α1-Giardin的原核表达与多克隆抗体制备  

Prokaryotic expression of Giardia α1-giardin and preparation of polyclonal antibodies

作　　者：李瑶[1] 张宏梅[1] 时文艳[1] 梁宸[1] 李垚艳 冯宪敏[1] 

机构地区：[1]吉林医药学院,吉林吉林132013

出　　处：《中国病原生物学杂志》2016年第4期334-337,共4页Journal of Pathogen Biology

基　　金：国家自然科学基金项目(No.81301450;31572262);吉林省科技厅国际合作项目(No.20160414050GH);吉林市杰出青年基金项目(No.201464037)

摘　　要：目的原核表达α1-Giardin并制备兔抗rα1-Giardin多克隆抗体。方法以蓝氏贾第鞭毛虫DNA为模板,PCR扩增α1-giardin编码基因片段,经双酶切后连入原核表达载体pET-41a(+),构建重组表达载体pET-41a(+)-α1-giardin,热激法转化大肠埃希菌E.coli BL21(DE3),经IPTG诱导后收集菌体并裂解,采用SDS-PAGE检测目的蛋白的表达,采用His-tag亲和层析柱纯化融合蛋白。将α1-Giardin蛋白与等体积弗氏完全佐剂乳化后免疫新西兰白兔,隔周以同样剂量蛋白加等体积弗氏不完全佐剂加强免疫2次。末次免疫后一周颈动脉取血,制备多抗血清,采用间接ELISA法测定抗体效价。结果成功构建原核表达载体pET-41a(+)-α1-giardin,转化大肠埃希菌后经IPTG诱导表达分子质量单位为34.8ku的可溶性蛋白;经His-tag亲和层析柱纯化获得高纯度的重组α1-giardin蛋白,制备的免疫兔抗血清ELISA滴度为1∶51 200。结论成功克隆、表达并纯化了贾第虫α1-giardin蛋白,制备了高滴度的抗α1-giardin多克隆兔血清,为以α1-Giardin为抗原的疫苗研究奠定了基础。Objectives To express Giardiaα1-giardin in E.coli BL21 and prepare polyclonal antibodies. MethodsThe gene encoding Giardiaα1-giardin was amplified with PCR using Giardia DNA as a template.The product was digested with a restriction enzyme and ligated into the plasmid pET-41a（＋）.The recombinant expression vector pET-41a（＋）-α1-giardin was transformed into E.coli BL21（DE3）for expression via induction with IPTG.Expression of the soluble protein and the molecular weight of that protein were analyzed with SDS-PAGE.The recombinant protein was purified using His-tag affinity chromatography.New Zealand white rabbits were immunized with purified rα1-Giardin protein with an equal volume of Freund＇s complete adjuvant（CFA）and boosted twice（every other week）with the same dose of protein plus an equal volume of Freund＇s incomplete adjuvant（IFA）.A week after final immunization,serum was collected and the antibody titer was determined with ELISA. Results The recombinant expression vector pET-41a（＋）-α1-giardin was successfully constructed and rα1-giardin was efficiently expressed as a soluble protein in E.coli BL21.In SDSPAGE,purified rα1-giardin produced a single band with a molecular mass of 34.8ku.ELISA indicated the titer of anti-α1-giardin rabbit serum was 1：51 200. Conclusion This study successfully cloned,expressed,and purified Giardiaα1-giardin protein and it prepared antiα1-giardin polyclonal rabbit serum.These results have laid the foundation for further vaccine research usingα1-Giardin as antigen.

关 键 词：蓝氏贾第鞭毛虫 α1-贾第素 原核表达 多克隆抗体 

分 类 号：R382.2[医药卫生—医学寄生虫学]