犬贾第鞭毛虫PDI-4基因的表达及定位  

Prokaryotic expression of PDI-4 from Giardia canis and determination of its location

作　　者：吴娜[1] 吴威[1] 李玲丹[1] 宫鹏涛[1] 李建华[1] 杨举[1] 李赫[1] 张西臣[1] 

机构地区：[1]吉林大学动物医学学院,吉林长春130062

出　　处：《中国病原生物学杂志》2015年第1期50-53,共4页Journal of Pathogen Biology

基　　金：国家自然科学基金项目(No.31101804;30970322)

摘　　要：目的克隆、表达二硫键异构酶4基因,并研究其在犬贾第鞭毛虫滋养体中的定位。方法根据GenBank登陆的贾第鞭毛虫二硫键异构酶4基因(gPDI-4)(登录号XM_001710176)序列,通过分析序列,设计合成一对gPDI-4引物。以犬贾第鞭毛虫滋养体总RNA为模板,采用反转录PCR的方法扩增PDI-4基因并克隆至pMD-18T载体,构建pMD-gPDI-4载体,再把目的基因亚克隆至pET-28a载体,构建表达载体pET-gPDI-4,转化入大肠埃希菌表达菌DE3中,经IPTG诱导后用SDS-PAGE分析表达情况。同时用组氨酸(His)结合树脂柱纯化PDI-4蛋白。用纯化的PDI-4蛋白免疫小鼠,之后收集血清作为一抗,通过免疫荧光法检测PDI-4在贾第鞭毛虫滋养体中的定位。结果反转录PCR(RT-PCR)扩增出大小为1 065bp的PDI-4基因,构建的pET-gPDI-4能高效表达gPDI-4蛋白,SDS-PAGE显示表达蛋白分子质量为36ku。免疫荧光试验显示贾第鞭毛虫PDI-4蛋白除定位于细胞表面、内质网和腹部吸盘外,主要存在于腹部鞭毛。结论成功克隆、表达了犬贾第鞭毛虫PDI-4基因,并确定其主要在腹鞭毛表达。Objectives To clone and express the protein gPDI-4and to determine its location in Giardia canis trophozoites. Methods A pair of primers（gPDI-4）was designed and synthesized in accordance with the gPDI-4sequence in GenBank（XM_001710176）.The total RNA of trophozoites was extracted to amplify gPDI-4using RT-PCR.The identified gene was cloned into a pMD-18 Tvector to generate pMD-gPDI-4,and the gPDI-4fragment was then sub-cloned into apET-28 avector to construct the expression vector pET-gPDI-4.The recombinant plasmid pET-gPDI-4was transformed into BL21（DE3）for expression of the protein gPDI-4following induction with IPTG.Purified gPDI-4was obtained with a histidine-binding resin column.The purified gPDI-4protein was verified to be correct with Western blot,and this protein was used to generate polyclonal antibodies that were then used to locate gPDI-4in G.canis trophozoites based on immunofluorescence. Results The gPDI-4gene was amplified using RT-PCR and was successfully cloned into pET-28 a.Expression of gPDI-4protein was efficiently induced with IPTG.The protein had a relative molecular weight of about 36 ku.Immunofluorescence results indicated that gPDI-4was predominately located in the ventral flagella. ConclusionThe plasmid pET-gPDI4 was successfully constructed and expressed in DE3.The location of gPDI-4mainly in ventral flagella was verified with immunofluorescence.

关 键 词：犬贾第鞭毛虫 蛋白二硫键异构酶4 滋养体 免疫荧光定位 腹鞭毛 

分 类 号：R383.14[医药卫生—医学寄生虫学]