猫importin基因生物信息分析及其在犬细小病毒感染F81细胞表达的动态变化

Bioinformatics Analysis of Feline importin Gene and Its Dynamic Expression in F81 Cells Infected with Canine Parvovirus

试验旨在系统研究猫源不同亚型importin(importinα1、importinα3、importinα4、importinα5、importinα6、importinα7、importinα8和importinβ)基因序列和蛋白基本特性,探讨其在犬细小病毒(canine parvovirus,CPV)感染F81细胞表达的动态变化。本研究利用RT-PCR方法扩增F81细胞中improtin基因全长序列,应用生物学软件预测其氨基酸序列及编码蛋白的结构及功能;进一步利用实时荧光定量RT-PCR技术检测importin基因在CPV感染F81细胞后24、48和72 h表达水平。结果显示,不同亚型importinα基因全长约在1500 bp,而importinβ基因全长为2600 bp;预测importin亚型蛋白序列发现,其等电点均在4.60左右,且发现importinα1、importinα5、importinα6、importinα7和importinβ为不稳定蛋白;分析发现这些importin蛋白均无信号肽、无跨膜区、无细胞定位;采用Predictprotein软件预测蛋白二级结构,结果显示importin亚型蛋白序列主要以α-螺旋和无规则卷曲形式存在。实时荧光定量RT-PCR结果显示,CPV感染F81细胞后随着感染时间的延长importinα1(P<0.01)和importinβ表达量逐渐降低,而importinα3、importinα4、importinα5、importinα6(P<0.01)、importinα7(P<0.01)和importinα8(P<0.01)表达量逐渐提高,其中importinα8在病毒感染细胞后表达量最高。本研究结果为进一步分析CPV进入细胞核的转运机制及开发治疗药物相关研究奠定基础。

This study was aimed to systematically analyze the gene sequences and proteins of the importin subtype genes(importinα1,importinα3,importinα4,importinα5,importinα6,importinα7,importinα8 and importinβ),and the change of transcription level of importin subtype of F81 cells infected CPV.The full length of importin genes were amplified by RT-PCR techniques from F81 cells and the amino acid sequence and the structure and function of importin protein were subsequently analyzed by bioinformatics software.Meanwhile,the dynamic change of expression of importin genes in CPV-infected F81 cells were investigated at 24,48 and 72 h postinfection by Real-time RT-PCR.The results showed that full length of the importinαsubtype genes were about 1500 bp and the importinβwas 2600 bp.The physical chemical properties of importin proteins showed the theoretical isoelectric was about 4.60 and the importinα1,importinα5,importinα6,importinα7 and importinβbelonged to instable proteins.Meanwhile,there were no signal peptide,no transmembrane structures and no cellular localization in importin proteins.The secondary structure of importin proteins were predicted by Predictprotein software and mainly composed ofα-helix and random coil.Real-time RT-PCR results showed that the importinα1(P<0.01)and importinβmRNA level were significantly reduced in CPV-infected F81 cells,while other importin mRNA level were markedly up-increased,the importinα8 gene had the highest expressional level(P<0.01).The study laid a foundation for further study on the transport mechanism of CPV into the nucleus of the cell and developing drugs.