| ObjectiveTo observed differences of immunogenicity and antigenicity between prokaryotic SmDl proteins and eukaryotic SmDl proteins, which were both recombinant proteins using genetic engineering techniques, and explore the possibility of production of anti-dsDNA and other antinuclear antibodies induced by excessive SmD1protein in order to provide an experimental basis for seeking to start primary and pathogenesis of systemic lupus erythematosus. In addition, invitro reagents kit of anti-SmDl was developed using the main material of eukaryotic recombinant SmD1protein and evaluated by testing several clinical autoimmunitic desease serum sample.MethodsChemical synthetic SmD1DNA was amplified by PCR, which was cloned into vector pET-21a for prokaryotic expression and vector pFastBacTM-1for eukaryotic expression, respectively. After construction of plasmid pET-21a-SmD1and pFastBacl-SmDl successfully, the next procedure could be continued.(1) Prokaryotic expression:the plasmid pET-21a-SmD1was transformed into E. coli BL21, positive clones was cultured in the condition of16℃10hours and induced by1mM IPTG in order to express. SDS-PAGE electrophoresis was used to identify recombinant proteins which purified by Ni-NTA column affinity chromatography.(2) Eukaryotic expression:The plasmid pFastBacTM-1-SmDl was transformed into DHl10Bac. Recombinant shuttle plasmid Bacmid gained by blue-white screening positive clones was transfected into insect cells Sf9, following by amplification of recombinant baculovirus and protein expression. SDS-PAGE electrophoresis was used to identify recombinant proteins which purified by Ni-NTA affinity chromatography and western blot verification.(3) Immunogenicity study:4-weeks-old Balb/c female mouse were immunized into prokaryotic and eukaryotic protein and protein in the way of multi-point intradermal injection. Mouses were immunized once every two weeks, three times total. At the the time of preimmune, the second week, fifth week, and ninth week, mouses’serum were collected and measured anti-dsDNA antibody IgG by ELISA and ANA profile by LIA. Spleen T lymphocyte of nine weeks old mouse were gained and analyzed IFN-γ frequency by ELISPOT and T lymphocyte subsets CD3, CD4and CD8by Flow cytometry analysis.(4) Indirect ELISA was set up for anti-SmD1detection of553serum sample of several autoimmunitic desease, including230SLE. ResultsBoth plasmid pET-21a-SmDl and pFastBacl-SmD1were constructed correctly and confirmed by DNA sequence analysis restriction analysis, which expressed successfully in E. coli BL21and baculovirus/insect cell systems (AcMNPV/Sf9), respectively, and obtained the desired protein. The purity more than95% of recombinant protein SmDl with HIS tagged was purified by Ni-NTA affinity chromatography. Recombinant SmDl protein concentration from E. coli BL21and from AcMNPV/Sf9were0.62mg/mL and0.908mg/mL, respectively, which were both determined by the Bradford method. Immunogenicity studies showed that in the same period, anti-dsDNA antibody titer induced by SmDl protein from Sf9was higher than the one induced by SmDl protein from E. coli BL21as well as anti-Sm antibodies titer. anti-dsDNA antibody titer induced by SmD1protein from Sf9reached a peak at the fifth week, then decreased gradually, while anti-dsDNA antibody titer induced by SmD1protein from E.coli BL21increased with time during nine weeks, maybe longer. The difference of anti-dsDNA in each group were statistically significant. SmD1protein induced anti-dsDNA antibodies, as well as anti-Sm antibodies, anti-SSA/60antibody, anti-SSA/52antibody, anti-SSB antibodies, anti-Se170antibody and anti-UlsnRNP antibodies.In the case of excessive SmD1, CD3+CD4+/CD3+CD8+balance was disturbed. Comparing with normal control, CD3+CD4+of SmD1immunizing group decreased significantly, while CD3+CD8+increased. The IFN-γ secreting T cell frequency of prokaryotic proteins control and eukaryotic proteins control were significantly higher than the normal control. The difference above between the two proteins control groups was not statistically significant. Anti-SmD1ELISA kit made of SmD1from AcMNPV-sf9performanced in full compliance YY/T1183-2010enzyme-linked immunosorbent assay reagents industry standard, and applicated well in clinic with sensitivity of57.8%and specificity of96.4%to SLE. ConclusionBoth appearance of epitope spreading phenomenon and production of anti-dsDNA antibodies and other antinuclear antibodies suggested that the amount of increase SmD1autoantigen may induce a lupus-like autoimmune disease. Immunogenicity and antigenicity of SmD1from AcMNPV-sf9which could bind with anti-dsDNA were stronger than SmD1from E. coli BL21due to the conformational epitopes.Anti-SmD1ELISA kit was helpful in SLE diagnosis with valuable clinical application. |
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