阴道毛滴虫RRas基因序列的cDNA一步克隆、表达及鉴定  

One-step cloning,expression,and detection of Trichomonas vaginalis RRas cDNA

作　　者：王文柯[1] 贾鑫[1] 曹磊[1] 王黎[1] 薛长贵[1] 

机构地区：[1]郑州大学基础医学院人体寄生虫学教研室,河南郑州450001

出　　处：《中国病原生物学杂志》2016年第11期999-1003,共5页Journal of Pathogen Biology

摘　　要：目的一步克隆、表达及鉴定阴道毛滴虫RRas基因,以了解其在组织细胞定位和功能。方法用PCR方法从阴道毛滴虫cDNA文库中扩增RRas片段,用一步克隆法构建RRas-PQE-80L载体,进行菌落PCR和酶切鉴定,阳性克隆提取质粒,经测序鉴定后转化至大肠埃希菌(E.coli)BL21,经异丙硫代-β-D-半乳糖苷(IPTG)诱导。表达产物用NiAgarose His纯化试剂盒纯化,纯化产物经SDS-PAGE电泳鉴定和Western blot分析。结果从阴道毛滴虫cDNA表达文库中扩增的RRas基因片段长617bp,酶切及DNA测序证实RRas-PQE-80L重组质粒构建正确。表达的融合蛋白分子质量单位约为23.6ku,该蛋白能被抗His标签抗体识别。结论成功构建重组原核表达质粒RRas-PQE-80L载体,并能在E.coli BL21中表达,表达蛋白具有反应原性。Objective To clone,express,and identify the RRas gene of Trichomonas vaginalis in order to further study its location and functions in tissues and cells. Methods A cDNA fragment of T.vaginalis RRas was amplified with PCR.In one step,the fragment was cloned into a PQE-80 LEasy vector.Clones with the PQE-80L/T.vaginalis RRas gene were identified with PCR and restriction enzyme digestion and then sequenced.E.coli BL21 cells were transformed with the recombinant plasmid.IPTG was used to induce expression of the plasmid in the host bacterium.The plasmid was purified with an Ni-Agarose His Purification Kit and identified with SDS-PAGE and Western blotting. Results A TvRRas cDNA fragment that was 617 bp in length was amplified with PCR and cloned into a PQE-80 LEasy vector.The constructed vector for expression of PQE-80L/T.vaginalis RRas was identified through double restriction enzyme（HindIII and BamHI）digestion and nucleotide sequencing.The fusion protein（about 23 600u）was expressed in E.coli and purified. Conclusion The vector PQE-80L/T.vaginalis RRas was constructed,the protein of interest was successfully expressed in BL21 and recognized by anti-His-tagged monoclonal antibody.

关 键 词：阴道毛滴虫 RRas基因 一步克隆 原核表达 WESTERN BLOT 

分 类 号：R382.21[医药卫生—医学寄生虫学]