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目的:利用AdEasyTM system构建携带小鼠p38MAPK基因的重组腺病毒,感染成骨细胞系MC3T3-E1,检测外源p38MAPK在细胞中的表达。方法:用PCR的方法扩增p38MAPK基因,将其克隆到pMD18-T载体中,进行测序。经BglⅡ和HindⅢ双酶切后接入pShuttle-CMV穿梭载体,构建重组腺病毒的穿梭质粒pShuttle-CMV-p38MAPK。将经PmeI线性化的pShuttle-CMV穿梭载体与pAdEasyTMDNA电穿孔共转化BJ5183重组细菌,获取重组腺病毒质粒Ad-p38MAPK,再将经PacI线性化的Ad-p38MAPK重组病毒骨架质粒转染AD293包装细胞,包装并扩增病毒。用Ad-p38MAPK感染小鼠MC3T3-E1成骨样细胞,以Western blot法检测p38MAPK在小鼠成骨细胞中的表达。结果:构建并包装表达p38MAPK蛋白的重组腺病毒,该重组腺病毒在体外能有效感染小鼠成骨细胞系MC3T3-E1并高表达p38MAPK蛋白。结论:成功地构建了携带小鼠p38MAPK基因的重组腺病毒,为研究p38MAPK在成骨细胞中的作用奠定了实验基础。
OBJECTIVE: To construct a recombinant adenovirus carrying mouse p38MAPK gene using AdEasyTM system and infect MC3T3-E1 osteoblastic cell line and detect the expression of exogenous p38MAPK in cells. Methods: The p38MAPK gene was amplified by PCR and cloned into pMD18-T vector for sequencing. The shuttle vector pShuttle-CMV was inserted into pShuttle-CMV shuttle vector to construct the shuttle plasmid pShuttle-CMV-p38MAPK of recombinant adenovirus. Recombinant adenovirus vector Ad-p38MAPK was obtained by electroporation of pShuttle-CMV shuttle vector and pAdEasyTM DNA electroporated by PmeI. The Pac-linear Ad-p38MAPK recombinant virus backbone plasmid was transfected into AD293 packaging cells, Package and amplify the virus. The mouse osteoblast-like cells were infected with Ad-p38MAPK and the expression of p38MAPK in mouse osteoblasts was detected by Western blot. Results: The recombinant adenovirus expressing p38MAPK protein was constructed and packaged. The recombinant adenovirus can effectively infect the mouse osteoblast cell line MC3T3-E1 and overexpress p38MAPK protein in vitro. Conclusion: The recombinant adenovirus carrying mouse p38MAPK gene was successfully constructed, which laid the experimental foundation for studying the role of p38MAPK in osteoblasts.