IL-18-PE38重组免疫毒素的原核表达及其鉴定

Construction and Prokaryotic Expression of a Recombinant Immunotoxin Fused with Mouse Interleukin 18 and Truncated Pseudomonas Exotoxin

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表达。方法 首先采用 RT- PCR获得小鼠 IL - 18基因 ,再通过适当的酶切及连接反应 ,构建小鼠 IL - 18与 PE38融合基因的原核表达载体 PRKL - IL 18- PE38,重组载体经限制性内切酶、PCR及 DNA序列测定等证实连接片段的正确性后 ,再转染感受态大肠杆菌 BL 2 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 酶切分析、PCR鉴定和 DNA测序等证实 ,IL - 18- PE38免疫毒素的原核表达载体被成功构建 ,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 IL - 18- PE38融合基因原核表达载体的获得 ,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 new recombinant immunotoxin expression vector by fusion of mouse interleukin18(mIL-18) gene and a truncated form of A (PE38) gene, and examine the expression of IL-18-PE38 fusion protein in Escherichia coli. Methods mIL-18 cDNA was cloned from mouse liver tissue through reverse transcription-polymerase chain reaction (RT-PCR). The mIL-18 cDNA was subcloned into a PE38 gene inserted fusion protein expression plasmid. The recombinant vector was identified by restriction endonucleases digestion, PCR and DNA sequencing. After transformed into E.coli BL21 and induced by IPTG, the expressed product was obtained and detected the molecular weight and specificity by SDS-PAGE and Western-blotting. Results The new recombinant immunotoxin expression vector was constructed successfully. The IL-18-PE38 fusion protein was expressed in E. coli BL21, and the molecular weight of the expression product was identical to the expected value. In addition, the protein expressed could react with the specific antibody against mIL-18. Conclusion IL-18-PE38 recombinant immunotoxin expression vector will provide the basis for study on the targeted cytotoxic activity to Th1 cell and may have some potential value in the treatment of autoimmune disease and T cell leukemias.