12、IL-27 和脾脏INF-γ 的表达; [0011] (1) Expression of a fusion immunoglobulin can reduce chronic EAE mice brain complement C3 and C5a, MHCI, MHCII molecules, B7-l, Fas, IL-12, IL-27 and INF-γ in spleen;

[0012] (2)融合免疫球蛋白可以增加Fe γ RIIb的表达;表现出对APC活化的多重阻遏作用。 [0012] (2) increase the expression of a fusion immunoglobulin can be of Fe γ RIIb; exhibit multiple repression of APC activation.

[0013] 根据本发明,所述融合免疫球蛋白是具有序列表中SEQ IDN2 :2氨基酸残基序列的蛋白质,或者是将SEQ IDN2 :2氨基酸残基序列经过一个或几个氨基酸残基的取代、缺失或添加具有与SEQ ID Ne : 2氨基酸残基序列相同活性的由SEQ ID Ne : 2衍生的蛋白质。 [0013] According to the invention, the fusion immunoglobulin is having the sequence of SEQ IDN2: 2 Protein sequence of amino acid residues, or is SEQ IDN2: 2 amino acid residue sequence through one or more substituted amino acid residues , deletions or additions having SEQ ID Ne: 2 of the same active amino acid residue sequence of a SEQ ID Ne: 2 derived proteins.

[0014] 根据本发明,所述融合免疫球蛋白是序列表中的SEQ IDN2 :2。 [0014] According to the invention, the fusion immunoglobulin is a sequence listing SEQ IDN2: 2. 序列表中序列2的氨基酸残基序列由554个氨基酸残基组成的蛋白质。 2 protein amino acid residue sequence of a 554 amino acid residues in the sequence table.

[0015] 根据本发明,所述融合免疫球蛋白编码基因,是下列核苷酸序列之一: [0015] According to the invention, the immunoglobulin fusion encoding gene, is one of the following nucleotide sequences:

[0016] 1)序列表中SEQ ID Ns : 1的DNA序列; [0016] 1) the Sequence Listing SEQ ID Ns: DNA sequence of;

[0017] 2)编码序列表中SEQ IDNe :2蛋白质序列的多核苷酸; [0017] 2) the coding sequence listing SEQ IDNe: 2 protein sequence of the polynucleotide;

[0018] 3)与序列表中SEQ IDN2 :1限定的DNA序列具有95%以上同源性,且编码相同功能蛋白质的DNA序列。 [0018] 3) the Sequence Listing SEQ IDN2: 95% or more homology to the DNA sequence defined in 1, and the DNA sequences encoding the same functional protein.

[0019] 根据本发明,所述融合免疫球蛋白编码基因是序列表中的SEQ IDN2 :1。 [0019] According to the invention, the gene fusion immunoglobulin coding sequence listing SEQ IDN2: 1. 序列1中的DNA序列由1665个碱基组成。 DNA sequence by 1 in 1665 bases.

附图说明 BRIEF DESCRIPTION

[0020] 图1为干预时间与分组因素交互效应图。 [0020] Figure 1 is the intervention time and interaction factors packet FIG.

[0021] 图2为实验小鼠脑HE染色(X 200)。 [0021] FIG. 2 is a HE staining mice brain (X 200).

[0022] 图3为实验小鼠脑LFB染色(X 200)。 [0022] FIG. 3 is a LFB staining mice brain (X 200).

[0023] 图4为实验小鼠脾脏IFN- γ免疫荧光组化(X 200)。 [0023] FIG. 4 is a spleen IFN- γ immunofluorescence staining (X 200) mice.

[0024] 图5为实验小鼠脑Fas免疫荧光(X 400)。 [0024] FIG. 5 is a brain Fas immunofluorescence (X 400) mice.

[0025] 图6为实验小鼠脑IL-12免疫荧光组化(Χ400)。 [0025] FIG. 6 is a IL-12 mice brain immunofluorescence staining (Χ400).

[0026] 图7为实验小鼠脑Ki-67、MBP双标免疫荧光组化(X 400)。 [0026] FIG. 7 is the experimental mouse brain Ki-67, MBP double-labeling immunofluorescence staining (X 400).

[0027] 图8为实验小鼠脑Ki-67、Ibal双标免疫荧光组化(X 200)。 [0027] FIG. 8 is experimental mouse brain Ki-67, Ibal double-labeling immunofluorescence staining (X 200).

[0028] 图9为各组脑内EB含量的变化。 [0028] FIG. 9 is a change in the content of each group EB brain.

[0029] 图10(1)、10⑵为各组不同时期脑内Fe YRIIb (CD32)的表达情况。 [0029] FIG. 10 (1), 10⑵ expression of each group is different periods Fe brain YRIIb (CD32) is.

[0030] 图11 (1)和11 (2)为EAE组小鼠在不同时期补体C3mRNA的变化。 Change [0030] FIG. 11 (a) and 11 (2) complement in EAE mice C3mRNA of different periods.

[0031] 图12 (1)和12⑵为EAE组小鼠不同时期脑内MHCI类分子mRNA的表达。 [0031] FIG. 12 (1) and the expression of mRNA molecules 12⑵ EAE brain of mice at different times MHCI class.

[0032] 图13⑴和13⑵为EAE组小鼠在不同阶段脑内MHCII类分子mRNA的表达。 [0032] FIG 13⑴ 13⑵ to EAE mice and the expression of mRNA molecules in the brain at different stages MHCII class.

[0033] 图14⑴和14⑵为EAE组小鼠在不同时期脑内IL-12的含量变化。 [0033] FIG 14⑴ 14⑵ and changes in brain tissues of EAE mice of IL-12 at different times.

[0034] 图15为各组小鼠脑内TGF-β、Fas和C5a在不同时期的表达。 [0034] FIG. 15 is the brain of mice in each group expressed TGF-β, Fas and C5a at different times.

[0035] 图16为各组小鼠脑内FoxP3、IL-27和B7分子在不同时期的表达。 [0035] FIG. 16 is the brain of mice in each group FoxP3 expression of B7 and IL-27 at different times. 图17为现有技术中融合蛋白作用示意图。 FIG 17 is a schematic diagram of the prior art the role of the fusion protein.

具体实施方式 detailed description

[0036] 下面结合具体实施例,对本发明的融合免疫蛋白在制备治疗多发性硬化症药物中的应用作进一步详细说明。 [0036] In particular embodiments in conjunction with the following embodiments, an immune fusion protein of the invention for use in the manufacture of a medicament for the treatment of multiple sclerosis further detail.

[0037] 一、实验材料: [0037] I. Experimental Materials:

[0038] 1、融合免疫球蛋白(专利号:CN200410006498. 9)和甲泼尼龙琥珀酸钠,甲泼尼龙琥珀酸钠由辉瑞制药有限公司生产。 [0038] 1, the fusion immunoglobulin (Patent No:. CN200410006498 9), and methylprednisolone sodium succinate, methylprednisolone sodium succinate produced by Pfizer.

[0039] 2、168只健康8~10周龄的雌性野生型C57BL/6小鼠,体重18-22g,由武汉大学实验动物中心提供,在室温18-25°C、相对湿度(50-60) %,人工12小时昼/夜循环照明环境中用全价营养饲料分笼饲养,小鼠能自由摄食及饮水。 [0039] 2,168 healthy 8-10 week old female wild type C57BL / 6 mice, weighing 18-22g, provided by Experimental Animal Center of Wuhan University, at room temperature, 18-25 ° C, relative humidity (50-60 )%, with 12 hours artificial day / night cycle of illumination environment nutritionally complete diets were housed, mice are free access to food and water. 适应性饲养1-2周后予以编号分组。 After feeding adaptation packets may be numbered 1-2 weeks.

[0040] 二、动物分组及模型建立: [0040] Second, the Animal grouping and model:

[0041] 本研宄在设计时考虑到EAE为一种免疫系统疾病,所需检测指标大多为免疫细胞自身表达或免疫反应过程中的产物,对于正常机体在没有免疫应答时表达较为恒定,也不存在生理性波动周期,所以在对发病不同时期各组比对时均以对照组作为基准,不再设立相应时间段的空白对照。 [0041] In this study based on the EAE is designed with an immune system disease, mostly required for detection index own immune cells or immune responses in the expression product of normal body for expression in the absence of an immune response is more constant, and physiological fluctuation cycle does not exist, so the onset of the control group are compared to each group at different times as a reference, no blank control corresponding period.

[0042] (A组)对照组:随机分出24只小鼠作为空白对照组,正常饲养,不给予任何干预。 [0042] (A group) Control group: 24 mice were randomly separated as a control group, normal feeding, do not give any interference. 分别再与C组相对应的时间点处死动物进行相关检测。 Then each time point and group C animals were sacrificed corresponding correlation detection.

[0043] (C组)EAE自然病程组:随机分出72只小鼠用M0G35-55肽段及免疫佐剂制作慢性EAE动物模型。 [0043] (Group C) EAE natural course group: 72 mice were randomly separated M0G35-55 immunoadjuvant peptide and produce an animal model of chronic EAE. 每只小鼠分2点腹股沟皮下共注射0. 2ml混合乳剂,其中含人工合成的M0G35-55肽段250 μ g,完全弗氏佐剂2mg,免疫0天和2天时经腹腔各注射百日咳毒素200ng。 2:00 min per mouse mixed emulsion inguinal 0. 2ml subcutaneous co-injection, which contains a synthetic peptide M0G35-55 250 μ g, Freund's complete adjuvant 2mg, immunized intraperitoneally on day 0 and day 2 each injection of pertussis toxin 200ng. 一周后重复免疫注射一次。 Repeat once a week after the immunization. 从免疫当天第0天起,至第42天实验终止前,每天观察动物行为活动,采用盲法,每天2人至少一次在同一时间依据Benson评分标准做EAE临床症状严重程度评估。 From the day of immunization day 0, day 42 to the front end of the experiment, observe animal behavior every day activities, blinded, at least once a day for 2 people at the same time to do the assessment of the severity of clinical symptoms of EAE according to Benson score. 分别在动物固有免疫期(免疫后3天)、发病前期(免疫后10-12天)、 发病初期(免疫后15-20天)、发病高峰期(免疫后24-28天)和慢性维持期(免疫后第42天)以每组12只处死动物,取组织进行相关检测。 Innate immunity of an animal, respectively (3 days after immunization), early onset (10-12 days after immunization), the early stages (15-20 days after immunization), the peak incidence (24-28 days after immunization) and chronic sustain period (day 42 post immunization) with 12 animals in each group were sacrificed, tissues taken for correlation detection. 未发病或死亡标本自动剔除。 No morbidity or mortality specimens automatically removed.

[0044] (D组)糖皮质激素治疗组:随机分出36只C57BL/6小鼠,与EAE组同时制作动物模型,未发病或死亡样本自动剔除,成功者于发病高峰期给予腹腔注射甲泼尼龙5mg/kg,每日1次,连续治疗7天并继续观察1周评价疗效。 [0044] (Group D) glucocorticoid therapy group: randomly separated 36 C57BL / 6 mice with EAE animal model group at the same time, the sample is not automatically removed morbidity or mortality, successful intraperitoneally injected to A peak incidence of prednisolone 5mg / kg, once daily, continuously for 7 days and continue to observe one week evaluated. 于免疫后第42天处死动物进行相关检测。 After immunization animals were sacrificed on day 42 performs correlation detection. 未发病或死亡样本自动剔除。 No morbidity or mortality sample is automatically removed.

[0045] (E组)融合蛋白治疗组:随机分出36只C57BL/6小鼠,与EAE组同时制作动物模型,未发病或死亡样本自动剔除。 [0045] (E group) fusion protein treatment groups: 36 randomly separated C57BL / 6 mice with EAE animal model group at the same time, the sample is not automatically removed morbidity or mortality. 制作动物模型成功小鼠于发病高峰期给予腹腔注mGE2mg/ kg,每日1次,连续治疗7天并继续观察1周评价疗效。 Success in the mouse model produced the peak incidence of intraperitoneal injection mGE2mg / kg, once daily, continuously for 7 days and continue to observe one week evaluated. 于免疫后第42天处死动物进行相关检测。 After immunization animals were sacrificed on day 42 performs correlation detection. 未发病或死亡样本自动剔除。 No morbidity or mortality sample is automatically removed.

[0046] 三、实验结果及分析: [0046] Third, the experimental results and analysis:

[0047] 以下对比采用统计学处理:所有数据均以均数土标准差表示并输入SPSS 14. 0统计软件,对照组和实验组以P < 〇. 05为差异,表示有统计学意义。 [0047] The following comparison using statistical analysis: All data were expressed as mean and standard deviation of soil indicates statistical software SPSS 14. 0 input, the control and experimental groups. P <05 square difference was statistically significant.

[0048] 实施例1 :实验小鼠的发病率及不同阶段平均临床评分 [0048] Example 1: The incidence of mice at different stages of mean clinical score and

[0049] 本实验于第42天终止时,A组所有小鼠始终无一发病;C组C57BL/6J模型小鼠除去发病前处死的24只小鼠,累计有41/48 (85. 4%)小鼠发病,C组于接种后15 - 20天陆续发病,24-28天达高峰,维持5-7天之后,自然病程组部分临床症状可有缓解,临床评分相对降低,但症状持续存在,此后进入慢性维持期,疾病呈典型的慢性非缓解性过程;D组共有29/36(80. 5 % )只小鼠发病,E组有28/36(77. 8 % )只小鼠发病;症状表现同EAE组相同。 [0049] The present experiment was terminated when, A group always all mice on day 42, none of incidence; group C C57BL / 6J mouse model mice was removed 24 prior to the onset of death, a total of 41/48 (85.4% ) mice the incidence of group C after inoculation at 15 - 20 days in succession onset, peak at day 24-28, maintained 5-7 days after clinical symptoms natural course group portion may alleviate, relatively lower clinical scores, but the symptoms persist thereafter maintained into the chronic phase, in a non-disease-modifying typical chronic course; group D total 29/36 (80 5%) the incidence of mice, E group 28/36 (77 8%) the incidence of mice ; EAE group with the same symptoms. E组有1只小鼠在未发病时死亡,可能与注射时操作不当有关;D组无小鼠死亡。 A group E when no incidence of death in mice, may be related to improper operation of the injection; group D No mice died. 激素治疗组在停药后有8/28(28.5 % )只小鼠发生停药后的反弹现象,表现短暂一过性瘫痪症状加重,持续2-3天后又明显好转,这与临床使用甲泼尼龙观察到的现象相一致;融合蛋白治疗组未见此现象。 Hormone therapy group after stopping 8/28 (28.5%) mice only after the withdrawal rebound phenomenon, showing a short transient paralysis symptoms, sustained and significant improvement in 2-3 days, which is in clinical use A splash nylon consistent with the observed phenomena; fusion protein treatment group, no this phenomenon.

[0050] 如表1及图1所示,在经过激素和融合免疫球蛋白治疗后,两组临床功能评分均有明显下降,相对于EAE自然病程组具有统计学差异(P < 0. 05),表明治疗能够缓解神经功能的损伤程度。 [0050] As shown in Table 1 and 2, and after hormone fusion immunoglobulin treatment, both groups were significantly reduced clinical score function 1, with respect to the natural course of EAE group having a significant difference (P <0. 05) indicating that treatment can alleviate the extent of damage to nerve function. 为剔除EAE组症状自然缓解的因素影响,实验结束时的评分对比表明,两治疗组较EAE组慢性缓解期仍有好转,统计学有差异(P < 0. 05);但两治疗组之间没有统计学差异(P > 0· 05)。 Excluding the impact factor for the natural relief of the symptoms of EAE group, compared to scores at the end of the experiment showed that the treatment group than in the two groups of chronic EAE remission still improved statistical difference (P <0. 05); but between the two treatment groups no significant difference (P> 0 · 05).

[0051] 表1实验小鼠的发病率及不同阶段平均临床评分(X土s) [0051] Table 1 the incidence of the different stages of laboratory mice and mean clinical score (X Soil s)

Figure CN104922670AD00091

[0053] 实验结果:(1)与EAE组发病高峰期比较,P < 0. 05 ; (2)与EAE组慢性维持期比较,P < 0. 05。 [0053] Experimental results: (1) Compared with the peak incidence of EAE group, P <0. 05; (2) the group of chronic EAE sustain period comparison, P <0. 05.

[0054] 实施例2 :实验小鼠脑组织病理学及免疫组化改变 [0054] Example 2: mice brain tissue pathological changes and immunohistochemistry

[0055] 经过治疗后,小鼠脑HE和LFB染色在光镜下可见到(见图2和图3)。 [0055] After treatment, the mouse brain and LFB HE staining can be seen in the light microscope (see FIG. 2 and FIG. 3). 炎性细胞浸润较EAE组发病高峰期和慢性维持期有所好转,髓鞘脱失也有缓解。 Inflammatory cell infiltration, and the peak incidence of chronic EAE group compared with the maintenance phase has improved, demyelination have eased. 未见到由于轴索损伤而引起的APP在局部聚集形成的异常染色。 APP not seen since axonal injury caused by an abnormal local accumulation of dye formed. 脑内小胶质细胞活化程度两治疗组均明显减轻,甲泼尼龙治疗后几乎已恢复正常;表明甲泼尼龙和融合蛋白具有抗炎和减轻髓鞘损伤的作用,尤以甲泼尼龙的作用更明显。 Brain microglia activation level of the two treatment groups were significantly reduced after methylprednisolone therapy has almost returned to normal; and methylprednisolone showed that the fusion protein has anti-inflammatory and reduce the role of myelin damage, especially in the role of methylprednisolone more obvious.

[0056] 图2中的A为对照组小鼠脑HE染色;B为EAE组小鼠固有免疫期脑HE染色;C为EAE组小鼠发病前期脑HE染色;D为EAE组小鼠发病初期脑HE染色;E为EAE组小鼠高峰期脑HE染色;F为EAE组小鼠慢性维持期脑HE染色。 In [0056] FIG 2 A is a control group of mice brain HE staining; B is the innate immune mice EAE HE staining of the brain; C mice early onset of EAE brain HE staining; D is the early onset of EAE mice HE staining brain; E is a peak of HE staining in EAE mouse brain; F. HE staining of brain maintained chronic EAE mice.

[0057] 图3中的A为对照组脑LFB染色;B为EAE组小鼠固有免疫期脑组织LFB染色;C 为EAE组发病前期脑LFB染色;D为EAE组发病初期脑LFB染色;E为EAE组发病高峰期脑LFB染色;F为EAE组慢性维持期脑LFB染色。 In [0057] FIG 3 A is a group of brain LFB staining; B is the innate immune LFB staining of brain tissue EAE mice; C to early onset in EAE brain LFB staining; D is the early onset of EAE LFB staining of brain; E peak incidence of EAE brain LFB staining groups; F. sustain LFB staining of cerebral group of chronic EAE.

[0058] 如图4-8所示,免疫荧光组化显示,经过甲泼尼龙治疗后,小鼠脾脏内IFN-γ和脑Fas的荧光显色较EAE组发病高峰期和慢性维持期有显著减少;经mGE治疗后显色差异不及甲泼尼龙治疗组显著。 [0058] shown in Figure 4-8, immunofluorescence staining revealed methylprednisolone after treatment, the IFN-γ mouse spleen and brain Fas fluorescent color than the peak onset in EAE and chronic sustain period significantly reduction; color difference after mGE methylprednisolone therapy treatment was significantly less. mGE和甲泼尼龙治疗后IL-12的荧光显色均有明显减少。 Fluorescent color of IL-12 were significantly reduced after treatment mGE and methylprednisolone. 甲泼尼龙和mGE治疗后均能抑制炎性细胞增殖和MBP破坏、吞·,但甲泼尼龙作用表现得更明显。 After treatment, methylprednisolone and mGE can inhibit proliferation of inflammatory cells and destruction of MBP, swallow-but methylprednisolone effect is even more marked. 其中: among them:

[0059] 图4中的A为EAE组发病高峰期脾脏IFN- γ免疫荧光组化;B为EAE组慢性维持期脾脏IFN-γ免疫荧光染色;C为甲泼尼龙治疗组脾脏IFN-γ免疫荧光染色;D为融合蛋白治疗组脾脏IFN-γ免疫荧光染色。 In [0059] FIG 4 A peak incidence of EAE group of IFN- γ in spleen immunofluorescence staining; B sustain period spleen IFN-γ immunofluorescence staining chronic EAE group; C is treated methylprednisolone immunized spleen IFN-γ staining; D is a fusion protein treatment group, IFN-γ in spleen immunofluorescence staining.

[0060] 图5中的A为EAE组发病高峰期脑内Fas免疫荧光染色;B为EAE组慢性维持期脑内Fas免疫荧光染色;C为甲泼尼龙治疗组脑组织Fas免疫荧光染色;D为融合蛋白治疗组脑组织Fas免疫荧光染色。 [0060] FIG. 5 A peak incidence of immunofluorescence staining of EAE Brain Fas group; the group B is maintained as chronic EAE Brain Fas immunofluorescence staining; C for the treatment of brain tissue Fas methylprednisolone immunofluorescence staining; D for the treatment of brain tissue fusion protein Fas immunofluorescence staining.

[0061] 图6中的A为EAE组发病高峰期脑IL-12免疫荧光染色;B为EAE组慢性维持期脑IL-12免疫荧光染色;C为甲泼尼龙治疗组脑IL-12免疫荧光染色;D为融合蛋白治疗组脑内IL-12免疫荧光染色。 In [0061] FIG. 6 A set of EAE onset of cerebral IL 12-immunofluorescence staining peak; B sustain period brain IL 12-immunofluorescent staining of chronic EAE group; C is treated methylprednisolone brain IL-12 immunofluorescence staining; D is a fusion protein treatment group intracerebral IL-12 immunofluorescence staining.

[0062] 图7中的A为EAE组发病高峰期脑Ki-67、MBP双标免疫荧光组化;B为EAE组慢性维持期脑Ki-67、MBP双标免疫荧光组化;C为甲泼尼龙治疗组脑组织Ki-67、MBP双标免疫荧光组化;D为融合蛋白治疗组脑Ki-67、MBP双标免疫荧光组化。 In [0062] FIG 7 A is in EAE incidence peak Brain Ki-67, MBP double-labeling immunofluorescence staining; B maintain cerebral Ki-67 as EAE group of chronic, MBP double-labeling immunofluorescence staining; C to A prednisolone treatment of brain tissue Ki-67, MBP double-labeling immunofluorescence staining; D is a fusion protein treatment of brain Ki-67, MBP double-labeling immunofluorescence staining.

[0063] 图8中的A为EAE组发病高峰期脑Ki-67、Ibal双标免疫荧光组化;B为EAE组慢性维持期脑Ki-67、Ibal双标免疫荧光组化;C为甲泼尼龙治疗组脑组织Ki-67、Ibal双标免疫荧光组化;D为融合蛋白治疗组脑Ki-67、Ibal双标免疫荧光组化。 In [0063] FIG 8 A is in EAE incidence peak Brain Ki-67, Ibal double-labeling immunofluorescence staining; B maintain cerebral Ki-67 as EAE group of chronic, Ibal double-labeling immunofluorescence staining; C to A prednisolone treatment of brain tissue Ki-67, Ibal double-labeling immunofluorescence staining; D is a fusion protein treatment of brain Ki-67, Ibal double-labeling immunofluorescence staining.

[0064] 实施例3 :血-脑屏障通透性变化 [0064] Example 3: blood - brain barrier permeability changes

[0065] 如图9和表2所示,自EAE组发病高峰期和慢性维持期始终保持高水平,经融合免疫球蛋白治疗后未观察到明显变化,较发病高峰期和慢性维持期无统计学差异(P > 0. 05),甲泼尼龙组血-脑屏障通透性有所降低,较EAE组有统计学差异(P < 0. 05 ;表明甲泼尼龙对血-脑屏障通透性升高有抑制作用,而融合免疫球蛋白未显示出此作用。 [0065] Table 2 and FIG. 9, since the peak incidence of EAE group and chronic sustain period always maintain a high level, significant change is not observed after treatment by an immunoglobulin fusion, compared with the peak incidence of chronic sustain period and no statistical Science difference (P> 0. 05), methylprednisolone group blood - brain barrier permeability decreased, there is significant difference (P <0. 05 compared with EAE group; methylprednisolone showed blood - brain barrier permeability inhibition of the increase, and showed no fusion immunoglobulin this effect.

[0066] 表2实验小鼠海马组织内EB含量(μ g/g脑组织)的变化 [0066] Table 2 Experimental mouse hippocampus EB content (μ g / g brain tissue) changes

Figure CN104922670AD00101

[0068] 实施例4 :Werstern_Blot 结果 [0068] Example 4: Werstern_Blot Results

[0069] (I)Fcy RIIb (CD-32b)在脑内的表达 Expression in the brain of the [0069] (I) Fcy RIIb (CD-32b)

[0070] 如图10⑴和图10⑵所示,图10⑵中的*EAE组不同时期与对照组比较,P < 0. 05 ;#两治疗组与EAE组发病高峰期比较,P < 0. 05 ; Y两治疗组与EAE组慢性维持期比较P < 0. 05。 [0070] As shown in FIG 10⑴ and 10⑵, * compare different periods EAE group and control group in FIG 10⑵, P <0. 05; # peak incidence between the two treatment groups with EAE group, P <0. 05; treatment group and two Y group of chronic EAE Comparative sustain period P <0. 05. 可见⑶32b在脑内的表达在发病高峰期和慢性维持期明显增加,较对照组均有统计学差异(P< 0.05),提示随着病情进展,体内免疫抑制效应可以自发性调节免疫应答的强度。 ⑶32b visible expression of brain maintained at the peak incidence of chronic and significantly increased compared with the control group were statistically significant (P <0.05), suggesting as the disease progresses, immunosuppressive effects in vivo can spontaneously adjust the intensity of the immune response . 经治疗后,甲泼尼龙组表达较发病高峰期无明显变化,但较慢性维持期降低且存在统计学差异(P< 0.05);而融合蛋白治疗组变化明显,较发病高峰期和慢性维持期均增加,统计学差异具有显著性(P < 〇. 05)。 After treatment, methylprednisolone set of expression than the peak incidence of no significant change, but the sustain period of slow decrease and there was significant difference (P <0.05); and significantly change the fusion protein treatment group, compared with the peak incidence of chronic and sustain period were increased, the difference was statistically significant (P <square. 05). 提示Fc γ RIIb在发病高峰期和慢性维持期已经存在自发性表达增加,是机体对免疫反应调节的表现。 Tip Fc γ RIIb already increased expression in spontaneously and the peak incidence of chronic sustain period, is the body's immune response regulation performance. 融合蛋白可以通过增加Fc γ RIIb 的表达发挥免疫抑制作用,甲泼尼龙的治疗作用则可能通过其他机制。 The fusion protein may exert immunosuppressive effects by increasing the expression of Fc γ RIIb, methylprednisolone therapeutic effect could be obtained by other mechanisms.

[0071] ⑵补体C3在脑内表达 [0071] ⑵ complement C3 expression in the brain

[0072] 如图11 (1)和图11 (2),图11 (2)中的*ΕΑΕ组不同时期与对照组比较,P < 0. 05 ; #两治疗组与EAE组发病高峰期比较,P < 0. 05 ; Y两治疗组与EAE组慢性维持期比较P < 0. 05。 [0072] As shown in FIG. 11 (a) and 11 (2), 11 (2) * ΕΑΕ during Different control group, P <0. 05; compare two treatment groups # EAE onset and peak group , P <0. 05; Y treatment group and two group of chronic EAE Comparative sustain period P <0. 05. 可见补体C3在脑组织中的的含量在固有免疫期有所升高,之后呈逐渐下降趋势,至发病高峰期维持较低水平,激素治疗组补体C3水平较发病高峰期无明显变化(Ρ > 0. 05),较慢性维持期有所升高。 Visible complement C3 content in brain tissue of some of the innate immune increased after decreased gradually to the peak incidence remain low, hormone therapy group Complement C3 levels than the peak incidence of no significant difference (Ρ> 0.05), the slower of the maintenance phase was increased. 融合蛋白治疗组则较发病高峰期和慢性维持期补体C3含量降低,有统计学差异(P < 〇. 05),表明融合蛋白能够减少补体C3的生成。 Fusion protein treatment group compared with the peak incidence of chronic and sustain reduction of complement C3, statistically significant (P <square. 05) indicates that the fusion protein can be reduced to generate complement C3.

[0073] (3) MHCI分子在脑内表达变化 [0073] (3) MHCI molecule expression in the brain

[0074] 如图12⑴和12⑵所示,图12⑵中的*EAE组不同时期与对照组比较,P < 0. 05 ;#两治疗组与EAE组发病高峰期比较,P < 0. 05 ; Y两治疗组与EAE组慢性维持期比较P < 0. 05。 [0074] FIG 12⑴ and shown in FIG 12⑵ * Comparative 12⑵ different periods of EAE group and the control group, P <0. 05; # two treatment groups compared with the peak incidence of EAE group, P <0. 05; Y treatment group and two group of chronic EAE Comparative sustain period P <0. 05. 脑内MHCI类分子在慢性维持期仍处于高水平表达,经过甲泼尼龙和融合蛋白治疗后表达下降,与慢性缓解期有统计学差异(P< 0.05);说明甲泼尼龙和融合蛋白均可以降低脑内MHCI类分子的表达。 Brain MHCI molecules chronic sustain period is still in high level expression of fusion proteins and after methylprednisolone treatment decreased expression, and chronic remission were significantly different (P <0.05); methylprednisolone and fusion proteins described can be brain reduced expression of MHCI molecules.

[0075] (4)MHCII分子在脑内表达变化 [0075] (4) MHCII molecule expression in the brain

[0076] 如图13⑴和13⑵所示,图13⑵中的*EAE组不同时期与对照组比较,P < 0. 05 ;#两治疗组与EAE组发病高峰期比较,P < 0. 05 ; Y两治疗组与EAE组慢性维持期比较P < 0. 05。 [0076] FIG 13⑴ and shown in FIG 13⑵ * Comparative 13⑵ different periods of EAE group and the control group, P <0. 05; # two treatment groups compared with the peak incidence of EAE group, P <0. 05; Y treatment group and two group of chronic EAE Comparative sustain period P <0. 05. 脑内MHCII类分子同样在慢性维持期仍处于高位,表明MHCII类分子在EAE中存在持续高表达,这与病程迀延不愈相一致。 MHCII molecules in the brain the same chronic maintenance phase remains high, indicating the presence of sustained high MHCII molecules expression in EAE, which is consistent with the course Gan delay healing. 经过干预治疗后,甲泼尼龙组和融合蛋白组均较慢性维持期含量下降,有统计学意义(P< 0.05);提示甲泼尼龙和融合蛋白都有抑制MHCII类分子表达的作用。 After intervention, and fusion proteins methylprednisolone group compared with the group of chronic sustain period was decreased, there was significant (P <0.05); methylprednisolone tips and fusion proteins both inhibit the expression of MHCII molecules.

[0077] (5)IL_12在脑内表达的变化 [0077] The expression (5) IL_12 the brain

[0078] 如图14 (1)和图14⑵所示,图14⑵中的*EAE组不同时期与对照组比较,P < 0. 05 ;#两治疗组与EAE组发病高峰期比较,P < 0. 05 ; Y两治疗组与EAE组慢性维持期比较P < 0. 05。 [0078] FIG. 14 (1) and shown in FIG. 14⑵, * compare different periods EAE group and control group in FIG 14⑵, P <0. 05; # two treatment groups compared with the peak incidence of EAE group, P <0 . 05; Y treatment group and two group of chronic EAE Comparative sustain period P <0. 05. IL-12在EAE组发病高峰期时处于最高值,而后变化不明显,经甲泼尼龙和融合蛋白治疗后下降,与EAE组高峰期和慢性维持期比较IL-12表达降低均有统计学意义(P < 0. 05),提示甲泼尼龙和融合蛋白都可以抑制IL-12的生成。 IL-12 is at the highest peak in EAE incidence values, and then did not change significantly decreased after treatment with methylprednisolone and fusion proteins, the sustain period and the peak and chronic EAE group comparison reduced IL-12 expression was statistically significant (P <0. 05), suggesting methylprednisolone and fusion proteins can inhibit the production of IL-12.

[0079] 实施例5 :RT-PCR结果 RT-PCR results: Example 5 [0079] Embodiment

[0080] (1)各组小鼠不同时期TGF-β、Fas和C5a在脑内的表达变化 [0080] (1) changes in TGF-β in each group of mice at different times, Fas expression in the brain and C5a

[0081] 如图15和表3所示,激素治疗组TGF-β脑内的表达较发病高峰期和慢性维持期均升高,有统计学意义(P< 0.05);融合蛋白治疗组TGF-β脑内的表达与发病高峰期比较降低,有统计学意义(P < 0.05),与慢性维持期比较无统计学意义(P > 0.05)。 [0081] FIG. 15 and Table 3, expression of TGF-β treated groups compared with the peak incidence of brain and chronic sustain period are increased, there is statistically significant (P <0.05); TGF- fusion protein treatment group β expression in the brain of the peak incidence of Comparative reduced statistically significant (P <0.05), and chronic sustain period no significant difference (P> 0.05). 激素治疗组脑内Fas的表达降低,较发病高峰期和和慢性维持期均有统计学意义(P < 0. 05); 融合蛋白治疗组Fas的表达也有一定降低,与发病高峰期和慢性维持期有统计学差异(P < 0. 05)。 Fas expression in the brain hormone therapy group is reduced, compared with the peak incidence of chronic and sustain period and were statistically significant (P <0. 05); Fas fusion protein treatment group there is a certain reduction, and maintain peak incidence of chronic there were significant differences of (P <0. 05). C5a的表达在激素治疗组无明显降低,与发病高峰期和慢性维持期比较无统计学差异(P> 0.05);在融合蛋白组的表达下降,与发病高峰期和慢性维持期比较有统计学意义(P < 0· 05)。 C5a expression did not decrease significantly in the treated groups, with the peak incidence of chronic sustain period and no statistical difference (P> 0.05); decreased expression of the fusion protein group, the sustain period and the peak incidence of chronic and were statistically significance (P <0 · 05).

[0082] 其中,图15中*两治疗组与EAE组发病高峰期比较P < 0. 05 ;#两治疗组与EAE 组慢性维持期比较P < 0. 05。 [0082] wherein, in the two treatment groups * in FIG. 15 and comparing the peak incidence of EAE group P <0. 05; # two treatment groups with the group of chronic EAE Comparative sustain period P <0. 05.

[0083] 表3EAE组不同时期与对照组脑内TGF- β、Fas和C5a mRNA表达水平变化 [0083] Table 3EAE group and the control group in different periods TGF- β mRNA expression levels of the brain changes, Fas and C5a

[0084] [0084]

Figure CN104922670AD00121

[0086] (2)各组小鼠不同时期FoxP3、IL-27和B7-1在脑内的表达变化 [0086] (2) at different times the mice FoxP3, IL-27 expression in the brain and changes in B7-1

[0087] 如图16所示,*两治疗组与EAE组发病高峰期比较P < 0. 05 ;#两治疗组与EAE组慢性维持期比较P < 0. 05。 As shown in [0087] 16, two treatment groups * group and the peak incidence of EAE Comparative P <0. 05; # two treatment groups with the group of chronic EAE Comparative sustain period P <0. 05. 两治疗组都可以明显下调脑内B7-1的表达,与EAE组发病高峰期和慢性维持期比较有统计学差异(P < 〇. 05);甲泼尼龙治疗组还可明显降低脑内IL-27 的表达,与EAE组发病高峰期和慢性维持期比较有统计学差异(P < 0. 05),融合蛋白治疗组的表达较发病高峰期下降,有统计学差异(P < 0. 05),但较慢性维持期无显著差异(P > 〇. 05);两治疗组对FoxP3表达未见明显影响。 Two treatment groups are significantly down-regulated the expression of B7-1 in the brain, with the peak incidence of EAE group and chronic sustain period were significantly different (P <square 05); methylprednisolone treatment group also significantly decreased Intracerebral IL -27 expression, with the peak incidence of EAE group and chronic sustain period were significantly different (P <0. 05), expression of fusion protein in the treatment group compared with the peak incidence decreased statistically significant (P <0. 05 ), but no significant difference (P> 05 billion) of the sustain period is slow;. expression of FoxP3 two treatment groups were not significantly affected.

[0088] 表4 FoxP3、IL-27和B7-lmRNA在EAE组小鼠脑内的表达 [0088] Table 4 FoxP3 expression, IL-27, and B7-lmRNA group EAE in mouse brain

Figure CN104922670AD00122

[0090] 从以上5个实施例可知:融合蛋白治疗组和甲泼尼龙治疗组都具有下调脑内APC 提呈相关主要因子MHCI、MHCII类分子、B7-1的表达,降低APC分泌的活性因子IL-12、 IL-27水平,抑制单核巨噬细胞活化、增殖;减轻MBP的破坏和吞噬。 [0090] Example 5 From the above found: Fusion protein treatment group and the treatment group methylprednisolone have reduced brain APC presenting the main factors associated MHCI, MHCII molecules, the expression of B7-1, reducing the activity of APC factor secretion IL-12, IL-27 levels, inhibition of monocyte macrophage cell activation, proliferation; MBP reduce destruction and phagocytosis. 融合蛋白对能够促进APC提呈功能的补体C3和C5a的生成具有抑制作用,上调抑制性受体Fc γ RIIb的表达;表现出对APC活化的多重阻遏作用,但未见对T淋巴细胞相关免疫应答有显著作用。 Fusion proteins inhibit the generation of complement can promote APC presenting function of C5a and C3, inhibitory receptor upregulation of Fc γ RIIb; exhibit multiple repression of APC activation, but no associated immune T lymphocytes answer has a distinct role. 即融合免疫球蛋白可以通过抑制APC的MHC分子表达,抑制其致炎细胞因子释放而缓解慢性EAE的神经功能损伤症状;融合免疫球蛋白具有明确的缓解慢性EAE小鼠临床功能损害的作用, 治疗过程中没有出现激素样停药反弹现象,可作为单独或联合其他药物制备治疗多发性硬化症的药物。 That fusion immunoglobulin can inhibit APC expression of MHC molecules and inhibit proinflammatory cytokine release and cell relieve symptoms of chronic EAE neurological damage; the role of fusion immunoglobulin has a clear clinical remission of chronic EAE in mice dysfunction, treatment hormone-like withdrawal rebound phenomenon, may be used as alone or in combination with other drugs to treat multiple sclerosis preparation of pharmaceutical process does not appear.

Claims (5)

  1. 1. 融合免疫球蛋白在制备治疗多发性硬化症药物中的应用。 1. Application of the fusion immunoglobulin in the manufacture of a medicament for the treatment of multiple sclerosis.
  2. 2. 如权利要求1所述的应用,其特征在于,所述融合免疫球蛋白是具有序列表中SEQ IDN2 :2氨基酸残基序列的蛋白质,或者是将SEQIDN2 :2氨基酸残基序列经过一个或几个氨基酸残基的取代、缺失或添加具有与SEQIDN2 :2氨基酸残基序列相同活性的由SEQ IDN2 :2衍生的蛋白质。 2. The use according to claim 1, wherein the fusion immunoglobulin is having the sequence of SEQ IDN2: 2 amino acid residue sequence of the protein, or the SEQIDN2: 2 amino acid residue sequence through one or substitution of several amino acid residues, deletion or addition has SEQIDN2: identical activity by a 2 amino acid residue sequence of SEQ IDN2: 2 derived proteins.
  3. 3. 如权利要求2所述的应用,其特征在于,所述融合免疫球蛋白是序列表中的SEQID Ns :2〇 3. The use according to claim 2, wherein the fusion immunoglobulin is SEQID Ns Sequence Listing: 2〇
  4. 4. 如权利要求1所述的应用,其特征在于,所述融合免疫球蛋白编码基因,是下列核苷酸序列之一: 1) 序列表中SEQIDNs: 1的DNA序列; 2) 编码序列表中SEQIDN2 :2蛋白质序列的多核苷酸; 3) 与序列表中SEQIDN2 :1限定的DNA序列具有95%以上同源性,且编码相同功能蛋白质的DNA序列。 1) the sequence table SEQIDNs::; 2) the coding sequence of the DNA sequence in Table 1 4. The use according to claim 1, wherein said immunoglobulin fusion encoding gene, is one of the following nucleotide sequence in SEQIDN2: 2 protein sequence of the polynucleotide; 3) the sequence table SEQIDN2: 1 DNA sequence having a defined homology of 95% or more, and the DNA sequence encoding the same functional protein.
  5. 5. 如权利要求4所述的应用,其特征在于,所述融合免疫球蛋白编码基因是序列表中的SEQIDNs: 1。 5. The use according to claim 4, wherein the fusion immunoglobulin coding gene sequence listing SEQIDNs: 1.
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