白色念珠菌谷氧还蛋白的表达纯化及多克隆抗体的制备  

Expression,purification,and preparation of polyclonal antibodies against Candida albicans glutaredoxin

作　　者：王巧[1] 谢霞 常帅[1] 周佳[1] WANG Qiao;XIE Xia;CHANG Shuai;ZHOU Jia(Department of Ultrasound Imaging,the First Affiliated Hospital of University of South China,Hengyang 421000,Hunan,China;Department of Infection Diseases,the First Affiliated Hospital of University of South China)

机构地区：[1]南华大学附属第一医院超声影像科,湖南衡阳421000 [2]南华大学附属第一医院感染科

出　　处：《中国病原生物学杂志》2020年第4期377-381,共5页Journal of Pathogen Biology

基　　金：湖南省技术创新引导计划项目(No.2017SK50205);2019湖南省卫计委课题项目(No.20190051)。

摘　　要：目的纯化白色念株菌(Candida albicans)谷氧还蛋白(glutaredoxin,Grx)并制备多克隆抗体。方法从NCBI网站搜索白色念株菌Grx蛋白序列,采用利用生物信息学方法分析其生物学特性。以白色念珠菌DNA为模板,PCR扩增Grx基因,双切后与质粒pET28a相连,连接产物pET28a-Grx转入表达菌BL21(DE3),使用IPTG诱导目的蛋白表达,使用亲和层析纯化目的蛋白。用Grx蛋白免疫小鼠,制备抗血清,进行Western blot和ELISA检测。结果白色念珠菌Grx蛋白含有一个CXXC结构域,属跨膜蛋白。其二级结构中58.8%的氨基酸为α-螺旋,12.6%为β片层,23.5%为无规卷曲,三级结构与酵母Grx蛋白结构相似。Grx蛋白具有多个B细胞表位。获得Grx基因并成功构建重组质粒pET28a-Grx,转染DE3后表达15.2×103的重组蛋白。用纯化的重组Grx蛋白免疫小鼠,获得的多克隆抗体ELISA效价为1:10240。结论成功表达白色念株菌Grx蛋白并制备了高滴度的多克隆抗体,为Grx的活性研究奠定了实验基础。Objective To purify recombinant Candida albicans glutaredoxin(Grx)and prepare polyclonal antibodies.Methods The amino acid sequence of the Grx protein of C.albicans was obtained from the NCBI website,and its biological characteristics were analyzed bioinformatically.The Grx gene was amplified using PCR with C.albicans DNA serving as a template.The gene was double digested and ligated to the plasmid pET28ato produce pET28a-Grx.The recom-binant plasmid pET28a-Grx was transformed into the expression strain E.coli BL21(DE3),and expression of the target protein was induced with IPTG.The protein of interest was purified using affinity chromatography.Mice were immunized with the Grx protein,serum was separated,and the antibody titer was determined.Results The C.albicans Grx protein contains a CXXC domain and is a transmembrane protein.Its secondary structure consists ofα-helices(58.8%),β-sheets(12.6%),and random coils(23.5%).Its tertiary structure is similar to that of yeast Grx protein.The Grx pro-tein also has multiple B-cell epitopes.The Grx gene was obtained,and the recombinant plasmid pET28a-Grx was successfully constructed;the plasmid codes for a 15.2-kDa recombinant protein.Highly pure recombinant Grx protein was obtained and was able to induce a high titer of polyclonal antibodies in mice.Conclusion The Grx protein of C.albicans was successfully expressed and quality polyclonal antibodies were prepared.These results provide an experimental basis for study of the activity of Grx.

关 键 词：白色念珠菌 谷氧还蛋白 蛋白纯化 多克隆抗体 

分 类 号：R379.4[医药卫生—病原生物学]