H5N1禽流感病毒感染小鼠后内参基因的筛选

Selection of Reference Genes in Mice Infected with H5N1 Avian Influenza Virus

本研究旨在评定H5N1禽流感病毒(H5N1 avian influenza virus,H5N1 AIV)感染小鼠后,脾脏组织中6个候选内参基因酪氨酸3/色氨酸5-单加氧酶激活蛋白(tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein,Ywhaz)、次黄嘌呤磷酸核糖转移酶1(hypoxanthine-guanine phosphoribosyltransferase 1,Hprt1)、琥珀酸脱氢酶A亚基(succinate dehydrogenase flavoprotein subunit,Sdha)、β-肌动蛋白(β-actin,Actb)、肽基脯氨酰异构酶A(peptidyl prolyl isomerase A,Ppia)和18S核糖体RNA(18S ribosomal RNA,18S rRNA)的稳定性。通过H5N1 AIV强毒株A/DK/GD/378/08感染小鼠,采用Real-time PCR方法分别于12、24、48、72、96、120和144 h检测感染组与对照组中6个候选内参基因的表达水平,然后用Genorm软件对内参的稳定性进行评价。结果表明,正常小鼠脾脏组织中6个内参基因稳定度由高到低分别为Ywhaz/Hprt、Sdha、Actb、Ppia、18S rRNA;H5N1 AIV感染的小鼠脾脏组织中6个内参基因稳定度由高到低分别为Ppia/Sdha、18S rRNA、Hprt、Ywhaz、Actb;综合评价正常和H5N1 AIV感染后小鼠脾脏组织中6个内参基因的表达稳定度由高到低分别为Sdha/Hprt1、Ywhaz、Ppia、Actb、18S rRNA。因此,小鼠脾脏组织中Ppia和Sdha是研究H5N1 AIV在机体中复制时最理想的2个内参基因;Sdha和Hprt1则是在研究H5N1 AIV与机体相互作用时最理想的2个内参基因。

This experiment was conducted to assess the stability of six candidate reference genes （Ywhaz,Hprt1,Sdha,Actb,Ppia and 18S rRNA） in spleen of mice infected with H5N1 avian influenza virus （H5N1 AIV）. The mice were infected with the virulent strain A/DK/GD/378/08 of H5N1 AIV and harvested at 12,24,48,72,96,120 and 144 h post-infection. The expression levels of the six reference genes in infected and uninfected mice were determined by Real-time PCR and assessed the stability of reference genes by Genorm software. The results showed that the stability of six reference genes in normal mouse spleen tissue descended as following： Ywhaz/Hprt,Sdha,Actb,Ppia,18S rRNA, the stability of six reference genes in mouse spleen tissue infected with H5N1 AIV descended as following： Ppia/Sdha,18S rRNA,Hprt,Ywhaz,Actb. Furthermore,based on comprehensive evaluation of the normal and the infected H5N1 AIV,the stability of six reference genes in mouse spleen tissue descended as following： Sdha/Hprt1,Ywhaz,Ppia,Actb,18S rRNA. Therefore,the Ppia and Sdha were the best reference genes to use in the study of replication levels of H5N1 AIV in spleen of mice. However,for the study of organism response to H5N1 AIV infection,the Sdha and Hprt1 were the best reference genes.