寨卡病毒NS4A蛋白影响小鼠大脑皮质神经元的迁移

NS4A protein of Zika virus influences the neuronal migration of mouse cortex

目的在体研究寨卡病毒(Zika virus)NS3和NS4A蛋白对大脑皮质神经元迁移的影响。方法通过c DNA末端快速扩增技术(RACE)及反转录PCR测定Zika病毒SZ01株基因组的开放阅读框,确定NS3和NS4A蛋白编码序列,构建融合Flag标签的p CIG蛋白表达载体。通过子宫内电转技术在E13.5 d小鼠的大脑皮质的神经前体细胞中表达病毒蛋白。免疫组化检测Flag标签、TBR1与e GFP,观察表达NS3和NS4A蛋白的细胞在E18.5 d大脑皮质中的分布,评估Zika病毒蛋白对大脑皮质神经元迁移的影响。结果 1)NS4A的氨基酸序列与NCBI数据库一致,NS3存在1处氨基酸位点突变。2)Flag标签荧光信号与e GFP荧光共定位,提示e GFP可以指示病毒蛋白在皮质中的表达。3)TBR1荧光显示在体表达NS4A后,神经元的分布与p CIG对照组及表达NS3相比有显著差异(P<0.001)。结论在体表达Zika病毒的NS4A蛋白可能影响神经元迁移。

Objective To determine the effect of NS3 and NS4A proteins of Zika virus on the neuronal migration in vivo. Methods To identify the coding sequence of NS3 and NS4A, the genome of Zika SZ01 was sequenced by rapid amplification of cDNA ends （RACE） and reverse-transcription PCR, then NS3 and NS4A was constructed in pCIG vector fused with Flag-tag to express these proteins. And then these plasmids was transfected into the embryo brain of E 13.5 mice by in utero electroporation, the distribution of the cells which express these proteins in the cor- tex was detected by Flag, eGFP and TBR1 fluorescence in E18.5 mice through immunohistochemistry so as to as- sess the influence of viral proteins on the neuronal migration of mouse cortex. Results 1 ） Sequence results showed that the amino acid sequence of NS4A is consistent with NCBI data, while NS3 has 1 amino acid mutant. 2） As the fluorescence of Flag and eGFP can co-localization, the eGFP fluorescence signal marks the cells that have expressed these virus proteins in cortex. 3） significantly different from pCIG TBR1 fluorescence shows the distribution of control and NS3 （P〈0. 001 ）. Conclusions affect the neuronal migration in vivo.