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目前基因治疗研究遇到的普遍问题是转染效率低下及目的基因蛋白在活体表达水平较低,其中解决目的基因表达较低的一个重要手段便是基因载体的改造。1990年,Jallat等人在转基因小鼠研究中发现:hFIX cDNA的表达量低,而其余一系列带有完整内含子Ⅰ或中间缺失4.8kb的内含子Ⅰ的转基因小鼠,其表达都和完整的Ⅸ因子表达相仿。这提示Ⅸ因子内含子Ⅰ顺序对其表达有一定的促进作用。1995年,Kurachi等人用含内含子Ⅰ片断的表达质粒转化HepG2细胞,发现瞬间表达要比cDNA高出7倍,其凝血活性和细胞内mRNA水平也有相应数量的提高。本实验室也进行了hFIX intron Ⅰ的研究工作,构建了逆转录病毒载体GlNaCi′IX,但对病毒上清进行RT-PCR检测,发现intron Ⅰ常被不同程度剪切。本文以SNMBAAIXm为蓝本,构建了反向表达载体GlNaPAi′IXBAM,其中Ⅸ因子转录方向与LTR转录方向相反,以期避免病毒包装中将intron Ⅰ剪切掉,使intron Ⅰ结构能够正确引入到靶细胞中,从而提高Ⅸ因子的表达量。
At present, the general problems encountered in gene therapy research are the low efficiency of transfection and the low level of expression of the target gene protein in vivo, and an important means to solve the problem of low target gene expression is the transformation of the gene vector. In 1990, Jallat et al. Found in transgenic mice that the expression of hFIX cDNA was low, while the rest of a series of transgenic mice with intact intron I or intron I with a deletion of 4.8 kb in the middle Similar to intact factor IX expression. This suggested that the factor Ⅸ intron Ⅰ sequence of its expression has a certain role in promoting. In 1995, Kurachi et al. Transformed HepG2 cells with the expression plasmid containing intron I and found that the transient expression was 7 times higher than that of cDNA. The clotting activity and the intracellular mRNA level were also increased by a corresponding amount. In our laboratory, hFIX intron Ⅰ was also constructed and retroviral vector GlNaCi’IX was constructed. However, RT-PCR of virus supernatants showed that intron Ⅰ was often sheared to varying degrees. In this paper, based on SNMBAAIXm, an inverted expression vector GlNaPAi’IXBAM was constructed, in which the direction of transcription of factor IX was opposite to that of LTR in order to avoid intron Ⅰ cut-off in virus packaging and intron Ⅰ structure can be correctly introduced into target cells , Thereby increasing the expression of factor Ⅸ.