可调控性鼠TGF-β1重组腺病毒载体的构建和表达

目的:构建和表达可调控性鼠TGF-β1重组腺病毒载体。方法:采用常规分子生物学方法, 从刀豆A刺激的小鼠脾细胞抽提总RNA, 克隆鼠TGF-β1基因;经穿梭载体与可调控性腺病毒载体骨架连接, 鉴定后在HEK293细胞包装。获得高滴度病毒后, 取上清采用ELISA方法鉴定目的基因的表达。结果:TGF-β1重组腺病毒经测序、限制性内切酶酶切分析、PCR等鉴定, 与预期结果一致;病毒感染细胞的上清液经ELISA检测, TGF-β1的表达量为(91.16±3.70)ng/L。结论:构建的可调控性鼠TGF-β1重组腺病毒载体得到了正确表达, 为进一步的研究和应用奠定了基础。

Construction and regulated expression of mouse TGF-β1 recombinant adenovirus vector

AIM:To construct a recombinant adenovirus vector which regulated expression of mouse transforming growth factor beta (TGF-β1).METHODS: Total RNA was extracted from Balb/c mice spleen pre-treated with Con A to clone TGF-β1. pTRE-shuttle vector was used as mediator to ligate TGF-β1 gene and the backbone of the replication-incompetent adenoviral vector. The constructed recombinant adenovirus contains tetracycline-responsive element which can regulate the expression of inserted genes. Identifying the desired recombinant adenovirus, it was packaged in HEK 293 cells. Supernatant of high titer adenovirus was collected to detect the TGF-β1 gene expression by ELISA kit. RESULTS:The constructed recombinant adenovirus had been identified by restriction endonucleases cutting, sequencing and PCR. In the supernatant of infected cells, it expressed the mice TGF-β1 in high concentration to 91.16 ng/L.CONCLUSION:The mice TGF-β1 recombinant adenovirus has been built which make a basis for further research and use in gene therapy.