基因重组免疫毒素白细胞介素18-PE38融合基因治疗类风湿性关节炎的初步研究

目的构建白细胞介素18(interleukin18,IL-18)-PE38融合基因的真核表达载体,并研究其在小鼠软骨细胞及3T3细胞中的表达,探讨类风湿性关节炎的基因治疗方法. 方法经限制性内切酶双酶切从质粒PRKL459K-IL18-PE38中获取IL-18-PE38融合基因, 将其与真核表达载体PsecTag2B连接,转化感受态菌,挑取单克隆培养并提取质粒,EcoRⅠ单酶切鉴定.脂质体转染法将构建的真核表达载体转染入3T3细胞及小鼠软骨细胞中,分别设置空载体对照,通过荧光免疫细胞化学法鉴定转染后瞬时表达情况. 结果 EcoRⅠ单酶切后电泳鉴定显示,所构建的真核表达载体PsecTag2B-IL-18-PE38片段长度约为6 000 bp.荧光免疫细胞化学法、荧光显微镜摄片均显示转染融合基因组荧光表达强,空载体对照组荧光表达微弱. 结论 IL-18-PE38融合基因真核表达载体构建成功,并在小鼠软骨细胞及3T3细胞中表达,为类风湿性关节炎的基因治疗研究奠定基础.

PRIMARY STUDY OF RECOMBINANT IMMUNOTOXIN IL-18-PE38 IN TREATING RHEUMATOID ARTHRITIS

Objective To establish a kind of gene therapy method of rheumatoid arthritis, to construct the interleukin-18-PE38 fusion gene expression vector and to explore the expression of the fusion gene in the chondrocytes and 3T3 cells. Methods Interleukin-18-PE38 fusion gene was cleaved from plasmid PRKL459k-IL-18-PE38 by restriction enzyme digestion,then linked with vectors PsecTag2B and transformed into competence bacteria, positive clones were selected and confirmed by restrictive enzyme(EcoRI) digestion assay. The rearrangement plasmid PsecTag2B-IL-18-PE38 was transfected into 3T3 cells and mouse chondrocytes by liposome protocol(experimental group),null vector was used as negative control, and the transient expression was identified by fluorescence immunocytochemical assay. Results Restrictive enzymes digestion analysis revealed that the length of the interleukin-18-PE38 fusion gene was 6 000 bp. Fluorescence immunocytochemical method showed that fluorescence intensity of the experimental group is strong,while fluorescence intensity of the control group is weak. Conclusion the eukaryotic expression vector PsecTag2B-IL-18-PE38 is established successfully which can be expressed in the 3T3 cells and mouse chodrocytes. Our results lay a foundation for the further investigation for rheumatoid arthritis therapy.