【作者】 帅宗文；

【导师】 张奉春； 唐福林； 张煊；

【作者基本信息】 中国协和医科大学 ， 内科学风湿病， 2007， 博士

【摘要】 【研究背景】60kD SSA/Ro(Ro60)抗原广泛分布于正常人体的肝、肾、淋巴细胞、成纤维细胞、上皮细胞等组织细胞的胞浆和胞核中,抗Ro60抗体存在于多种自身免疫性疾病如:系统性红斑狼疮(SLE)、干燥综合征(SS)、亚急性皮肤型红斑狼疮(SCLE)、新生儿狼疮(NLE)和系统性硬化症(SSc)等,并与这些疾病中的多种临床损害如皮疹、反复腮腺肿大、肺损害、白细胞减少、心脏传导阻滞等相关。研究表明,Ro60抗原拥有20个独立的抗原表位,不同表位的自身抗体可能会导致不同的临床损害,表现为不同的自身免疫病出现不同表位的自身抗体。本课题组的初步临床研究发现,不同的自身免疫病患者,抗Ro60抗体所识别的表位组成不同,且不同表位自身抗体与不同的临床损伤相关。有研究证实,根据Ro60蛋白20个抗原表位氨基酸序列,人工合成的多肽仍保留其相应表位的抗原活性。为进一步探讨Ro60不同表位自身抗体与临床损害间的关系,本课题组首先成功构建了抗Ro抗体单链可变区(Single-chain Fv,ScFv)噬菌体抗体库,根据课题组初步临床研究的结果,人工合成Ro60抗原3个表位多肽(P1表位:482～493,P2表位:310～323,P3表位:230～241),用3个多肽从ScFv噬菌体抗体库中筛选获得3个ScFv单抗。为了能直接观察这3个单抗可能导致的不同组织器官损害,了解它们在不同自身免疫病发生及发展中的作用,需要通过动物实验,分别用它们作用正常和各种自身免疫病的模型动物。缺少正常抗体Fc片断的ScFv单抗,只具有特异性抗原结合能力,没有免疫损害效能。将ScFv单抗与铜绿假单胞菌外毒素A(Pseudomonasaeruginosa exotoxin A,PE)中的PE40片断重组融合为免疫毒素ScFv-PE40,该免疫毒素兼有类似天然抗体的特异性抗原结合能力和对组织细胞定点损害功能。以此作用于实验动物,探讨Ro60抗原不同表位的自身抗体是否具有导致不同损害的潜能。【研究目的】1.获取Ro60抗原3个不同表位的ScFv单抗与毒素PE40的重组融合蛋白(免疫毒素EP1P、EP2P和EP3P)、实验对照用重组蛋白(单抗P1、P2、P3及毒素蛋白PE40),并鉴定它们应有的生物活性。2.Ro60抗原3个不同表位的自身抗体,能否导致正常BABL/c鼠的重要脏器损害。3.正常BABL/c鼠体内,抗Ro60抗原3个不同表位的自身抗体,能否导致各自特异的组织细胞损害。【方法】1.用PCR方法扩增Ro60抗原3个表位的ScFv单抗序列、PE40序列,并将扩增的3个ScFv分别与PE40连接(ScFv-PE40),将3个ScFv-PE40、3个ScFv、1个PE40片断分别插入载体pET32a(+),获得7个表达克隆,DNA测序确定插入序列及插入载体的位点是否正确。2.IPTG诱导7个克隆分别在大肠杆菌内表达,通过改变诱导剂IPTG的浓度、表达温度和表达时间,探索每种重组蛋白可溶性表达的最佳条件,并在此条件下诱导其大量表达。3.用NTA-Ni亲和层析柱纯化7种可溶性表达的载体融合蛋白,并用肠激酶切除其中4种蛋白(3个ScFv-PE40、PE40)中的载体标签蛋白,从酶切体系中纯化不含标签蛋白的相应重组蛋白:EP1P、EP2P、EP3P、EPE40。4.使用ELISA方法鉴定6个重组蛋白(P1、P2、P3、EP1P、EP2P和EP3P)的特异性抗原结合功能,采用蛋白转染试剂将4个重组蛋白(EP1P、EP2P、EP3P、PE40)转入体外培养的血管内皮细胞(EA.hy 926),MTT法测定4种蛋白的细胞毒活性。5.48只正常BALB/c鼠随机平均分为8组,分别间隙尾静脉注射7种经鉴定的重组蛋白和PBS共2周,眼静脉放血处死小鼠,取其脑、腮腺、肺脏、心脏、肝脏、肾脏制作组织切片,常规HE染色后病理检查,并分析检查结果。【结果】1.获得7种重组蛋白(EP1P、EP2P、EP3P、EPE40、P1、P2、P3),经鉴定,EP1P/P1、EP2P/P2、EP3P/P3分别特异性识别Ro60抗原表位482～493、310～323、230～241,重组蛋白EP1P、EP2P、EP3P和EPE40均具有明显的细胞毒活性。2.动物实验的病理检查显示:全部实验小鼠的脑、腮腺、心脏、肝脏、肾脏组织切片HE染色检查结果正常,但EP1P处理组小鼠较其他实验组出现明显的肺脏出血。【结论】1.成功获得Ro60抗原3个不同表位的免疫毒素(EP1P、EP2P和EP3P)、实验对照用重组蛋白(P1、P2、P3单抗及PE40毒素蛋白),并证实他们具备应有的特异性抗原结合能力和/或细胞毒活性。2.初步动物实验的结果显示:所研究的3个Ro60抗原表位自身抗体可能不能独立导致正常个体始发自身免疫病,但部分Ro60表位自身抗体在慢性炎症的背景下,可以成为损害组织器官的致病抗体,在这种背景下,不同Ro60表位的自身抗体,可能导致不同的组织器官损害。3.实验获得的3个Ro60表位免疫毒素及其实验对照用重组蛋白克隆、重组它们的方法、条件以及初步动物实验结果,为本课题进一步深入研究提供了工具和参考手段。更多还原

【Abstract】 【Background】The 60kD SSA/Ro(Ro60) antigen is present in a wide variety of human tissues, including kidney and liver parenchymal cells,lymphocytes,fibroblasts,epithelioid cells and etc.In various autoimmune diseases(AIDs),such as systemic lupus erythematosus(SLE),Sjogren’s syndrome(SS),subacute cutaneous lupus erythematosus(SCLE),neonatal lupus erythematosus(NLE),systemic sclerosis and so on,antoantibodies against Ro60 were found to be associated with different clinic lesions in these diseases.The clinic lesions involved skin rash,recurrent parotid gland enlargement,pulmonary damage,hypoleukia,heart block and etc.It was proved that the antigenicity of Ro60 was comprised of 20 epitopes,which might be responsible for the associations between various clinic pictures and distinctive profiles of antoantibodies against different Ro60 epitopes in AIDs.Our prelimillary clinic investigaton showed patients with different antoimmune diseases had their distinctive profile of antoantibodies against different Ro60 epitops,and antibodies to different Ro60 epitopes had relationships with different clinic manifestations.Based on our prelimillary clinical findings above,and the discovery that every monoantigen peptide(MAP),synthetized according to amino-acid residue sequences of the 20 epitopes on Ro60 antigen,remained its antigenicity,we synthetized 3 MAPs which were comprised of Ro60 amino-acid residue 482～493,310～323,230～241 named as epitope P1,epitope P2 and epitope P3,respectively.By panning with the 3 MAPs from Ro60 phagmid ScFv antibodies library which we had successfully constructed before,three ScFv monoclone antibodies(McAb) called P1,P2 and P3 against epitope P1,epitope P2 and epitope P3,respectively,were obtained.In order to demonstrate if the 3 ScFv McAb could cause their distinctive damages and to understand their exact roles in the onset and development of AIDs,it is necessary to get direct evidences whether the 3 different ScFv McAbs could result in different damages in the health or AIDs model animals.However,whithout Fc fragment,any ScFv McAbs only remained the function to combind with their specific antigen and losted their damage-causing functions caused by Fc fragment.Recombinant immunotoxin in which the cell-binding domain of pseudomonas exotoxin was replaced with ScFv McAb,possesed both the antigen-binding and local damage abilities,which was similar to and might represent of intact antibodies. Recombinating 3 immunotoxins by every one of the 3 ScFv McAb with PE40,and using them as 3 native autoantibodies targeting the 3 epitopes on Ro60 in animal experiments,we could detect if each of the 3 autoantibodies had its potency to induce its characteristic lesions.【Objectives】1.To obtain and identify 3 recombinant immunotoxins(EP1P,EP2P and EP3P) which are made from each of the 3 ScFv McAbs against different epitopes of Ro60 antigen and PE40,and 4 other recombinant proteins including 3 McAbs(P1,P2 and P3) and PE40 which served as controls.2.To detect if each of the 3 immunotoxins(represent of related native antoantibodies) can independently make damages on the main organs from health BALB/c mice.3.To explore if each of the 3 autoantibodies targeting the different epitopes of Ro60 can result in its specific lesions profiles in normal BALB/c mice.【Methods】1.After the DNA sequences of PE40 and the 3 ScFv McAbs were amplificated by polymerase chain reactions(PCR),Three immunotoxins(ScFv-PE40) DNA sequences were obtained by ligating each of the DNA sequences of the 3 ScFv McAbs with the DNA segment of PE40.All the 7 amplificated DNA sequences for the 3 ScFv,the 3 ScFv-PE40 and PE40 were put into vector pET32a(+) to construct 7 clones for the 7 RP expressions.Sequence analyses to all the 7 clones constructed were employed to find whether each of the 7 inserted sequences and their inserted sites were correct.2.To detect the optimum conditions of soluble expression for each of the 7 RPs encoded by the 7 constructed clones in Escherichia toli,various concentrations of IPTG,several protein-expressive temperatures and different time-span for protein expression were tried.Then,each of the 7 RPs was induced to abundantly express at its optimum conditions.3.When the 7 soluble expressive RPs were purified by NTA-Ni affinity column,all of them actually consisted of the recombinant interest protein and the tag protein from the expressive vectoer pET32a(+).For further experiments,the tag proteins were cut off from the 3 purified recombinant immunotoxins and PE40, which(EP1P,EP2P,EP3P and EPE40) then were purified again from their enterokinase(EK) cleavage reaction systems.4.For identification of the 7 recombinant protiens,ELISA process was employed to detect whether the 6 RPs(P1,P2,P3,EP1P,EP2P and EP3P),each of which contained one of the 3 ScFv McAbs constituents,had the abilities to combine with their specific epitopes from Ro60 antigen.After all the 4 RPs(EP1P,EP2P,EP3P and EPE40),each of which contained PE40 constituents,were separately transfected into one strain of vascular endothelial cell(EA,hy 926) cultured in vitro,MTT assay were taken to evaluate the cytotoxicity of the 4 proteins to EA. hy 926.5.48 health BALB/c mice were randomly divided into 8 goups,and each of groups had 6 mice.Each groups interstitially received one of the identified RPs or phosphate buffered solution(PBS) intravenously for 2 weeks.After all the mice were killed by exanguinating from ophthalmic vein,tissue slices from brain, parotid,lung,heart,liver,kidney of every mice were made and stained routinely by hematoxylin and eosin(HE).Pathologic examinations for all slices were performed and the datum obtained were analysized statistically.【Results】1.7 RPs including EP1P,EP2P,EP3P,EPE40,P1,P2 and P3 were obtained.It was identified that EP1P and P1,EP2P and P2,EP3P and P3 could selectively combine with the Ro60 epitopes 482～493,310～323,230～241,respectively,and that EP1P、EP2P、EP3P and EPE40 owned cytotoxicities to eukaryocytes.2.Pathological inspections on the histological sections from the mice showed none of the slices from brain,parotid,heart,liver and kidney were suspected to be abnomal.But only the pulmonary slices from the group treated by EP1P displaied severer pneumorrhagia than that from any other groups.【Conclusions】1.Three recombinant immunotoxins(EP1P,EP2P,EP3P) against 3 different epitopes of Ro60 antigen,3 recombinant ScFv McAbs(P1,P2,P3) targeting the same epitops as the 3 immunotoxins,and recombinant EPE40 were successfully obtained.It was proved that all of them had their proper physiologic functions.2.The preliminary results from the experiments with health BALB/c mice showed that it is very likely that none of the 3 autoantibodies against different epitopes of Ro60 antigen could independently induce health individuals to have AIDs as an initial factor,but in certain circumstances,for instance,in an occurrence of chronic imflammations,autoantibodies targeting some Ro60 epitopes might act as harmful factors to damage individuals.At these circumstances,autoantibodies against different epitopes on Ro60 antigen might result in different lesions in different situs in AIDs,which might be responsible for different clinic manifestations.3.The clones obtained in this experiment for expression of the 3 recombinant immunotoxins and the other RPs serving as controls,and the processes and the conditions to recombine them,as well as the preliminary results from the health mouse experiment would be valuable for further studies in this field.更多还原