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目的:构建能介导结缔组织生长因子(CTGF)基因RNA干扰的复制缺陷型腺病毒表达载体。方法:以大鼠CTGF基因为靶序列,设计并合成含编码短发夹RNA序列的寡核苷酸,构建腺病毒穿梭质粒p-shuttle-CTGF,酶切及测序分析正确后,与腺病毒骨架质粒pAdEasy-1共转染AD-293细胞,进行病毒包装,得到腺病毒载体Ad.H1-CTGF,用该载体感染HSC-T6细胞,观察其对CTGF基因表达抑制的效果。结果:构建的腺病毒穿梭质粒p-shuttle-CTGF经酶切、测序分析证实正确;包装的病毒载体滴度为4×1010PFU/mL,感染HSC-T6细胞后,Western印迹证实CTGF表达显著减少。结论:构建的腺病毒载体Ad.H1-CTGF可有效抑制HSC-T6中CTGF的表达,为抗纤维化研究提供了有力的工具。
OBJECTIVE: To construct a replication-deficient adenovirus expression vector capable of mediating RNA interference of connective tissue growth factor (CTGF) gene. Methods: The rat CTGF gene was used as the target sequence to design and synthesize the oligonucleotide containing the short hairpin RNA sequence. The adenovirus shuttle plasmid p-shuttle-CTGF was constructed. After digestion and sequencing, The plasmid pAdEasy-1 was cotransfected into AD-293 cells and packaged in a virus to obtain the adenovirus vector Ad.H1-CTGF. The vector was used to infect HSC-T6 cells to observe the inhibitory effect on CTGF gene expression. Results: The recombinant adenovirus shuttle plasmid p-shuttle-CTGF was confirmed by restriction analysis and sequencing. The titer of the packaged virus vector was 4 × 1010 PFU / mL. After HSC-T6 cells were infected, the CTGF expression was significantly reduced. CONCLUSION: The constructed adenovirus vector Ad.H1-CTGF can effectively inhibit the expression of CTGF in HSC-T6 and provide a powerful tool for anti-fibrosis research.