十二指肠钩虫巨噬细胞迁移抑制因子基因的克隆和表达

Cloning and expression of AduMIF-1,a macrophage migration inhibitory factor from Ancylostoma duodenale

目的克隆、表达十二指肠钩虫巨噬细胞迁移抑制因子(MIF)AduMIF-1基因。方法设计、合成特异引物,以十二肠钩虫成虫cDNA为模板,通过PCR扩增AduMIF-1基因。将获得的AduMIF-1编码序列克隆至原核表达载体pET32a,构建重组表达质粒pET32a/AduMIF-1。重组表达质粒转入大肠埃希菌BL21(DE3)中,IPTG诱导表达并分离纯化重组AduMIF-1。结果成功扩增到AduMIF-1全长编码序列,完整阅读框长度为360bp,编码119个氨基酸。构建了重组表达质粒pET32a/AduMIF-1,经IPTG诱导表达和分离、纯化,获得了重组AduMIF-1,融和蛋白分子质量单位约为33ku。结论本研究从十二指肠钩虫中分离到MIF基因,并成功进行了重组表达、分离与纯化,为进一步研究AduMIF-1的生物学功能奠定了基础。

Objective To clone and express AduMIF-1,a macrophage migration inhibitory factor（MIF）,from the hookworm Ancylostoma duodenale.Methods The nucleotide sequence encoding AduMIF-1 was amplified by PCR from the adult A.duodenale cDNA library and cloned to construct the recombinant plasmid pET32a/AduMIF-1.The recombinant AduMIF-1 fusion protein was expressed in E.coli BL21（DE3） and purified by Ni-NTA affinity chromatography.Results Full-length cDNA encoding AduMIF-1 was obtained from A.duodenale.The open reading frame of AduMIF-1 consisted of 360 nucleotides that encoded 119 amino acids.The AduMIF-1 fusion protein with a MW of 33 ku.was successfully expressed in E.coli after induction with IPTG and purification by Ni-NTA affinity chromatography.Conclusion A MIF from A.duodenale was cloned,expressed,and purified in this study,thus contributing to the further study of the biological function of AduMIF-1.