【摘要】 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。更多还原

【Abstract】 Objective To clone and express heat shock protein 60(AduHSP60) of the hookworm Ancylostoma duodenale.Methods The nucleotide sequence encoding AduHSP60 was amplified by PCR from adult A.duodenale cDNA and ligated into vector pETHF to construct the recombinant plasmid pETHF/AduHSP60.The recombinant plasmid was transformed into E.coli BL21(DE3) and induced with IPTG.The recombinant AduHSP60 was purified with Ni-NTA affinity chromatography.Results Full-length cDNA encoding AduHSP60 was successfully amplified.AduHSP60 consisted of 1,701 nucleotides and encoded 566 amino acids.The recombinant plasmid pETHF/AduHSP60 was constructed and the recombinant protein was expressed with IPTG.A protein with a molecular weight of about 60 ku was obtained and mainly existed in a soluble form.Conclusion The HSP60 gene in A.duodenale was isolated in this study and the recombinant AduHSP60 was expressed and purified in E.coli.This work has laid a foundation for further study of the function of AduHSP60.更多还原