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The objective of the experiment is to explore the purification and production of immunotoxin. The chimeric toxin, which is composed of 40 peptides of interleukin 10 (from amino acids 18 to 57) fused to a mutant form of Pseudomonas extoxin (PE) devoid of its native cell recognition domain. Two kinds of prokaryotic expression vector containing the chimeric toxin IL-10(18-57)-PE40 were constructed respectively. After induction of IPTG for 3 hours, IL-10(18-57)-PE40 was expressed highly in cytoplasmic fraction in Rosettablue(DE3), and was directed to periplasmic space as soluble form in E. coli BL21(DE3)pLysS . Western -blotting showed that the expressed protein could react with the specific rabbit sera against LHRH-PE40. With the application of salting out of (NH4)2SO4, hydrophobic interaction chromatography, Cu-affinity chromatography and anion exchange chromatography, the purity of IL-10(18-57)-PE40 was about 96%. The cytotoxicity assay, Cell-ELISA and fluorescent antibody test support the hypothesis that IL-10(18-57) based ligand-mediated cytotoxicity can serve to target cytotoxic agents in vitro.

Objective: To construct the interleukin-18-PE38 fusion gene expression vector and explore the expression of the fusion gene in the chondrocyte., Methods: The recombinant eukaryotic expression vector PsecTag2B-IL-18-PE38 was constructed by inserting interleukin-18-PE38 fusion gene into eukaryotic expression vector PsecTag2B with molecular cloning technique. It was confirmed by restrictive enzymes (EcoR I) digestion assay and PCR. The vector was transfected into primary chrondrocyte by liposome protocol, and the transient expression was identified by fluorescence immunocytochemical assay., Results: Restrictive enzymes digestion analysis and PCR revealed that the interleukin-18-PE38 fusion gene was cloned into the eukaryotic expression vector PsecTag2B successfully. Immunofluorescence photograph of fluorescence immunocytochemical method confirmed that the fusion gene can be expressed in the cytomembrane and cytoplasm., Conclusion: The results confirmed that PsecTag2B-IL-18-PE38 fusion gene can be expressed in the chondrocyte, which could serve as a foundation for the study on rheumatoid arthritis therapy.

IL-15 and IL-15 receptors (IL-15R) play a crucial role in the pathogenesis of adult T-cell leukemia (ATL), multiple myeloma and inflammatory autoimmune diseases. To develop a novel therapeutic agent capable of eliminating IL-15R-over-expressing abnormal cells, the gene coding for human IL-15 antagonist (IL-15M) was fused with a DNA fragment coding for the mutated form of Pseudomonas exotoxin, PEdelta293. The resulting gene fusion was cloned into pET16b under the control of T7 promoter, giving rise to the expression plasmid pET-IL15M-PEdelta293. Using Ni2+ -NTA affinity chromatography, IL15M-PEdelta293 was purified from E. coli BL21 (DE3) pLysS transformed with pET-IL15M-PEdelta293. The fusion toxin showed cytotoxicity to IL-15R-bearing myelogenous leukemia cell line K562 and K562-derived multidrug resistant cell line K562/AO2. However, IL-15R negative cell line Jurkat was insensitive to IL15M-PEdelta293. In addition, the toxic effect of IL15M-PEdelta293 on K562 was completely blocked by excessive amount of recombinant human IL-15. These results demonstrated that the selective cytotoxicity of IL15M-PEdelta293 correlated with the appropriate IL-15R expression on target cells. The present data suggest that the chimeric toxin constructed in this report may have therapeutic potential in the treatment of diseases associated with abnormal expression of IL-15/IL-15R, even in the treatment of chemotherapy refractory tumors.

Aim: To investigate the inhibitory effect of translocating peptide/granzyme B fusion protein on cell growth., Methods: The translocating peptide gene of Pseudomonas exotoxin A (PE) was fused with active granzyme B gene by recombinant PCR to construct PE II-GrBa fusion protein gene. PE II-mGrBa with a mutation of serine to cystein at active center of GrB was used as negative control. The resulting PE II-GrBa and PE II-mGrBa genes were transiently transfected into mammalian cells via lipofectamine mediation. The effects of expression of PE II-GrBa gene on morphology and growth of transfected cells were detected by MTT colorimetry, TUNEL assay and indirect immunofluorescence staining., Results: Transient expression of PE II-GrBa resulted in cytoskeleton abnormality, cell growth inhibition, and apoptosis in some cells., Conclusion: Expression of PE II-GrBa fusion protein can inhibit cell growth.

A recombinant immunotoxin named CEA/PE38/KDEL was constructed, which was composed of anti-CEA single-chain Fv and the truncated and modified form of Pseudomonas exotoxin (PE38/KDEL). The CEA/PE38/KDEL immunotoxin was expressed in the E. coli strain BL21 (DE3)-star as inclusion bodies. The denatured inclusion bodies were purified with Ni-NTA chelate agarose, then the constant gradient dialysis was used to perform the refolding of the CEA/PE38/KDEL immunotoxin. Results of FACS and MTT assay indicate that the refolded immunotoxins keep potent and specific cytotoxicity to tumor cells bearing CEA antigens.