【作者】 高辉；

【导师】 仇华吉； 冉多良；

【作者基本信息】 新疆农业大学 ， 预防兽医学， 2007， 硕士

【摘要】 随着携带哺乳动物细胞启动子的重组杆状病毒逐渐成为基因功能研究、非复制型载体疫苗研制以及开发基因治疗策略的一个非常有效的工具,寻找一种在昆虫细胞和哺乳动物细胞之间的穿梭启动子对于杆状病毒介导的基因转移方面的研究非常必要。在本研究中,我们利用对虾白斑综合症病毒(white spot syndrome virus,WSSV)的ie1启动子和杆状病毒ETL启动子以及杆状病毒多角体启动子,分别构建了含有不同启动子调控下的EGFP报告基因表达盒的重组杆状病毒,然后转导不同哺乳动物细胞,以检测不同启动子在细胞中的转导和表达效率。结果显示,WSSV的ie1启动子在昆虫细胞sf9中具有很强的启动子活性,能够控制报告基因的高效表达,同时在哺乳动物细胞中,ie1启动子与ETL启动子一样在不同哺乳动物细胞中均有活性,能够介导EGFP不同水平的表达,并且丁酸钠能够提高ie1启动子在哺乳动物细胞中的转导和表达效率。转移载体瞬时转染结果证实,ie1启动子能够直接启动外源基因在哺乳动物细胞中的表达,而不依赖于杆状病毒基因表达。本研究首次发现ie1是一种杆状病毒非依赖性、在昆虫细胞和哺乳动物细胞之间的新型穿梭启动子,该穿梭启动子对于杆状病毒表达载体在基因治疗和非复制型载体疫苗方面的研究将具有重要应用价值。更多还原

【Abstract】 As recombinant baculoviruses containing mammalian cell-active promoters are becoming a promising tool for studying gene functions and developing non-replicative vector vaccines and gene therapy strategies, a shuttle promoter that mediates gene expression in both insect cells and mammalian cells will facilitate the development of a baculovirus vector-based mammalian cell gene delivery vehicle.In this report, three recombinant baculoviruses were generated carrying the reporter EGFP gene under the control of white spot syndrome virus (WSSV) ie1 promoter, or either of two control promoters, baculovirus early to late (ETL) promoter and polyhedrin promoter. Several mammalian cell lines were transduced with the resulting recombinant baculoviruses to test the expression of EGFP. The results showed that WSSV ie1 promoter showed a strong promoter activity in insect cells and mammalian cells. The promoter activity of the ie1 promoter but not ETL promoter can be enhanced by sodium butyrate, a histone deacetylase inhibitor. Transient plasmid transfection assay indicated that ie1 promoter activity in mammalian cells is independent on baculovirus gene expression, which is different from ETL promoter, a baculovirus-dependent shuttle promoter.This study demonstrated, for the first time, that ie1 is a baculovirus-independent shuttle promoter between insect cells and mammalian cells. This novel shuttle promoter will facilitate the application of baculovirus-based vectors in gene expression, gene therapy and non-replicative vector vaccines.更多还原