摘要
目的制备具有高免疫原性的白念珠菌重组烯醇化酶蛋白质。方法以白念珠菌C1标准株基因组DNA作为模板,用PCR法扩增烯醇化酶的全长DNA序列,以pET28a(+)为载体,构建重组表达质粒,转化大肠埃希菌BL21(DE3),IPTG诱导重组融合蛋白质表达。用抗his标签的单克隆抗体和白念珠菌抗体阳性的病人血清进行western blot鉴定,亲和层析柱纯化重组蛋白质。结果获得了含白念珠菌烯醇化酶全长基因的重组表达载体和相应工程菌株,经IPTG诱导后能高效表达重组融合蛋白质。经已含抗体的病人血清进行western blot鉴定,表明诱导的重组蛋白质有良好的免疫原性。结论成功克隆了白念珠菌烯醇化酶全长基因并在大肠埃希菌中获得高效表达;重组蛋白质具有良好的免疫原性。
Objective To generate a highly antigenic recombinant enolase of the human opportunistic pathogen Candida albicans.Methods The full-length coding sequence of enolase was amplified by PCR from the genomic DNA of C.albicans and cloned into the prokaryotic expression vector pET28a（＋ ） .A His6-tagged enolase was produced under the induction of IPTG in E.coli BL21（DE3）,analyzed by SDS-PAGE,and purified by TALON metal affinity resins.The antigenicity of the recombinant protein was evaluated by western blotting with sera from systemic candidiasis patients.Results The full-length gene of C.albicans enolase was cloned and recombinant enolase was highly expressed in E.coli.Western blotting showed that the recombinant protein specifically reacted with the antibody in sera from systemic candidiasis patients.Conclusions A satisfactory antigenic recombinant enolase of C.albicans has been expressed and purified,which will be helpful to develop novel specific diagnostic reagents.
出处
《临床检验杂志》
CAS
CSCD
北大核心
2010年第5期353-355,共3页
Chinese Journal of Clinical Laboratory Science
基金
江苏省科技支撑计划-社会发展项目(BE2009673)
江苏省博士后基金资助项目(0802032c)
关键词
白念珠菌
烯醇化酶
基因克隆
原核表达
Candida albicans
enolase
gene cloning
prokaryotic expression
