【摘要】 目的克隆十二指肠钩虫谷胱甘肽转移酶(GST)AduGST-1基因,并在大肠埃希菌中表达获得重组AduGST-1。方法设计特异引物,以十二肠钩虫成虫cDNA为模板,通过PCR扩增AduGST-1基因。将获得的AduGST-1编码序列克隆至原核表达载体pETHF,构建重组表达质粒pETHF/AduGST-1。重组质粒转化至大肠埃希菌BL21(DE3),用IPTG诱导表达、Ni亲和层析分离纯化重组AduGST-1,SDS-PAGE分析重组蛋白表达及纯化情况。结果成功扩增到AduGST-1全长编码序列,并登记到GenBank(accession no.JQ812812)。AduGST-1编码序列长度为624bp,编码307个氨基酸残基。成功构建了重组表达质粒pETHF/AduGST-1,在BL21(DE3)中表达并纯化了重组AduGST-1。结论首次报道从十二指肠钩虫中分离到GST基因,该基因可在大肠埃希菌中高效表达,并分离纯化了重组GST蛋白,为进一步研究AduGST-1功能与应用奠定了基础。更多还原

【Abstract】 Objective To clone and express AduGST-1,a glutathione S-transferase(GST) from the human hookwrom Ancylostoma duodenale.Methods The nucleotide sequence encoding AduGST-1 was amplified by PCR from adult A.duodenale cDNA and used to construct the recombinant plasmid pETHF /AduGST-1.The recombinant AduGST-1 was expressed in E.coli BL21(DE3) and purified by Ni-NTA affinity chromatography.Results The full length of cDNA encoding AduGST-1 was obtained from A.duodenale and deposited in GenBank(accession no.JQ812812).It was composed of 624 nucleotides and encoded 307 amino acids.The recombinant plasmid pETHF /AduGST-1 was successfully constructed.The recombinant AduGST-1 was successfully expressed in E.coli,purified by Ni-NTA affinity chromatography and analyzed by SDS-PAGE.Conclusion It was the first time cloning,expression and purification of a GST in A.duodenale.This study will contribute to the further study on the functions and applications of AduGST-1.更多还原