中文摘要：

目的：构建Flag-RNF8真核表达质粒，证实融合蛋白在前列腺癌细胞内的表达与定位。方法：提取人前列腺癌细胞CWR22RV1总RNA，反转录为cDNA。PCR扩增RNF8全长编码区cDNA序列，亚克隆至含有Flag标签的真核表达载体中。将构建的重组质粒测序并转染到前列腺癌细胞CWR22RV1中，提取细胞蛋白进行Western blot检测，同时利用共聚焦激光扫描显微镜观察Flag-RNF8在CWR22RV1细胞内定位。使用免疫沉淀的方法纯化RNF8蛋白。结果：RNF8蛋白全长编码区cDNA克隆到了真核表达载体pcDNA3-Flag中，酶切鉴定片段约为1500bp。Western blot检测到了融合蛋白Flag-RNF8的表达，分子量约为57kDa。Flag-RNF8在CWR22RV1细胞中表达并定位在细胞核中。成功纯化RNF8蛋白。结论：成功构建了Flag-RNF8全长编码区cDNA的真核表达质粒；鉴定了Flag-RNF8融合蛋白的表达并纯化RNF8蛋白；在CWR22RV1细胞中，Flag-RNF8蛋白主要定位在细胞核中。为进一步研究RNF8的生物学特性及其功能奠定了基础。

英文摘要：

Objective：To construct the expression plasmid of Flag- RNF8 and identify the expression and localization of this recombinant protein,and purify the RNF8 protein, nethods：Total RNA was extracted from human prostate cancer CWR22RV1 cells. The RNF8 coding sequence was amplified by polymerase chain reaction （PCR） method and subcloned into pcDNA3 - Flag vector. After the target region was sequenced, the plasmid was transfected into CWR22RV1 cell line. The expression of the recombinant plasmid in CWR22RV1 cells was proved by Western blot. The localization of Flag - RNF8 in CWR22RV1 ceils was observed by using laser scanning confocal microscopy. Purify the RNF8 protein by immunopreeipitation. Results： RNF8 had been constructed into expressing vector pcDNA3 Flag successfully. The length of the fragment was 1500bp,identified by restriction enzymes digestion. The expression of Flag - RNF8 fusion protein was detected by Western blot with a molecular weight 57kDa. Flag - RNF8 localized in the nucleus. Conclusion：The recombinant plasmid was successfully cloned into eukaryotic expressing vector. The expression of Flag - RNF8 fusion protein was identified and pulled down by Flag antibody . Flag - RNF8 was expressed in nucleus.