| Immunotoxins have the potential in disease therapy for its selective killing of some cell populations. Since activated T cells play a central role in transplantation rejection, an immunosuppressive strategy is to produce immunotoxin that could selectively delete activated T cells. CTLA-4 is inductively expressed on surface of activated T cells. Therefore, CTLA4-mediated immunotoxins might be developed as novel immunosuppressant due to their ability to selectively delete activated T cells.Based on this concept, a novel immunotoxin called mST was constructed by fusing a pore-forming antifungal peptide T to the C-terminal of the anti-CTLA4 scFv. And the gene encoding mST was expressed in prokaryotic as well as eukaryotic system. The recombinant protein was purified and its cytotoxicity to CTLA4-positive EL4 cells was detected. The main results include:1) In this research, we constructed the expression vectors pQE-mST for prokaryotic expression in E.coli M15. Immunotoxin mST was mainly expressed as insoluble inclusion bodies. Only a small amount, approximately 0.1mg/L of soluble mST was purified from the cytoplasm of mST-producing cells after induced by 0.1mM IPTG at 28℃for 5h.. Whears, several microgams of mST inclusion bodies was purified from precipitate of producing bacteria after sonicated. The inclusion bodies were dissolved in 6M guanidine-HCl and purified by Ni-NTA under denatured condition. Subsequently, the inclusion bodies were refolded in refolding buffer containing 50 mM Tris-HCl, 1M Urea, 0.8 M L-Arginine, 2mM GSH, 0.2mM GSSH, pH8.0,at 4℃for 48h. About 70% inclusion bodies could be refolded into soluble protein. However, in the further process of removing denaturant by dialysis agains PBS, considerable proportion of protein precipitated. And we get the soluble protein at the concentration of 0.1mg/ml finally.2) We further constructed the expression vectors pPIC9K-mST for eukaryotic Pichia Pastoris expression to avoid the problem of inclusion body protein refolding. Yeast cells are transformed by electroporation and then multi-copies of inserts were screened on Geneticin? -YPD plates. After small-scale methanol induction, expression conditions are optimized. Finally, we get 1 mg mST from 1 liter culture after induced by 3% methonal, 30℃for 96h.3) Cultured mouse CTLA-4 positive cell EL4 was used to detect the cytotoxicity of recombinant mST. The soluble mST from E.coli M15 shows killing rate of 36%±11.2%, 44%±3.8%, 64%±9.1%to EL4 at concentration of 0.5, 1 and 2 uM, respectively. The mST refolded from inclusion bodies produced by E.coli M15 shows killing rate of 24%±13.1%, 45%±10.1%, 62%±10% to EL4 at concentration of 0.5, 1 and 2 uM individually. And the killing rate of soluble protein from Pichia pastoria to EL4 cells is 37.5%±2.2% at concentration 1 uM. However, no obvious cytotoxicity of ScFv, namely mS, at the same concentration was detected in this research.The above results demonstrate that small amount of soluble mST could be produced by both E.coli and Pichia pastoria. And high level of mST could be expressed as inclusion bodies in E.coli. The cytotoxicity of mST to CTLA4-positive EL4 suggests that mST might delete activated T cells. But the efficiency and specificity of mST need to be investigated in future.
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