| Objective Currently, there are no specific treatments for autoimmune diseases in humans. All treatments, most steroids, are based on general suppression of entire T-cell compartment and therefore only offer temporary symptomatic relief and can not be used for prolonged periods. The side effects of entire T-cell suppression mainly include opportunistic infection and tumour genesis. Experimental allergic encephalomyelitis (EAE) is a model of T cell-mediated autoimmune disease, with many clinical and histopathologic similarities to the human diease mutiple sclerosis (MS). EAE is most commonly induced in experimental animals by immunization with purified myelin proteins such as myelin basic protein (MBP), myelin proteolipid protein (PLP), and myelin oligodendrocyte glycoprotein (MOG). EAE is mediated by CD4~+ T cells of the Thl phenotype that recognize myelin peptide determinants in the context of major histocompatibility complex (MHC) class II molecules and initiate a cascade of events leading ultimately to paralysis accompanied by perivasular mononuclear infiltrates and demyelination in CNS white matter. EAE serve as a useful model for learning more about the etiopathogenesis of MS and for evaluating treatment protocols. A more useful and clinically applicable approach would therefore be to induce the specificelimination and/or inactivation of the T cell clone(s) that mediate EAE or MS in the host. This approach became feasible because of recent advancements in the understanding of the mechanisms leading to T-cell activation. Numerous studies demonstrated that some cytokine and chemokine receptors such as IL-2R, IL-12R and IL-18R are expressed on the surface of the activating T cells. In particular, recent data suggested that IL-18 receptor is expressed selectely by Thl cells, but not Th2 cells. On the basis of these findings, we choosed IL-18 receptor (IL-18R), the specific surface marker of activated T cell, as a target, constructed expression plasmid of recombinant immunotoxin IL-18-PE38, and detected the expression of target gene and identified the products activation in vitro. We also inoculated recombinant plasmid into EAE animal model for gene therapy, then we observed the amelioration of clinical symptom and modulation related cytokines production, evaluated treatment effects and further explored the mechanism of autoimmune diseases treatment with recombinant immunotoxin.Methods At first, we extracted the total RNA from mouse liver tissue and mIL-18 gene was amplified by RT-PCR. Recombinant plasmid PRKL-IL18-PE38 was constructed by ligating the mIL-18 gene into the prokaryotic expression vector containing PE38 gene named PRKL-IL18-PE38. The plasmid PRKL-IL18-PE38 was identified by restriction endonuclease digestion, PCR and sequence analysis. After transfered recombinant plasmid into Escherichia coli BL21, the expression of recombinant plasmid was detected by SDS-PAGE and Western blot analysis.In the second part, we constructed the recombinant plasmid pSecTag2B-IL18-PE38 by cloning the mIL-18 and PE38 fusion gene into the eukaryotic expression vector pSecTag2B. The constructed plasmid wasconfirmed by the restriction endonuclease digestion and PCR analysis. NIH3T3 cells were transfected with purified recombinant plasmid by Lipofection strategy in order to identify the transcription and expression of pSecTag2B-IL18-PE38 plasmid in mammalian cells. The expression of products was detected by immunofluorescence assay. Supernatants of the transfected cell culture were collected and used to detect the bioactivity of IL-18-PE38 protein in vitro.In the third part, EAE was induced by subcutaneous immunization of in 6 to 8-week-old C57BL/6 male mice. Each mouse received one injection with 200ul of 400ug of MBP in the complete freund's aduvant containing 5mg/ml of BCG. Mice were injected through peritoneal injection with in 2xlO5 B. pertussis at the same day of immunization. Mice immunized with MBP were divided into 4 groups randomly. They were recieved injection intramuscularly with nake pSecTag2B-IL18-PE38 plasmid or wrapped pSecTag2B-IL18-PE38 plasmid with liposomes in different dose respectively. Another group of the mice recieved pSecTag2B control vector using the same administration protocol. After treatment, body weight and clinical score were recorded daily, and related cytokine production were tested with diease ongoing.Results We obtained 488bp mIL-18 gene fragment with RT-PCR from the template of total RNA of BABL/c mice liver. Prokaryotic expression recombinant plasmid PRKL-IL18-PE38 was identified by restriction analysis and sequence analysis. It was showed the single fragment which size was about 4.6Kb through EcoRI or Ndel digestion; two fragments which sizes were about 480bp and 4 lOObp through Ndel and Hindlll digestion. Another two fragments with 1 640bp and 2 900bp in size were observed through Ndel and EcoRI digestion. The sequencing for insertional gene of PRKL--IL18-PE38 showed that it was in accordance with the portion of mIL-18 gene and PE38 gene and their ORF was not changed. The results suggested that the IL-18 gene and PE38 gene had been inserted correctly into prokaryotic expression vector. Recombinant plasmid PRKL-IL18-PE38 was transformed into Escherichia coli BL21 and the expression product in BL21 was detected by Western blot. It showed that the protein with Mr of 56 KD was able to react with the specific anti-mIL-18 goat sera. The results might be related to the expression of fusion gene in prokaryotic cells.Eukaryotic expression recombinant plasmid pSecTag2B-IL18-PE38 was confirmed by restriction endonuclease digestion and PCR analysis. It was showed that the IL-18-PE38 segment was inserted correctly into the eukaryotic expression vector pSecTag2B. After NIH3T3 cells were transfected by purified pSectag2B-IL18-PE38 with Lipofection strategy, the cells with expression of objective protein could be observed by immunofluorescence. In the bioactivity detection assay, the supernatant of the transfected cell culture was of obvious selective cytotoxicity to activated T lymphocyte.EAE model had been established by active immunization with MBP in C57BL/6 mice successfully. After received injection using the administration protocol, the pSecTag2B-IL18-PE38-treated mice showed the disease starting day was delayed and the EAE scores were lower significantly compared with the control mice. Related cytokines production in mice serum and spleen cell culture supernatants were tesed by ELISA assay. The results showed pSecTag2B-IL18-PE38-treated mice were lower in levels of IFN-y and higher in levels of IL-4. The desirable results showed in both groups with nake plasmid pSecTag2B-IL18-PE38 and wrapped plasmid pSecTag2B-IL18-PE38 with liposomes in lower dose. pSecTag2B-treated control mice showed thetypical course of the diease without any clinical or weight improvement after vector DNA injection. The result suggested that the treatment effects might be related to improvement of Thl/Th2 balance. Conclusion(1) We constructed the prokaryotic expression plasmid of recombinant immunotoxin IL-18-PE38 and obtained the target gene expression in Escherichia coli BL21 successfully.(2) We constructed the eukaryotic expression plasmid of recombinant immunotoxin IL-18-PE38 successfully and obtained effective expression in NIH3T3 cell. The supernatant of the transfected cell culture was of obvious cytotoxicity to activated T lymphocyte in vitro.(3) The data from EAE model indicated that the gene therapy protocol of pSecTag2B-IL18-PE38 in EAE mice lead to amelioration of clinical and pathological signs of EAE. The results suggested that the gene therapy of recombinant immunotoxin might be a potential useful approach for the treatment of autoimmune diseases. |
PDF Full Text Request