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目的构建一种新型免疫毒素DT390-mIP10(白喉杆菌毒素390-小鼠γ干扰素诱导性蛋白10)基因的真核表达质粒,并对其功能进行初步研究。方法通过RT-PCR扩增mIP10基因,并插入到含有DT390基因片段的真核质粒SRα中,构建重组质粒。以PolyFect脂质体转染NIH3T3细胞,用免疫荧光检测重组质粒的表达;用MTT比色法测定重组免疫毒素质粒的生物学活性。结果构建了DT390-mIP10的真核表达载体SRα-DT390-mIP10,并在NIH3T3细胞中获得表达。表达的DT390-mIP10在体外能有效地杀伤活化的T细胞。结论免疫毒素基因重组真核表达载体DT390-mIP10的构建成功并在真核细胞中表达,为其在肿瘤及自身免疫性疾病治疗方面的进一步应用研究奠定了基础。
Objective To construct a eukaryotic expression plasmid of novel immunotoxin DT390-mIP10 (diphtheria toxin 390-mouse interferon-inducible protein 10) gene and to study its function. Methods The mIP10 gene was amplified by RT-PCR and inserted into the eukaryotic plasmid SRα containing the DT390 gene fragment to construct a recombinant plasmid. NIH3T3 cells were transfected with PolyFect liposomes, and the expression of the recombinant plasmids was detected by immunofluorescence. The biological activity of the recombinant immunotoxin plasmids was determined by MTT colorimetric assay. Results The eukaryotic expression vector SRα-DT390-mIP10 of DT390-mIP10 was constructed and expressed in NIH3T3 cells. The expressed DT390-mIP10 effectively killed activated T cells in vitro. Conclusion The construction of recombinant eukaryotic expression vector DT390-mIP10 of immunotoxin gene and its expression in eukaryotic cells lay the foundation for its further application in the treatment of tumors and autoimmune diseases.