小鼠LRRC10腺病毒载体构建及其在心肌细胞中的表达

Construction of Mouse Lrrc10 Adenovirus Vector and Analysis of Its Expression in Cadiomyocytes

目的:构建含有小鼠Lrrc10(Leucine-rich Repeat Containing protein10)基因的重组腺病毒表达载体。方法:设计小鼠Lrrc10特异性引物,以小鼠cDNA为模板,通过PCR扩增出mLrrc10的编码区,并引入HA标签蛋白和SalΙ酶切位点。该片段经凝胶电泳纯化后插入pMD-18T载体。测序后,用SalΙ和Hind III酶切,将目的片段亚克隆至pAd-track-cmv穿梭载体中。用PmeΙ线性化后,用100 ng转化细菌BJ5183,在细菌内同源重组后得到pAd-Lrrc10质粒。pAd-Lrrc10经PacⅠ线性化后用LipofectamineTM 2000转染293A细胞,包装得到含Lrrc10基因的病毒重组子。将病毒重组子在293A细胞中扩增后,反复冻融得到滴度较高的含Lrrc10的病毒液。将收集的病毒液感染心肌细胞,绿色荧光观察GFP、免疫印迹检测Lrrc10-HA蛋白的表达。结果:用病毒液感染原代心肌细胞,24小时后在荧光显微镜下可观察被感染的细胞发出绿色荧光,提取心肌细胞总蛋白,Western可检测到Lrrc10-HA融合蛋白的表达。结论:小鼠Lrrc10腺病毒载体构建成功,并可将编码Lrrc10-HA的目的片段导入心肌细胞中表达。

Objective：To construct the recombinant adenovirus expression vector of mouse Leucine-rich Repeat Contai-ning protein 10 （Lrrc10）.Methods：Specific primers for the mouse Lrrc10 gene were designed and were employed to amplify the coding sequence using mouse cDNA as template.HA-tagged protein and a SalΙrestriction enzyme site were induced into the PCR primers.The PCR product was purified after gel electrophoresis and inserted into the pMD-18T vector.After sequencing,the positive plasmid was digested with Sal Ιand Hind III and the target fragment was sub-cloned into pAd-track-cmv shuttle vector.The recombinant plasmid was linearized with restriction enzyme PmeΙ.About 100 ng of the purified linear fragment was transformed into bacteria BJ5183.After homologous recombination in the bac-teria,plasmid pAd-Lrrc10 was obtained.pAd-Lrrc10 was linearized with restriction enzyme PacⅠ and transfected into 293A cells with LipofectamineTM2000.Then,the recombinant virus containing Lrrc10 was obtained after packaging. The virus was amplified in 293A cells and then the cells were repeatedly freezed and thawed to get higher titers virus flu-id containing Lrrc10.Cardiomyocytes were infected with the collected virus fluid.To detect the expression of Lrrc10-HA,green fluorescence protein （GFP）was observed by green fluorescence and immunoblotting was performed.Re-sults：Green fluorescent could be detected by fluorescence microscope in the primary cardiomyocytes infected with the vi-rus fluid before 24 h.The total protein of myocardial cells was extracted and the expression of Lrrc10-HA fusion protein could be detected by Western blot..Conclusion：The mouse Lrrc10 adenovirus vector was constructed successfully,and it could induce the sequence coding Lrrc10-HA into cardiomyocytes.