结缔组织生长因子基因RNA干扰复制缺陷型腺病毒载体的构建及鉴定

Construction and Identification of Replication-Deficient Adenovirus Vector for RNA Interference with CTGF Gene

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 recombinant adenovirus vector expressing small interfer- ence RNA（siRNA） against the connective tissue growth factor（CTGF） gene. Methods： The siRNA sequence target- ing CTGF mRNA was synthesized and shuttle plasmid p-shuttle-CTGF was constructed, which was co-transfected into AD-293 cells with the bone plasmid pAdEasy-1 to obtain the homologous recombination adenovirus vector Ad.H1-CTGF. The concentration of CTGF protein in HSC-T6 cells infected with adenovirus vector Ad.H1-CTGF was determined by Wetsern blot. Results： The p-shuttle-CTGF was identified by endonuclease and sequencing. The adenovirus vector Ad.H1-CTGF was successfully constructed with the titre of 4×1010 PFU/mL by purification. The concentration of CTGF protein in HSC-T6 ceils was decreased observably after Ad.H1-CTGF infected by Western blot. Conclusion： The constructed Ad.H1-CTGF can effectively inhit the expression of CTGF in HSC-T6 ceils, which provided a powerful tool for anti-fibrosis study.