中性白细胞抑制因子在大肠杆菌中的表达与纯化

Expression and purification of neutrophil inhibitory factor in E.coli

利用PCR技术扩增编码钩虫中性白细胞抑制因子(NIF)成熟肽的cDNA,克隆于表达载体pET-21a(+),序列分析表明与文献报道一致。经IPTG诱导,在大肠杆菌BL21(DE3)plys中实现高效可溶性表达。SDS-PAGE分析结果表明,外源蛋白(相对分子质量28900)约占全菌蛋白的20%。菌体用溶菌酶处理,上清经Q-SepharoseFF阴离子交换、羟基磷灰石层析、SephacrylS-100凝胶过滤,得到纯度约95%的重组NIF。活性测定结果表明,大肠杆菌表达的重组NIF能有效地抑制中性白细胞粘附。这些结果为利用大肠杆菌制备重组NIF奠定了基础。

The cDNA encoding mature protein of neutrophil inhibitory factor isolated from hookworm(Ancylostoma caninum)was amplified by PCR and cloned into expression vector pET-21a(+)between the sites of NdeI and Bam HI.Its cDNA sequence was determined.The recombinant plasmid was transformed into E.coli BL21(DE3)plys.The over-expression of recombinant neutrophil inhibitory factor,accounting for approximately20%of the total cell protein,was obtained after three hour induction with IPTG.After three step chromatography comprising of Q-Sepharose FF anion exchange,hydroxyapatite affinity and Sephacryl S-100gel filtration,the expressed protein was purified to approximately95%.It is indicated by neutrophil-plastic adhesion assay that recombinant neutrophil in-hibitory factor expressed in E.coli can efficiently inhibit the adhesion of neutrophils.It shows possibility that recom-binant neutrophil inhibitory factor can be produced on a large scale using E.coli.