阴道毛滴虫Rab1a重组蛋白的表达和细胞内定位

Expression of Recombinant TvRab1a and Intracellular Location of TvRab1a in Trichomonas vaginalis

目的制备阴道毛滴虫(Trichomonasvaginalis,Tv)Rab1a重组蛋白及其多克隆抗体,并对TvRab1a蛋白进行阴道毛滴虫的细胞内定位。方法将我们已构建的pQE80L/TvRab1a重组表达质粒转入大肠埃希菌E.coliM15,在IPTG诱导下表达重组蛋白,经Ni-NTA亲和柱层析后获得纯度较高的TvRab1a的重组蛋白;用经复性处理的重组蛋白免疫动物,获得TvRab1a重组蛋白抗血清,免疫印迹WesternBlot鉴定抗血清。采用荧光免疫细胞化学对TvRab1a蛋白进行细胞内定位。结果WesternBlot分析显示,TvRab1a重组蛋白可与豚鼠的抗TvRab1a血清反应,同时该抗血清在滴虫提取蛋白中检测到与预测TvRab1a分子量一致的条带;免疫荧光化学检测发现TvRab1a分布于细胞核周围的高尔基复合体与内质网。结论获得的抗TvRab1a蛋白的多抗血清可用于TvRab1a基因功能的研究;TvRab1a功能场所位于高尔基复合体与内质网。

Objective To express recombinant TvRabla protein and analyze the intracelluar location of native TvRabla in Tricomonas vaginalis. Method The TvRabla recombinant protein was expressed in E.coli strain M15 ceils. The fusion protein was purified by affinity chromatography on a Ni-NTA agarose column and analyzed by SDS-PAGE. The antiserum was prepared by immunizing a guinea pig with purified TvRabla protein and tested by Western-blot, The intra-cellular location of TvRabla was determined by fluoroimmunocytochemistry with the anti-serum. Result SDS-PAGE analysis showed that the fusion protein had a molecular weight of 23 000 u. Western-blot showed that the anti-serum reacted with the TvRabla fusion protein, and the native protein of T.vaginali. Immunofluorescence analysis demonstrated that the TvRabla protein was localized in the region of ER and Glogi apparatus of the protist ceils, which was consistent with previous studies. Conclusion The TvRabla antiserum could be used for studying the role of Rabla in T.vaginalis The native protein of Rabla was mainly expressed in the regions of ER and Golgi apparatus of the protist cells.