十二指肠钩虫热休克蛋白HSP60基因的克隆及重组表达  

Cloning and expression of AduHSP60,a heat shock protein from Ancylostoma duodenale

作　　者：许琴英[1] 邵正[1] 邓莉[1] 何庆丰[1] 彭礼飞[1] 

机构地区：[1]广东医学院寄生虫学暨临床寄生虫检验学教研室,广东湛江524023

出　　处：《中国病原生物学杂志》2013年第7期624-627,共4页Journal of Pathogen Biology

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。Objective To clone and express heat shock protein 60 （AduHSP60） of the hookworm Ancylostoma duode- nale. Methods The nucleotide sequence encoding AduHSP60 was amplified by PCR from adult A. duodena＆ 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 af finity chromatography. Results Full-length cDNA encoding AduHSP60 was successfully amplified. AduHSP60 consis ted of 1,701 nucleotides and encoded 566 amino acids. The recombinant p[asmid 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. duodena＆ was isolated in this study and the re- combinant AduHSP60 was expressed and purified in E. coll. This work has laid a foundation for further study of the function of AduHSP60.

关 键 词：十二指肠钩虫 热休克蛋白60 克隆 原核表达 

分 类 号：R383.13[医药卫生—医学寄生虫学]