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目的构建绿脓杆菌外毒素PE38融合鼠IL-18基因的原核表达载体,并鉴定其产物在大肠杆菌中的表达。方法首先经逆转录2聚合酶链反应(RT-PCR)获得小鼠IL-18基因,再通过酶切及连接反应,构建小鼠IL218与PE38融合基因的原核表达载体PRKL-IL18-PE38,重组载体经PCR、限制性内切酶及DNA序列测定证实连接片段正确后,转染感受态大肠杆菌BL-1,经IPTG诱导表达,表达产物用SDS-PAGE和蛋白免疫印迹法分别测定其相对分子质量及特异性。结果成功构建了IL-18-PE38免疫毒素的原核表达载体,重组载体在大肠杆菌中获得了稳定的表达,表达产物的相对分子质量与预期值一致,且所表达蛋白可被抗IL-18的特异性抗体所识别。结论获得了IL-18-PE38融合基因在原核系统的表达,为进一步研究其对Th1细胞的靶向细胞毒性及临床应用奠定了基础。
Objective To construct a prokaryotic expression vector for IL-18 gene of Pseudomonas exotoxin PE38 fusion mouse and to identify its expression in E. coli. Methods The mouse IL-18 gene was obtained by reverse transcription-polymerase chain reaction (RT-PCR). The prokaryotic expression vector PRKL-IL18-PE38 was constructed by restriction enzyme digestion and ligation, The vector was confirmed by PCR, restriction endonucleases and DNA sequence analysis. The correct fragment was transfected into competent E. coli BL-1 and induced by IPTG. SDS-PAGE and Western blotting were used to determine the relative molecular weight Quality and specificity. Results The prokaryotic expression vector of IL-18-PE38 immunotoxin was successfully constructed. The recombinant vector was stably expressed in E. coli. The relative molecular mass of the expressed product was consistent with the expected value. The expressed protein could be expressed by anti-IL-18 Specific antibodies recognize. Conclusion The expression of IL-18-PE38 fusion gene in prokaryotic system was obtained, which laid the foundation for further study of its targeted cytotoxicity to Th1 cells and its clinical application.