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目的克隆十二指肠钩虫(Ancylostoma duodenale)热休克蛋白60(heat shock protein 60,HSP60)基因,并在大肠埃希菌中表达获得重组AduHSP60蛋白。方法以十二肠钩虫成虫cDNA为模板,PCR扩增AduHSP60基因。将获得的目的基因编码序列连接至原核表达载体pETHF,构建重组表达质粒pETHF/AduHSP60。重组质粒转化大肠埃希菌BL21(DE3),经IPTG诱导表达、Ni-NTA亲和层析分离纯化重组蛋白,SDS-PAGE分析重组蛋白的表达及纯化情况。结果成功扩增到AduHSP60全长编码序列,编码序列长度为1 701bp,编码566个氨基酸。构建了重组表达质粒pETHF/AduHSP60,经诱导表达、分离纯化获得了分子质量单位为60ku的可溶性重组AduHSP60蛋白。结论本研究从十二指肠钩虫中分离获得了HSP60基因,构建的pETHF/AduHSP60重组质粒能在大肠埃希菌中表达重组蛋白,为进一步研究AduHSP60的生物学功能奠定了基础。
Objective To clone the heat shock protein 60 (HSP60) gene of Ancylostoma duodenale and obtain the recombinant AduHSP60 protein in Escherichia coli. Methods The cDNA of AduHSP60 was amplified by PCR from the cDNA of adult C. elegans. The obtained target gene coding sequence was ligated to the prokaryotic expression vector pETHF to construct the recombinant expression plasmid pETHF / AduHSP60. The recombinant plasmid was transformed into Escherichia coli BL21 (DE3) and induced by IPTG. The recombinant protein was purified by Ni-NTA affinity chromatography and the expression of recombinant protein was analyzed by SDS-PAGE. Results The full-length coding sequence of AduHSP60 was successfully amplified. The coding sequence was 1 701 bp in length and encoded 566 amino acids. The recombinant expression plasmid pETHF / AduHSP60 was constructed and induced to express. The soluble recombinant AduHSP60 protein with molecular weight of 60 ku was obtained. Conclusion The HSP60 gene was isolated from the duodenal hookworm and the constructed recombinant plasmid pETHF / AduHSP60 could express the recombinant protein in Escherichia coli, which laid the foundation for further study on the biological function of AduHSP60.