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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 the truncated ubiquitin ligase TrCP-CC and its mutant △ F-CC, and to detect its expression. Methods The eukaryotic expression plasmids Migr1-TrCP-CC-HA and Migr1- △ F-CC-HA were constructed by PCR and overlap PCR. The recombinant plasmids were cloned into shuttle plasmid pAd-Track-CMV by PCR, Recombinant adenovirus plasmids pAd-TrCP-CC and pAd-ΔF-CC were obtained by homologous recombination with the backbone vector pAdeasy-1 and transfected into HEK293 cells by using the shuttle plasmid pAdTrack-TrCP-CC and pAdTrack- △ F- The recombinant adenovirus Ad-TrCP-CC and Ad-△ F-CC were amplified by PCR. The titers of the recombinant adenovirus Ad-TrCP-CC and Ad-△ F-CC were determined. The transcription level of the target gene was detected by RT- Results The recombinant plasmids pAd-TrCP-CC and pAd-ΔF-CC were constructed correctly by restriction enzyme digestion and sequencing. After HEK293 cells were packaged, the green fluorescence was observed under the microscope. The virus titers were 2.5 × 109 and 1.0 × 109pfu / L, the target gene can be effectively detected by RT-PCR. Conclusions The recombinant adenovirus Ad-TrCP-CC and Ad-△ F-CC have been successfully constructed and successfully expressed in HEK293 cells, which lays the foundation for further study of the mechanism of its targeting fusion protein Bcr-Abl ubiquitination and degradation.