高纯度白介素-3与假单胞菌外毒素融合蛋白的表达、纯化及质谱鉴定  

Renaturation,Purification and Identification of Fusion Toxin IL3-PE38KDEL

作　　者：廖乐乐[1] 娄世锋[1] 曾翰庆[2] 白凤霞[2] 

机构地区：[1]重庆医科大学附属第二医院血液内科,重庆400010 [2]重庆医科大学检验系重点实验室,重庆400016

出　　处：《生物技术通报》2010年第6期138-141,151,共5页Biotechnology Bulletin

摘　　要：旨在利用简单的表达纯化工艺,获得纯度较高、有活性的重组免疫毒素IL3-PE38KDEL并对其物理学及免疫学性质进行鉴定。利用PQE30-IL3-PE38KDEL/SG12009工程菌表达重组免疫毒素IL3-PE38KDEL,在提取包涵体后经一步强阳离子柱层析得到纯度较高的免疫毒素复性纯化产物,用Westenblotting、质谱、氨基测序等先进技术对产物鉴定。结果显示,发酵后目的蛋白表达量占总菌体的17.56%,经强阳离子柱层析纯化复性后的目的蛋白纯度达到90%以上,Western blotting、质谱、氨基测序结果均表明,纯化产物分子量为54.9kD且具有相应的免疫学活性,序列与预期序列完全一致。纯化复性方法对重组免疫毒素IL3-PE38KDEL的生物性质及纯度有较大影响,因此选择一种适当的纯化复性方法至关重要。It was to obtain IL3-PE38KDEL,a new recombinant immunotoxin consisting of a truncated pseudomonas exotoxin A（PE38KDEL）linked to human interleukin 3（IL3）in E.coli SG13009,with high purity and normal bioactivity.The recombinant plasmid PQE30-IL-3-PE38KDEL,was transformed into E.coli SG13009.After IPTG induction,the expressed protein products were verified by mass spectrometry and Western blotting.Purified by Ni 2＋-NTA agarose affinity chromatography,refolding and it was identified.Rerults showed that the recombinant immunotoxin was expressed in E.coli SG13009 clone strain containing PQE30-IL-3-PE38KDEL induced by IPTG in the form of inclusion body.SDS-PAGE analysis revealed that expression level of the product was up to 17.56%of the total bacterial proteins.It was a protein band about Mr above 54 900 in gel,which could be identified by PEA of anti-pseudomonas exotoxin A monoclonal antibody with Western blotting technique.The highly purified recombinantprotein by Ni 2＋-NTA agarose affinity chromatography and refolding.Mass spectrometry demonstrated that the recombinant protein was the recombinant immunotoxin consisting of a truncated pseudomonas exotoxin A linked to human interleukin 3.IL3-PE38KDEL was successfully expressed in E.coli.A suitable protocol for purification of the recombinant protein was set up.After purification,pure protein of the expressed IL3-PE38KDEL was obtained.It laid a solid foundation for the further research of function.

关 键 词：重组免疫毒素 IL3-PE38KDEL 纯化复性 质谱鉴定 

分 类 号：Q51[生物学—生物化学]