EoRab43参与游仆虫细胞内大核周围的物质运输  

EORAB43 REGULATING VESICULAR TRANSPORT AROUND THE MACRONUCLEUS IN EUPLOTES OCTOCARINATUS

作　　者：李江姣[1] 聂宇[1] 梁爱华[1] 党旭红[1] 王伟[1] 

机构地区：[1]山西大学生物技术研究所化学生物学与分子工程教育部重点实验室,太原030006

出　　处：《分子细胞生物学报》2009年第1期35-42,共8页Journal of Molecular Cell Biology

基　　金：国家自然科学基金(30770295;30670282);高等学校博士点专项科研基金(20050108003);山西省自然科学基金(2008011063)资助~~

摘　　要：Rab家族蛋白是真核细胞内膜泡运输途径中重要的调节因子。EoRab43是八肋游仆虫中一种编码非典型Rab蛋白的基因。本研究依据已获得的EoRab43基因序列设计引物,从八肋游仆虫大核DNA中扩增了EoRab43基因的3′端153bp片段,即EoRab43153bp(对应于EoRab43蛋白的C末端50个氨基酸,EoRab43C),构建重组表达质粒pGEX-EoRab43153bp转化大肠杆菌BL21(DE3)进行表达,纯化后的融合蛋白GST-EoRab43C免疫BALB/c小鼠制备多克隆抗体。经检测,制备的抗体具有较高的效价及良好的特异性。利用制备的抗体对EoRab43在游仆虫细胞内进行免疫荧光定位,结果显示该蛋白主要定位于该生物细胞内大核染色体的周围。Rab family proteins play a crucial role in regulating vesicular traffic in eukaryotic cells. EoRab43 is an atypical Rab gene identified in Euplotes octocarinatus. In order to understand the function of EoRab43, the 153bp fragment of the 3＇-end of EoRab43 gene was subcloned into expression vector pGEX-6P-1, and the recombinant plasmid pGEX-EoRab43153bp was transfered into E.coli BL21 （DE3） to express the fusion protein. The fusion protein GST-EoRab43C was expressed and purified by affinity chromatography. BALB/c mice were immunolized by purified GST-EoRab43C. The titer of anti-EoRab43C polyclonal antibody was detected by in- direct ELISA assay and the specificity of the antibody was detected by Western blot. Immunofluorescence experiments were performed using anti-EoRab43C antibody in the cells of Euplotes. The results showed that EoRab43 displayed a punctuate pattern in the cytoplasm around the macronuclear chromosome of Euplotes.

关 键 词：EoRab43 八肋游仆虫 大核 免疫荧光定位 膜泡运输 

分 类 号：Q51[生物学—生物化学]