Expression And Pathogenesis Of The Monoclonal Antibodies Against SSA/Ro60kDa Antigen Epitopes

BackgroundAnti-SSA/Ro 60kDa antibody is found in the sera of up to 50% of systemic lupus erythematosus (SLE) patients and a higher percentage of patients with Sjo"gren's syndrome(SS).Ro ribonucleoprotein is composed with SSA/Ro 60kDa,hYRNA,andSSA/Ro 52kDa.Originally identified by double immunodiffusion in the sera of SLE patients with no ANA , and later identified as SS-A in the sera of SS patients, anti-Ro is associated with several clinical aspects of the disease as well as with alleles of HLA and T cell receptor genes. All sera with a Ro precipitin have antibodies against 60-kd Ro, which is associated with one of several hYRNA, which are short and uridine rich. Antibodies to a 52-kd protein (anti-52-kd Ro) are found in some sera with anti-60-kd Ro. The physical relationship of this protein to the RoRNP particle (60-kd Ro plus hYRNA) remains a subject of controversy.Scofield et al studied antibody binding to short peptides (octapeptides overlapping by 7 residues) and found as many as 20 distinct primary epitopes .BALB/c mice immunized with 60-kDa Ro peptides developed an immune response directed against the entire Ro/La ribonucleoprotein particle that was similar to that found in humans with lupus or Sjo¨gren's syndrome. Functional studies showed a statistical decrease in salivary flow in immunized mice compared with controls. Furthermore, there were lymphocytic infiltrates in the salivary glands of immunized animals that were not present in controls.We synthesized 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 phonemic ScFv antibodies library which we had successfully constructed before, three ScFv monoclonal antibodies (McAb) called P1, P2 and P3 against epitope P1, epitope P2 and epitope P3, respectively, were obtained. Using monoclonal antibodies against Ro/SSA 60kDa, we investigate the expression of different epitopes in nephritis of animal models, and pathogenesis of autoimmunity. Objectives1,To obtain three single- chain fragment V (scFv) monoclonal antibody from the SSA phagemid antibody library,, express scFv against different epitopes of SSA,R060 kDa antigen from previously constructed vector pET32a(+) plasmid and to purify by NTA-Ni affinity column.2. Using monoclonal antibodies against Ro/SSA 60kDa, to investigate the expression of different epitopes in MRL/lpr mouse and BALB/c mouse kidney, and analysis epitopes expression and impairment in target organs.Methods1,Sequence analyses to all the 3 clones constructed , infect pET32a(+) into E.coli.ORIGAMI, induce protein expression by IPTG.. Westem-blot was performed to test 6×His tag。And purify protein by NTA-Ni affinity column.2,ELISA with mouse SSA/Ro 60kDa and immunofluorescence on Hep-2 cells was employed to detect whether the 3 proteins (P1, P2, P3), had the abilities to combine with their antigens.3,Immunohistochemical investigation to P1-P3 epitopes expression of MRL/lpr and BALB/c mouse organs such as kidney, spleen, liver,and heart. Analysis expression of SSA/Ro 60kDa epitopes and target organ impairment by Image-proplus software.Results1. Sequences of inserted genes of three clones were identified with which reported previously. Three plasmids were transfected into E.coli.3 BL21(DE3).Three proteins were further expressed by IPTG. SDS—PAGE display proteins around 45kDa were expressed with a great quantity. 6×His tag Western-blot process is positive of scFv P1,P2,P3.ScFv were purified by NTA-Ni affinity column.2. Monoclonal scfv P1,P2,P3 was positive respectively by ELISA,mouse SSA/Ro 60kDa was used.Monoclonal P1, P2 and P3 could combine with Hep-2 cells,. tested by IIF.Plasma and nuclear were both positive,similar to anti-SSA/Ro 60kDa antibody.3. MRL/lpr mouse of 20 weeks old mimic human SLE and secondary SS.Lymphocytes infilteate in MRL/lpr mouse kidney but BALB/c .4. A number of cytoplasma and muclear is positive in glomerlus,proximal renal tubule,distal renal tubule and collecting tube in both MRL/lpr and BALB/c mice.Measure the values of integral optical density(IOD),difference of P1,P2,P3 beteween two groups is not significant;difference of P1,P2,P3 in either group is of no significance.5. Nuclear positive ratio of P3 is higher in MRL/lpr than BALB/c.The difference is significant (Nuclear positive ratio of P1,P2,P3 in MRL/lpr is 21.2%,15.8% and 36% respectively,the difference is significant (p<0.05).6. Nuclear positive ratio of P3 is higher than P1 and P2 in MRL/lpr group,the difference is significant(F=4.74,/K0.05).Difference of nuclear positive ratio among P1,P2,P3 is of no significance.7. Spleen in MRL/lpr mouse is enlarged , but inmmunohistochemistry is negative in the two group.There is no serious lymphocytes infiltrate in other organs,such as liver and heart.Inimunohistochemistry is negative in nuclear,and low positive in cytoplasma in nearly all of the samples.Conclusions1. It was proved that monoclonal scFv anti-SSA/Ro 60kDa can be produced from phagemid library.Three recombinant ScFv monoclonal anbodies(anti-Pl, P2, P3) targeting different epitops of SSA/Ro 60kDa were obtained.2. All of scFv above can combine with human and mouse SSA/Ro 60kDa ,and used in animal models..3. SSA/Ro 60kDa epitopes expression may be upregulated by increased cell activation in autoimmune-mediated inflammation,epitope aa 230～241 may be involved in autoimmune disease.