An Investigation On The Pathogenic Mechanisms Of 3 ScFv Monoclone Antibodies Against The Different Epitopes On 60kDSSA/Ro Antigen In Related Autoimmune Disease

【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.