| Background and our goal: Organ transplantation is an important method to treat terminal organ failure. However, the rejection to donor grafts is the main barrier for organ transplantation. The ideal method to minimize rejection is to induce the long-term immune tolerance between patients and grafts, but currently no satisfied method is applied. In clinic, we commonly use immunosuppressors to control the rejection. However, long-term use of nonspecific immunosuppressors has obvious side effect, which will cause severe consequences. In order to control immune rejection and to sustain the normal organ function, we need to find out the effective ways to inhibit abnormally activated immunocytes. Because of their characteristics, immunotoxins are the optimal choices to achieve this goal. Immunotoxins, which is a combination of a vector and a toxin, can specifically kill target activated T cells but not damage normal tissues and cells. It has obvious advantages over other reagents in the therapy of tumor, autoimmune disease, and organ transplantation. The triggering of graft rejection is the outcome of the interaction among complicated surface molecules on T cell, cytokines and other relative factors.αchain of interleukin-2 receptor(CD25,IL-2R),an early expressed molecule on activated T cell, is an important marker representing the activation of help T cell(Th) and the priming of cell-mediated immune response. Only interleukin-2 receptor existingαchain is the high-affinity IL-2 receptor form. The interaction between T cells with IL-2R and IL-2 provides signals for activation and proliferation of T cells. This event plays an important part in graft rejection. IL-2Rαchain becomes an ideal target molecule for the IL-2 immunotoxins therapy. Immunotoxins, which contain IL-2 as their adaptor, can bind and kill activated T cell and then can inhibit graft rejection. Several immunotoxins, which target IL-2R, have been constructed. Some of them even have shown favorable therapeutic effects both in fundamental experiment and in clinical research. It is a new immunosuppressor with promising prospect. Because of the immunogenicity of the immunotoxins, the application of immunotoxins is currently limited. On one hand, most of the immunotoxins use non-human resources antibodys as targeting vectors, which are alien to recipient and cause immune response in body which accelerates the degradation of immunotoxins and increases the side effect. On the other hand, because of their large molecular weight, it is difficulty for immunotoxins to infiltrate into tissues. So the researchers try to use small immunotoxins with human resourses. The small immunotoxins can easily reach the target organ through the membrane of blood capillary and easily transfer into cells. The human resource immunotoxins can resolve the xenogeneic problem. Ribosome inactivating proteins (RIPs) derived from plant are potent toxins, which can powerfully inhibit the synthesis of proteins in cells. Some of the RIPs have been applied in construction of immunotoxins and show good effects in the therapy of anti-graft rejection, cancer and autoimmune disease. Luffin family is one kind of ribosome inactivating proteins (RIPs), which is extracted from the seeds of luffa. Luffin P1 is a new member of this family and is powerful in inhibiting protein synthesis. Its low molecular weight facilitates targeting vector to enter into the target cell while reduce the immunogenicity of immuontoxins. This character decreases the side effect and increases the therapeutic effect. Like a missile, Luffin P1 can only destroy the target cells without damage the normal cells. It has obvious advantages in clinical therapy. Therefore, we postulate that if we combine IL-2 with the Luffin P1 which has strong toxicity, low molecular and weak antigenicity, IL-2 can specifically recognize the IL-2R expressed on activated T cell, which is activated by allo-or xeno-graft antigens, then the Luffin P1 destroys the activated T cells. The survival time of the graft could be prolonged significantly. In our experiment, we have constructed and expressed a new immunotoxin through engineering technology, in which Luffin P1 functions as toxin and IL-2 as specific detector for activated T cells. We hope this new immunotoxin could specifically inhibit graft rejection and prolong the survival time of the grafts. Results According to the human hIL-2 gene sequence in gene bank, we designed two pairs of nest primers to clone human IL-2 from human PMBC. We used overlap extension PCR to combine hIL-2 cDNA with Luffin P1 cDNA together and then construct a wholehIL-2-Luffin P1 sequence and extend the sequence. The hIL-2-Luffin P1 cDNA was digested by Nco I and Xho I restriction enzyme and ligate via T4 DNA Ligase with pET20b (+) vector. The recombinant plasmid was transformed into E.coli DH5αand positive colonies were identified by gene sequencing. Recombinant plasmid pET-hIL-2-Luffin P1 and pET-Luffin P1 were transferred into expression host bacteria Origami (DE3) which carry chromosome T7 RNA polymerase gene, respectively. We used 0.4mmol/L IPTG to induce the expression of recombinant protein under 300C. The harvested proteins were loaded on SDS-PAGE gel and electrophoresis. A new protein band near 22.5kD was found on the gel. In order to further verify the proteins, we used anti-His McAb and anti-IL-2 McAb to carry out Western blot respectively. The results show the expressed protein is the protein we wanted. The proteins from recombinant plasmid pET-Luffin P1 were loaded on Tris-Tricine Gel and electrophoresis. Protein about 7kD was found on the gel. The location assay of expressed protein indicated that target protein could be expressed in periplasm. After verification and grading-up expression, recombinant plasmids were induced to express target proteins, and purified with His·Bind resin. We got 1.54mg hIL-2-Luffin P1 and 0.55mg Luffin P1 from a liter of bacterial liquid, respectively. In this experiment we observed the biological activity of the immunotoxins derived from gene engineering in vitro and in vivo. We carried out experiments of mouse mixed lymphocyte response and lymphocyte proliferation stimulated by ConA in vitro and allogeneic skin transplantation among mice and renal transplantation among rats in vivo, observing the impact of hIL-2-Luffin P1 on the survival of grafts. In our experiments aimed to inhibit the activity of lymphocytes in vitro, we added different concentrations of hIL-2-Luffin P1 to the MLR reactive system, while Luffin P1 was adopted as control group. The results showed that CPM declined corresponding to the gradually increased concentration of hIL-2-Luffin P1, and cell proliferation was inhibited obviously. The inhibition ratio gets to 97% at the concentration of 10-6mmol/L hIL-2-Luffin P1. On the contrary, the control group treated with Luffin P1 showed no obvious inhibition to lymphocyte proliferation. The inhibition ratio only reaches 14% at the same concentration. We also found that when we respectively added different concentrations of hIL-2-Luffin P1 and Luffin P1 to spleen cells stimulated by ConA, the group treated withhIL-2-Luffin P1 also showed strong ability to inhibit the proliferation of these spleen cells. The result was the same as that of the MLR. In general, our experiments indicated that the target protein could inhibit lymphocyte proliferation obviously. In our experimental model of allo-skin transplantation, we used C57 mice as skin donors and BALB/c mice as recipients. The results indicated that hIL-2-Luffin P1 could prolong the survival time of the allo-skin grafts. When the concentration of Luffin P1 is 70ug/kg, the Luffin P1 treated group shows no obvious difference from the control group. On the contrary, when hIL-2-Luffin P1 was at the concentrations of 2.25ug/kg, 22.5ug/kg and 225ug/kg respectively, the survival time of the skin grafts was prolonged to some different extent, about 14.4±1.58D, 15.8±1.69D and 18.2±2.53D correspondingly. Pathological analysis indicated that when the skin grafts were rejected in the control group, there were obvious lymphocyte infiltration and inflammatory reaction, while the lymphocyte infiltration in the experimental groups was relatively light。Allo-renal transplantation on rats was also used to observe the effect of hIL-2-Luffin P1 on the survival of grafts, we used Wistar rats as donors and SD rats as recipients, and divided the rats into 3 groups: the control group, Luffin P1-treated group and hIL-2-Luffin P1-treated group. The results indicated that the survival time of the hIL-2-Luffin P1-treated group was obvious longer than the control group, while Luffin P1-treated group showed no obvious difference from the control group. Pathological analysis showed that when the allo-renal grafts were rejected in the control group, the renal structure was destroyed and there was obvious lymphocyte infiltration, while the renal pathological changes of the experimental group were relatively lighter. Conclusions: 1. We have successfully constructed the recombinant expression plasmids of single chain ribosome inactivating protein Luffin-P1 and immunotoxin hIL-2-Luffin P1. The recombinant expression plasmids express proteins after the induction of IPTG. SDS-PAGE verifies the molecular weights of the proteins match up to what being expected and Western blot ascertains they are target proteins. We get purified immunotoxin hIL-2-Luffin P1 and toxin Luffin P1 through affinity chromatograph. 2. Experiments in vitro show that hIL-2-Luffin P1 can inhibit mixed lymphocyte reaction and lymphocyte proliferation stimulated by ConA, and the activity of inhibitinglymphocyte proliferation is proportional to the dose, while Luffin P1 has no obvious effect on the lymphocyte proliferation in the two reaction systems mentioned above. This indicates that recombinant immunotoxin hIL-2-Luffin P1 can specifically kill activated T lymphocytes expressing IL-2Rα. 3. In both animal models of allo-skin transplantation among mice and allo-renal transplantation among rats, hIL-2-Luffin P1 can prolong the survival time of the grafts. The survival time is proportional to the dose. This indicates that hIL-2-Luffin P1 can inhibit immunological rejection.
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