摘要
目的构建嵌合泛素连接酶TrCP-CC及其突变体△F-CC的重组腺病毒,并检测其表达。方法采用PCR和重叠PCR法构建真核表达质粒Migr1-TrCP-CC-HA和Migr1-△F-CC-HA,以此为模板,通过PCR将其克隆至穿梭质粒pAd-Track-CMV,构建重组腺病毒穿梭质粒pAdTrack-TrCP-CC和pAdTrack-△F-CC,再与骨架载体pAdeasy-1经同源重组得到重组腺病毒质粒pAd-TrCP-CC和pAd-△F-CC,转染HEK293细胞进行包装扩增,得到重组腺病毒Ad-TrCP-CC和Ad-△F-CC,测定其滴度,并经RT-PCR检测目的基因的转录水平。结果经酶切和测序证实重组腺病毒质粒pAd-TrCP-CC和pAd-△F-CC构建正确,经HEK293细胞包装后显微镜下可观察到绿色荧光,病毒滴度分别为2.5×109和1.0×109pfu/L,经RT-PCR检测目的基因可有效表达。结论已成功构建重组腺病毒Ad-TrCP-CC和Ad-△F-CC,并在HEK293细胞中成功表达,为进一步研究其靶向融合蛋白Bcr-Abl泛素化和降解的机制奠定了基础。
Objective To construct a recombinant adenovirus expressing chimeric ubiquitin-ligase TrCP-CC and its mutant △F-CC and determine the expressed products.Methods Eukaryotic expression vectors Migr1-TrCP-CC-HA and Migr1-△F-CC-HA were constructed by PCR and overlap PCR,and used as templates for amplification by PCR.The amplified target gene fragments were cloned into shuttle plasmid pAd-Track-CMV,and the constructed recombinant plasmids pAdTrack-TrCP-CC and pAdTrack-△F-CC were used for homologous recombination with skeleton vector pAdeasy-1.The obtained recombinant adenovirus vectors pAd-TrCP-CC and pAd-△F-CC were transfected to HEK293 cells for package and amplification.The obtained recombinant adenoviruses Ad-TrCPCC and Ad-△F-CC were determined for titers,in which the transcription levels of target genes were determined by RT-PCR.Results Both restriction analysis and sequencing proved that recombinant adenovirus vectors pAd-TrCP-CC and pAd-△F-CC were constructed correctly.Green fluorescence was observed in the HEK293 cells transfected with the recombinant adenovirus vectors.Recombinant adenoviruses Ad-TrCP-CC and Ad--△F-CC reached titers of 2.5 × 109 and 1.0 × 109 pfu /L respectively,in which target genes were expressed effectively as proved by RT-PCR.Conclusion Recombinant adenoviruses Ad-TrCP-CC and Ad-△F-CC were successfully constructed and expressed in HEK293 cells,which laid a foundation of study on the mechanism of targeting fusion protein Bcr-Ab1 for ubiquitination and degradation.
出处
《中国生物制品学杂志》
CAS
CSCD
2010年第6期561-565,共5页
Chinese Journal of Biologicals
基金
国家自然科学基金(30670901)
