波形蛋白对H9N2亚型AIV复制的影响研究  

Effects of vimentin on replication of H9N2 subtype avian influenza virus

作　　者：卢安然 殷贵虎 黄翔宇 黄静雯 胡嘉宁 蔡怡秦 冯秀丽[1] LU Anran;YIN Guihu;HUANG Xiangyu;HUANG Jingwen;HU Jianing;CAI Yiqin;FENG Xiuli(College of Veterinary Medicine,Nanjing Agricultural University/Key Laboratory of Animal Microbiology of China's Ministry of Agricultural and Rural Affairs/MOE Joint International Research Laboratory of Animal Health and Food Safety,Nanjing 210095,China)

机构地区：[1]南京农业大学动物医学院/农业农村部动物细菌学重点实验室/教育部动物健康与食品安全国际合作联合实验室,江苏南京210095

出　　处：《畜牧与兽医》2022年第4期93-101,共9页Animal Husbandry & Veterinary Medicine

基　　金：国家重点研发项目(2017YFD0500706);国家自然科学基金(31872458)。

摘　　要：为研究波形蛋白(vimentin)对H9N2亚型禽流感病毒(AIV)复制的影响,构建了真核表达载体FLAG-vimentin,并采用Western blot技术验证FLAG-vimentin重组载体和病毒NP蛋白的表达情况,用荧光定量PCR技术检测病毒HA基因水平。同时,设计合成siRNA,敲低内源性vimentin,检测其对病毒HA基因表达水平的影响。构建原核表达载pCold I-vimentin,蛋白纯化后孵育细胞,采用Western blot、荧光定量PCR技术检测病毒的蛋白表达和基因水平的变化。结果证实,H9N2亚型AIV感染FLAG-vimentin重组载体转染的Hela细胞12 h时,病毒NP蛋白表达减少;而在感染24和36 h时,病毒NP蛋白表达上升。荧光定量PCR结果亦显示,在AIV感染后12 h时,HA基因表达水平明显降低。siRNA敲低Hela细胞内源性vimentin后,H9N2亚型AIV的HA基因表达水平在病毒感染12 h时明显升高,24 h时差异较小,36 h时有所升高。纯化后的vimentin重组蛋白孵育细胞,在病毒感染36 h时,H9N2亚型AIV的NP蛋白表达显著被抑制。荧光定量PCR结果亦证实病毒HA基因在AIV感染后36 h时表达水平明显降低。上述结果表明,H9N2亚型AIV在人源Hela细胞上的早期复制中,vimentin能起到一定程度上的抑制作用,且其重组表达蛋白孵育细胞能够阻断H9N2亚型AIV对细胞的感染,为深入研究vimentin抗病毒复制的机制提供了重要的试验依据,同时为开展抗H9N2亚型AIV的药物研发提供了参考。In order to determine the effect of vimentin on the replication of H9 N2 subtype avian influenza virus(AIV), the eukaryotic expression vector FLAG-vimentin was constructed. The expressions of the recombinant vimentin and NP proteins were verified by Western blot, and the level of the HA gene was detected by qPCR. Meanwhile, endogenous vimentin was knocked down by siRNA, and the expression level of HA gene was detected. The prokaryotic expression vector pCold I-vimentin was constructed, and the cells were incubated with the recombinant vimentin protein. The protein expression and the gene level of NP and HA were detected by Western blot and qPCR. The results showed that at H9 N2 subtype AIV infected HeLa cells transfected with FLAG-vimentin for 12 h, the expression of the viral NP protein was decreased, and as 24 h and 36 h, the expression of the viral NP protein was increased. The results of qPCR confirmed that the expression level of the HA gene in the Hela cells transfected with FLAG-vimentin was decreased significantly at 12 h after the AIV infection. The HA gene expression of H9 N2 subtype AIV was increased significantly at 12 h after the knockdown of the endogenous vimentin protein in HeLa cells by siRNA. The NP protein expression of H9 N2 subtype AIV in the Hela cells incubated with the purified recombinant vimentin protein was significantly inhibited at 36 h after viral infection. Also, the expression level of the gene of virus HA was significantly decreased at 36 h after the AIV infection. The above results indicated that vimentin proteins could inhibit the early replication of H9 N2 subtype AIV on the Hela cells, and the recombinant vimentin protein incubated Hela cells could block the infection of H9 N2 subtype, which provided an important experimental basis for further research on the mechanism of antiviral replication of vimentin proteins, and serve as reference for research on and development of drugs against H9N2 subtype AIV.

关 键 词：波形蛋白 H9N2亚型AIV 重组蛋白表达 病毒基因表达 小干扰RNA 

分 类 号：S852[农业科学—基础兽医学]