摘要
目的对亚洲带绦虫真核翻译起始因子3(eukaryotic translation initiation factor3 subunit,EIFs 3)进行克隆及免疫学初步研究。方法利用在线生物信息学工具从亚洲带绦虫成虫cDNA文库中筛选出含有EIFs3基因,并将该基因克隆到原核表达质粒pET-28a(+)中,在大肠埃希菌BL21/DE3中诱导表达,表达产物通过十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)鉴定,重组后的蛋白用His-镍蛋白纯化柱纯化,纯化的重组蛋白用蛋白印迹(Western blotting)进行免疫学分析。结果 PCR,双酶切及DNA测序均显示重组体构建成功,用亲和层析法得到高纯度的蛋白,且该重组蛋白可被亚洲带绦虫及牛带绦虫及猪带病人血清识别,表明其具有免疫反应性。结论亚洲带绦虫成虫EIFs 33基因可在原核表达系统中获得具有免疫活性的高效表达。
Objective To clone and express the gene named as eukaryotic translation initiation factor 3 subunit（EIFs 3） of Taenia saginata asitica and to analyze the immunogenicity of its recombinant protein.Methods By online analysis of the gene at bioinfomatics websites,the gene from the Taenia saginata asiatica full-length cDNA plasmid libratory was identified and its coding region sequence was analyzed.Then the coding region of the gene was amplified with PCR.The recombinant protein was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis（SDS-PAGE） after being induced with isopropyl thiogalactoside（IPTG） in E coli BL21/DE3 and purified with NI-IDA affinity chromatography.In addition,the immunoreactivity of the purified recombinant proteins was analyzed with Western blotting.Results As demonstrated by PCR,double enzyme digestion and DNA sequencing of the recombinant plasmid was successively constructed.SDS-PAGE results showed that the gene was expressed in E coli BL21/DE3 and the recombinant protein could react with Taenia asiatica and taeniarhynchus saginatus infected patient＇ serum,which indicated its immunoreacticity.Condusion The EIFs 3 of Taenia saginata asitica was cloned and expressed,and the purified protein has immunogenicity.
出处
《中国公共卫生》
CAS
CSCD
北大核心
2010年第9期1146-1148,共3页
Chinese Journal of Public Health
基金
国家自然科学基金资助(30760227)
贵州省科技攻关项目(2008-3060)
贵州省科技攻关项目(2009-3101)
贵州省农业科技攻关项目[黔科合NY字(2009-3074)]
贵州省省长基金[黔省专合字(2009)82号]
贵阳市科技局社发攻关项目(2009-3005)
