中性白细胞抑制因子在大肠杆菌中的表达与纯化

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—Sepharose FF阴离子交换、羟基磷灰石层析、Sephacryl S-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。

Expression and purification of neutrophil inhibitory factor in E. coli

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.