内含子在人凝血Ⅸ因子反转录病毒载体中的表达及剪接作用

Splicing and Stability of Intron in the Expression Retroviral Vector with Human Clotting Factor Ⅸ

构建了带有内含子的反转录病毒表达载体，研究内含子对hFⅨ的表达影响。反转录病毒载体经过PA317细胞包装后，稳定保留内含子是进一步研究内含子对hFⅨ表达影响的关键。首先构建了GlNaC－i－Ⅸ、GlNaC－i'-Ⅸ表达元件正向插入的反转录病毒载体。其中，GlNaC－i－Ⅸ带有源于IL－2的异源内含子，GlNaC－i'－Ⅸ带有来自hFⅨ自身的第一内含子；其中间部分顺序缺失，只包括剪接供体和剪接受体位点。用RT－PCR方法发现，反转录病毒介导基因转移的载体中的内含子都被剪切掉。为避免内含子的剪切，又构建了表达元件反向插入的反转录病毒载体GlNaC－i'－ⅨR、GlNaPAi'ⅨBAM。用同样的方法证明，反向插入载体中的内含子被保留下来。证明表达元件反向插入的反转录病毒载体可以得到稳定的含有内含子的表达载体，ELISA检测证明内含子能提高hFⅨ表达。为进一步提高hFⅨ在体外细胞和体内的表达水平提供依据。

To study the role of intron in the expression of hFⅨ, retroviral vectors withnitron containing hFⅨ were constructed. It is fundamental for the intron studywhether the nitron constructed in retroviral vector can be steadily transferred intotarget cell. First we constructed two forward-orientation retroviral vectors: GlNaC-i-Ⅸcontains the exogenous intron from IL-2, and GlNaCu-i-Ⅸ contains the truncatednitron Ⅰ from hFⅨ gene, covering the splicing donor and acceptor sequences. RT-PCRresult indicated that nitron in the forward-orientation retroviral vector was spliced afterpackaging in PA317. Then, reverse-orientation retroviral vectors GlNaC-i'-ⅨR andGlNaPAⅨi' BAM were constructed, in which the reverse and complimentarysequences of hFⅨ gene with nitron appeared in retroviral RNA. RT-PCR assaycombined with ELISA test indicated that intron was retained after packaging and hFⅨgene with nitron constructed in the reverse-orientation retroviral vector can betransduced intact and expressed hFⅨ at a high level in vitro.