十二指肠钩虫谷胱甘肽转移酶AduGST-1基因的克隆和重组表达

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。

Cloning and expression of AduGST-1, a glutathione S-transferase from Ancylostoma duodenale

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.