免疫毒素基因DT390真核表达载体的构建及功能研究  

Study of the construction and function of an eukaryotic expression plasmid of immunotoxin DT390 gene

作　　者：陈文捷[1] 李虹[2] 贾怡[3] 张林[3] 吕梅励[3] 李明远[2] 蒋中华[2] 

机构地区：[1]四川大学华西基础医学与法医学院免疫学教研室,四川成都610041 [2]四川大学华西基础医学与法医学院微生物学教研室,四川成都610041 [3]四川大学华西基础医学与法医学院法医物证学教研室,四川成都610041

出　　处：《细胞与分子免疫学杂志》2006年第3期299-301,305,共4页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家高技术研究发展计划(863)资助(No.2002AA214101);国家自然基金委创新研究群体基金项目(No.30221001);四川省应用基础项目基金资助(No.04JY029-002)

摘　　要：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。AIM： To construct a new recombinant eukaryotic expression plasmid of mouse IFN-γ-inducible protein 10 （mIP10） gene and a truncated diphtheria toxin （DT390） gene and investigate its biological function. METHODS： mIP10 cDNA was cloned from mouse liver tissue by RT-PCR and was inserted into eukaryotic expression plasmid SRα containing DT390 gene to construct plasmid SRα- DT390-mIP10. After being identified with restriction endonucleases digestion analysis and DNA sequencing, the recombinant plasmid was transfected into NIH3T3 cells through PolyFect. The expression of recombinant plasmid was detected by immunofluorescence assay and the bioactivity of mIP10-DT390 in cultured supernatant of the transfected NIH3T3 cells was detected by MTT Colorimetry. RESULTS： A new recombinant immunotoxin expression plasmid SRα- DT390-mIP10 was constructed successfully and was expressed in NIH3T3 cells. The expressed DT390-mIP10 killed the activated T cells effectively. CONCLUSION： A new recombinant eukaryotic expression plasmid SRα-DT390-mIP10 has been constructed and expressed successfully, which is useful in further studying the treatment of cancer and autoimmune diseases.

关 键 词：免疫毒素 mIP10 DT390 真核表达 

分 类 号：R392.11[医药卫生—免疫学]