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

作　　者：郝春秋[1] 彭梅娟[1] 谢玉梅[1] 周云[1] 魏欣[1] 马力[1] 王素娜[1] 李瑞娟[1] 张岩[1] 白雪帆[1] 贾战生[1] 

机构地区：[1]第四军医大学唐都医院感染病诊疗中心,陕西西安710038

出　　处：《生物技术通讯》2013年第1期57-60,共4页Letters in Biotechnology

基　　金：国家自然科学基金(30571658)

摘　　要：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.

关 键 词：结缔组织生长因子 RNA干扰 腺病毒载体 

分 类 号：Q78[生物学—分子生物学]