摘要
通过构建原核表达载体、优化原核表达条件和纯化表达产物来获得人白细胞介素-2(IL-2)的融合蛋白(His-IL2)。将IL-2基因连接到pET100/D-TOPO载体中,经酶切鉴定和测序,构建了pET-IL2质粒。将质粒转化到大肠杆菌BL21(DE3)中,通过优化诱导温度、诱导时间及IPTG诱导终浓度来减少包涵体的生成,从而提高可溶性蛋白的产量,由SDS-PAGE和Western blot证明IPTG诱导终浓度为0.6 mmol/L、温度为25℃,诱导5 h时可溶性蛋白含量明显高于包涵体,且该融合蛋白能够被特异性抗体所识别。分别以葡聚糖凝胶层析和Ni-IDA树脂亲和层析纯化His-IL2融合蛋白,比较两种不同的纯化方法对其蛋白纯化效率的影响,SDS-PAGE分析得出Ni-IDA树脂亲和层析对此融合蛋白纯化的特异性高,纯度达到85%左右。His-IL2融合蛋白的优化表达和纯化为进一步研究融合蛋白的活性与功能奠定了基础。
The aim of this study is to construct prokaryotic expression vector of human interleukin-2 （ IL-2）, to optimize expression conditions and to purify the expressed product. The gene fragment of IL-2 was ligated into the vector pET100/D-TOPO, and the recombinant plasmid was successfully constructed according to restriction endonuclease analysis and DNA sequencing. The plasmid was transformed into Eschrichia coli BL21 and induced to express protein. The analyses of SDS-PAGE and Western blot showed that the recombinant E. coli produced higher proportion of soluble protein contrast to inclusion body when induced for 5h by 0. 6 mmol/L of IPTG at 25~C, and the fusion protein could be recognized by specific antibodies. The fusion protein could be highly purified by Ni-IDAresin affinity chromatography, and the purity was up to about 85%. The purification of fusion protein provides a basis for further research on the activity and function of IL-2.
出处
《畜牧与兽医》
北大核心
2013年第10期21-25,共5页
Animal Husbandry & Veterinary Medicine
基金
国家转基因生物新品种培育重大专项(2011ZX08008-004)
国家自然科学基金青年基金项目(31101871)
江苏省科技支撑计划-社会发展项目(BE2013679)
