KLF15基因表达特征及其腺病毒超表达载体的构建

Expression characteristics of KLF15 gene and construction of its adenovirus vector

【目的】分析锌指蛋白转录因子15(KLF15)在小鼠各组织及C2C12细胞分化过程中的表达情况,构建KLF15的腺病毒超表达载体。【方法】采集小鼠肝、脾、肾、脂肪、骨骼肌及培养的C2C12成肌细胞,提取不同分化时期(0,2,4d)的C2C12细胞及不同组织的RNA,检测KLF15的表达情况;过夜连接HindⅢ和XbaⅠ双酶切后的pAdTrack-CMV与KLF15真核表达质粒(pcDNA3.1-KLF15),获得pAdTrack-KLF15质粒。将该质粒经PmeⅠ线性化后转入BJ5183感受态细胞进行重组,构建pAdEasy-KLF15腺病毒超表达载体。将pAdEasy-KLF15质粒PacⅠ酶切线性化回收后,转染293A细胞进行包装、扩繁和纯化。将获得的KLF15重组腺病毒侵染C2C12细胞,实时荧光定量PCR检测其对KLF15表达的影响。【结果】KLF15在C2C12成肌细胞分化过程中表达显著升高,并在分化后期达最高值;KLF15在肝脏、肾脏和脂肪中的表达水平极显著高于骨骼肌和脾脏。成功获得了pAdEasy-KLF15腺病毒超表达载体,其侵染C2C12细胞后能显著上调KLF15mRNA的表达水平。【结论】KLF15基因在C2C12成肌细胞中高表达,并成功构建了KLF15重组腺病毒载体。

[Objective] The aims of this study were and differentiation of C2C12 cell of mice and conduct to explore the expression of KLF15 gene in tissues its adenovirus vector. [Method] Firstly, tissues of mice and C2C12 cells in different differentiation phases were collected to extract RNA for detecting expression of KLF15 gene. Secondly,the target fragment obtained from pcDNA3.1-KLF15 vector was connected to pAdTrack-CMV after being digested by Hind Ⅲ and Xba I to obtain pAdTrack-KLF15 vector. The pAdTrack-KLF15 vector was linearized by Pine I and transformed into BJ5183 competent cells with pAdEasy to construct pAdEasy-KLF15. After the pAdEasy-KLF15 vector was digested by Pac I ,the purified product was transfected into 293A cells using lip for viral packaging. Finally,q-PCR was used to detect the expression of KLF15 mRNA after pAdEasy-KLF15 vector was injected into C2C12 cells for 48 h. [Result] KLF15 showed significantly increasing expression during differentiation of C2C12 myoblasts and KLF15 expression levels in liver,kidney and fat were significantly higher than in the skeletal muscle and spleen. The recombinant adenovirus vector pAdEasy-KLF15 was successfully obtained,and the mRNA expression of KLF15 gene indicated an observably up-regulated trend after it was transfected C2C12 cells.[Conclusion] KLF15 gene showed high expression in C2C12 cells. The recombinant adenovirus vector expressing mouse KLF15 gene was successfully constructed.