hNoc4L基因重组慢病毒表达载体的构建与鉴定

Construction and identification of recombinant lentiviral vector of hNoc4L gene

摘要:

核糖体前体的形成和核运输需要多种核仁复合物的参与，hNoc4L是酿酒酵母S. cerevisiae的核仁复合物相关蛋白4的同源蛋白，并含有保守的Noc结构域，但其功能未知。为了构建hNoc4L基因过表达的慢病毒载体，本实验通过将EF1α启动子替换原shRNA慢病毒载体pll3.7的U6启动子，成功构建了慢病毒表达载体pll3.7-EF1，并进一步得到了hNoc4L基因过表达的慢病毒载体。利用慢病毒包装系统对不同物种的细胞进行感染，以此检测该重组慢病毒载体的包装效率，并通过构建的hNoc4L过表达的RAW264.7稳定细胞系检测了该载体的免疫原性和稳定转染能力。结果表明成功构建了高效、长期稳定表达和免疫原性低的hNoc4L特异性表达的慢病毒载体，为进一步研究hNoc4L蛋白在哺乳动物核糖体生物发生中的调控作用奠定了基础。

Abstract:

Formation and nuclear export of pre-ribosomes requires many nucleolar complexes. hNoc4L which contains a conserved Noc doman is a homolog of nucleolar complex associated 4 (S. cerevisiae), but its function is completely unclear. Here, we successfully got the recombinant lentiviral vector pll3.7-EF1-hNoc4L-Flag by replacing the U6 promoter in pll3.7 with EF1α promoter, and then inserted hNoc4L to down-stream of the EF1α prompter. We determined the transduction efficiency in different mammalian cell lines based on lentiviral packaging system. Subsequently, we analyzed the immunogenicity of the recombinant lentivirus and stable expression of hNoc4L in RAW264.7 cells. The results showed that the recombinant lentivirus characterized a high transduction efficiency, long-term expression and low immunogenicity. Therefore, we pave the way for further identification of the biological activity of hNoc4L protein during ribosome biogenesis in mammalian.