【摘要】 目的:构建含锌指蛋白ZBTB20基因的重组腺病毒载体,以研究锌指蛋白ZBTB20的生物学功能。方法:将带有FLAG标签的ZBTB20 cDNA片段(ZBTB20-FLAG)克隆至pAdTrack-CMV载体,将该载体与pAdEasy质粒进行细菌内同源重组从而获得重组腺病毒载体pAd-ZBTB20,之后在293细胞中进行包装以及扩增,并对病毒滴度进行检测;采用肝脏组织特异性ZBTB20基因敲除小鼠的原代肝细胞和肝脏组织模型,分别于离体和在体水平鉴定所制备的重组腺病毒Ad-ZBTB20介导的ZBTB20蛋白表达情况;并采用报告基因技术检测过表达的ZBTB20蛋白的功能活性。结果:成功制备了锌指蛋白ZBTB20重组腺病毒,该重组腺病毒感染原代肝细胞和小鼠肝脏组织能过表达ZBTB20蛋白,并且此ZBTB20蛋白能抑制其下游靶基因甲胎蛋白的转录。结论:所构建的ZBTB20重组腺病毒可以介导ZBTB20的过表达并具备转录调节活性,为今后研究ZBTB20的相关生物学功能奠定了基础。更多还原

【Abstract】 AIM: To construct the recombinant adenovirus vector containing zinc finger protein ZBTB20 gene for the further study of the biological function of ZBTB20. METHODS: The ZBTB20 cDNA fragment with FLAG tag was cloned into pAdTrack-CMV vector. The linearized vector was introduced into pAdEasy-1 / BJ5183 bacterial cells for generating homologous recombinant adenovirus plasmids,and was further transfected into HEK-293 packaging cells for the production of Ad-ZBTB20. The primary hepatocytes were used in the experiment. The expression and activity of ZBTB20 protein were determined by Western blotting and reporter gene assay after Ad-ZBTB20 infection. The expression of ZBTB20 protein was also examined in the liver tissue of liver-specific ZBTB20 knockout mice injected with Ad-ZBTB20 via the tail vein. RESULTS: Recombinant adenovirus Ad-ZBTB20 with high titer was successfully generated. Over-expression of ZBTB20 mediated by the recombinant was detected both in vitro and in vivo,with the activity of inhibiting the transcription of its target gene alpha-fetoprotein. CONCLUSION: Recombinant adenovirus Ad-ZBTB20 over-expresses ZBTB20 protein with the transcription regulation activity,which is a useful tool for investigating the biological role of ZBTB20.更多还原