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采用AdEasy系统构建带有标记基因GFP和FC的重组腺病毒AdCCN1,并感染人骨肉瘤细胞,观察外源性CCN1对肿瘤细胞生长和迁移的影响.PCR扩增FC编码序列标记的人CCN1,克隆到pAdTrack-TO4中获得重组穿梭载体pAdTrack-CCN1.PmeⅠ线性化该质粒并电转到有腺病毒骨架质粒的BJ/AdEasy-1菌中,同源重组获得腺病毒质粒pAdCCN1,PacⅠ线性化并转染293细胞,包装制备AdCCN1.卡那霉素抗性筛选、XbaⅠ与KpnⅠ酶切鉴定,荧光显微镜和Western印迹检测标记基因GFP和FC表达,鉴定获得的复制缺陷型重组腺病毒AdCCN1.AdCCN1与AdGFP分别感染人骨肉瘤细胞143B,通过MTT实验、胶原集落形成实验、划痕愈合和室移动实验,观察CCN1对143B增殖和迁移的影响.酶切鉴定表明,带有FC标记的CCN1被成功克隆到穿梭载体pAdTrack中.卡那霉素抗性筛选并酶切鉴定,获得重组腺病毒质粒pAdCCN1.Western印迹和荧光显微镜观察,确定与CCN1共表达的FC和GFP表达.AdCCN1感染骨肉瘤细胞,与对照比较细胞数量增多,集落形成增加,细胞划痕逐渐愈合,迁移至膜上的细胞数量明显增加.应用AdEasy系统高效快速构建了带有标记GFP和FC的重组腺病毒AdCCN1,为监控腺病毒转基因的效果提供了便捷的工具.AdCCN1感染骨肉瘤细胞后可促进细胞增殖和迁移,提示CCN1在肿瘤发生和转移中可能起到重要作用.
The AdEasy system was used to construct recombinant adenovirus AdCCN1 with marker genes GFP and FC and infected human osteosarcoma cells to observe the effect of exogenous CCN1 on the growth and migration of tumor cells.PCR was amplified by PCR and cloned into pAdTrack-TO4 recombinant shuttle vector pAdTrack-CCN1.Pme Ⅰ linearized the plasmid and electroporated into adenovirus backbone plasmid BJ / AdEasy-1 bacteria, homologous recombination adenovirus plasmid pAdCCN1, Pac Ⅰ linearized and transfected 293 Cells and packaging were prepared by AdCNN1. Kanamycin resistance screening, Xba Ⅰ and Kpn Ⅰ digestion, fluorescent microscopy and Western blotting were used to detect the expression of GFP and FC, and the replication-defective recombinant adenovirus AdCCN1.AdCCN1 and AdGFP were respectively infected Human osteosarcoma cell line 143B was used to observe the effect of CCN1 on the proliferation and migration of 143B cells by MTT assay, collagen colony formation assay, wound healing and cell migration assay.Identification by restriction enzyme digestion indicated that FCN1 was successfully cloned into the shuttle vector pAdTrack Kanamycin resistance screening and restriction enzyme digestion to obtain the recombinant adenovirus plasmid pAdCCN1.Western blot and fluorescence microscopy to determine the CCN1 co-expression of FC GFP expression.AdSCN1 infection of osteosarcoma cells, compared with the control cells increased, the colony formation increased, cell scratches gradually healed, the number of cells migrating to the membrane increased significantly.Using AdEasy system efficient and rapid construction with labeled GFP and FC Recombinant adenovirus AdCCN1 provides a convenient tool for monitoring the effect of adenovirus transfection.AdCCN1 infection of osteosarcoma cells can promote cell proliferation and migration, suggesting that CCN1 may play an important role in tumorigenesis and metastasis.