阴道毛滴虫rab1基因的克隆表达与蛋白质的细胞内定位

《阴道毛滴虫rab1基因的克隆表达与蛋白质的细胞内定位》由会员上传分享，免费在线阅读，更多相关内容在学术论文-天天文库。

1、汕头大学医学院03级研究生毕业论文目录中文摘要„„„„„„„„„„„„„„„„„„„„„„„„„„1英文摘要„„„„„„„„„„„„„„„„„„„„„„„„„„3前言„„„„„„„„„„„„„„„„„„„„„„„„„„5实验材料„„„„„„„„„„„„„„„„„„„„„„„„„„7实验方法„„„„„„„„„„„„„„„„„„„„„„„„„„9实验结果„„„„„„„„„„„„„„„„„„„„„„„„„„27讨论„„„„„„„„„„„„„„„„„„„„„„„„„„44结论„„„„„„„„„„„„„„„„„„„„„„„„„„50参考文献„„„„„„„„„„„„„„„„„„„„„„„

2、„„„51附录„„„„„„„„„„„„„„„„„„„„„„„„„„54致谢„„„„„„„„„„„„„„„„„„„„„„„„„„612汕头大学医学院03级研究生毕业论文阴道毛滴虫Rab1基因的克隆表达及蛋白质的细胞内定位2003级研究生徐晓园导师傅玉才教授摘要研究目的（1）克隆和分析原生动物阴道毛滴虫（Trichomonasvaginalis）两个Rab1鸟苷三磷酸酶（GuanosineTriphosphate,GTP）同源基因：TvRab1a和TvRab1-like；构建pQE80L/TvRab1a和pQE80L/TvRab1-like原核表达重组质粒并表达融合蛋白。（2）对

3、这两个TvRab1蛋白进行阴道毛滴虫细胞内定位，初步确定其功能。材料与方法（1）从已构建的阴道毛滴虫cDNA表达文库中分离出两个TvRab1的同源基因，测序并对其序列保守结构域进行同源性分析。用PCR方法扩增基因组DNA，并进行测序及序列分析。（2）将两段目的基因分别克隆至原核表达载体pQE80L中，并转化大肠杆菌E.coliM15感受态细胞，在异丙基硫代-β-D-半乳糖苷（isopropylthio-β-D-galactoside,IPTG）诱导下表达目的基因融合蛋白；Nｉ-NTA亲和层析纯化表达产物，SDS-PAGE鉴定表达产物的分子量及纯化效果。（3）用纯化并复性的融合

4、蛋白分别免疫豚鼠，获得重组蛋白抗血清；采用Westernblot法对抗体特异性进行分析鉴定。（4）利用所得抗体进行免疫荧光细胞化学实验，对TvRab1a及TvRab1-like蛋白进行阴道毛滴虫原虫细胞内定位。（5）用AvaⅡ、HhaⅠ和Hinp1Ⅰ核酸内切酶分别对阴道毛滴虫基因组DNA酶切，1%琼脂糖凝胶电泳检测酶切效果后，将所有酶切产物从凝胶转移至尼龙膜上，与标记的TvRab1a探针65℃进行Southern杂交。（6）提取阴道毛滴虫总RNA经1.2%FA琼脂糖凝胶电泳分离后，转移至尼龙膜，再与已标记的TvRab1a探针进行Northern杂交。结果（1）在已构建的cDN

5、A文库中发现2个Rab1鸟苷三磷酸酶同源基因。其中1个cDNA序列长707碱基对，读码框含606对碱基，推测蛋白质序列含有202个氨基酸。序列分析表明该氨基酸序列与Rab1a蛋白亚家族的同源性最高，我们将其命名为TvRab1a。另一cDNA序列长717对碱基，读码框含603对碱基，推测蛋白质序列具200个氨基酸。序列比对分析显示该氨基酸序列与Rab1a蛋白有较高的同源性，我们将其命名为TvRab1-like。两氨基酸序列都拥有Rab鸟苷三磷酸酶家族的所有保守结构域、特异性RabF结构域和典型的异戊烯化结构域。进化树分析提示毛滴虫的这两个基因系Rab1a的同源基因，在进化上它们

6、更接近-1-汕头大学医学院03级研究生毕业论文Rab1亚家族。以基因组DNA为模板扩增这两段序列并与其以ｃDNA序列进行比较发现这两个基因均含有一段25bp的内含子。（2）成功构建pQE80L/TvRab1a和pQE80L/TvRab1-like原核表达重组质粒，IPTG诱导表达，经SDS-PAGE凝胶电泳分析显示，表达重组质粒分别在宿主菌大肠杆菌M15内表达出预期大小的重组蛋白质。（3）经Ni-NTA亲和柱纯化并复性的两个重组蛋白分别免疫豚鼠，获得了重组蛋白的相应抗体。Westernblot反应发现获得的抗体分别能与相应的重组蛋白反应，并能在虫体蛋白提取液中检测到相应大小的

7、清晰条带，说明制备的抗体特异有效。（4）免疫荧光细胞化学实验结果表明两个蛋白均定位于原虫细胞的内质网及高尔基复合体区域。（5）SouthernBlot结果显示AvaⅡ酶切的基因组DNA杂交后有六条带，HhaⅠ酶切的基因组DNA杂交后有四条带。（6）NorthernBlot结果显示TvRab1amRNA大小为707bp。结论（1）成功分离和克隆出两个阴道毛滴虫Rab1a鸟苷三磷酸酶同源基因，并表达了这两个基因的重组蛋白，获得了这两个蛋白的多克隆抗体。（2）对这两个蛋白进行阴道毛滴虫细胞内定位，发现它们定位