The Effect Of Chimeric Molecules Of Recombinant Dsg3EC_1-2 With Toxin On T, B Lymphocytes In Pemphigus Vulgaris

Objective: Pemphigus vulgaris is an autoantibodies-mediated, organ-specific chronic autoimmune blistering disease of the skin and mucous membranes. Autoantibodies IgG found in PV patients bind to the cell surface of keratinocytes and cause loss of cell to cell adhesion with resultant blister formation. The autoimmune target in PV was identified to be a desmosomal cadherin, Dsg3. The disorders of cellular immunity and humoral immunity were found simultaneously exist in pemphigus patients in recent years. Compelling evidence has been accumulated for the pathogenic roles of IgG autoantibodies against Dsg3 produced by B cells. T cells are involved in anti-Dsg3 antibodies production in an antigen-special manner in PV patients. In our study, the recombinant protein of the Dsg3EC1-2 segment, including the pathogenic epitopes or immunodominant epitopes of PVA, and Pseudomonas Exotoxin, PE40 is produced in prokaryotic cells. Then we can observe the effect of the recombinant chimeric toxin on the PBMC separated from PV patients. This will lay a foundation for us to further study the biological treatment for PV.Methods: At first, the recombinant chimeric toxin Dsg3EC1-2PE40 was induced by IPTG and was expressed in BL21trxB(DE3) cells. It was identified by Western blotting assay and purified by IMAC. Then the PBMC and T cells were separated from PV patients. We detected and quantitated autoantibody production B cells in different concentration of recombinant chimeric toxin by ELISPOT assay. By MTT assay and 3H-TdR assay, we observed the effect of the recombinant chimeric toxin on the metabolism and proliferation of T cells in PV patients in vitro.Results: 1. Dsg3EC1-2PE40 was produced in solubility in BL21trxB(DE3) cells, while Dsg3EC1-2 was expressed in AD494(DE3)cells as an inclusion body. Dsg3EC1-2PE40 didn't increased in BL21trxB(DE3) cells as Dsg3EC1-2 did with the extended expression time. 2. The expressed proteins only can combine with IgG from PV patients but not did with IgG from the normal control. 3. The purity of Dsg3EC1-2PE40 is more than 80%. Its concentration is about 547μg/ml. 4. From the results of ELISPOT assay, comparing with Dsg3EC1-2, the number of anti-Dsg3 antibodies production B cell in PBMC of PV patients decreased about 40% with the effect of Dsg3EC1-2PE40 comparing with Dsg3EC1-2. 5. Comparing with Dsg3EC1-2, the metabolism and proliferation of T cells in PV patients was inhibited markedly with the action of Dsg3EC1-2PE40. There was significant difference between them. Conclusions: 1. The recombinant proteins were successfully induced and expressed in prokaryotic cells, and were purified with Talon Metal Affinity Resins. We got the purified recombinant proteins, which can combine with Pab IgG specially. 2. The recombinant proteins could specially combine with IgG from PV patients but not did with IgG from the normal control people. 3. The recombinant chimeric toxin Dsg3EC1-2PE40 inhibited B cell in PBMC in PV patients from producing anti-Dsg3 antibodies IgG. Comparing with Dsg3EC1-2, the number of anti-Dsg3 antibodies production B cell decreased about 40%. 4. Comparing with Dsg3EC1-2, the metabolism and proliferation of T cells in PV patients was inhibited markedly with the action of Dsg3EC1-2PE40. There was significant difference between them. This indicated that the recombinant chimeric toxin could inhibit or kill T cells in PV patients in vitro.