泛素化激酶Cbl蛋白对H9N2亚型禽流感病毒早期复制的抑制作用  

Inhibitory effect of the ubiquitin kinase Cbl protein on early replication of H9N2 subtype avian influenza virus

作　　者：陈佳静 李彤彤[1] 张则 卢安然 李陈飞 冯秀丽[1] CHEN Jiajing;LI Tongtong;ZHANG Ze;LU Anran;LI Chenfei;FENG Xiuli(College of Veterinary Medicine,Nanjing Agricultural University/Key Laboratory of Animal Bacteriology,Ministry of Agriculture 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》2021年第6期78-83,共6页Animal Husbandry & Veterinary Medicine

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

摘　　要：为研究Cbl蛋白(Casitas b-lineage lymphoma)对H9N2亚型禽流感病毒(AIV)复制的影响,采用PCR技术扩增Cbl基因片段并连接到FLAG-N载体中,Western blot技术验证FLAG-Cbl重组载体表达及病毒NP蛋白的表达情况,并采用荧光定量PCR技术检测病毒的血凝素(HA)、核蛋白(NP)和非结构蛋白(NS)基因水平;采用RNAi技术检测Cbl对病毒HA基因表达水平的影响。结果显示:EcoRⅠ、HindⅢ双酶切鉴定及测序分析发现,成功构建真核表达重组载体FLAG-Cbl;Western blot结果证实H9N2亚型AIV感染FLAG-Cbl重组载体转染的A549细胞6 h时,病毒NP蛋白表达降低;荧光定量PCR结果证实病毒NP、NS及HA基因在AIV感染FLAG-cbl重组载体转染的A549细胞的6和12 h时表达水平均明显降低;RNAi敲低A549细胞内源性Cbl蛋白后,H9N2亚型AIV的HA基因表达水平在病毒感染12、24和36 h明显升高。结果表明:Cbl蛋白能在早期抑制H9N2亚型AIV在A549细胞上的复制,为深入研究Cbl蛋白抗病毒复制的机制提供了重要的试验依据,同时为开展抗AIV的药物研发提供了参考。This study was to investigate the effect of Casitas b-lineage lymphoma(Cbl)on the replication of H9 N2 subtype avian influenza virus(AIV).The Cbl gene fragment was amplified by PCR and was linked to a FLAG-N vector.The expression of the FLAG-Cbl recombinant vector and the expression of the viral NP protein were verified by Western blot.The levels of HA,NP and NS genes were detected by quantitative PCR.Additionally,the RNAi technology was used to detect the effect of Cbl on the expression level of the HA gene of AIV.The results of double enzyme digestion of EcoRⅠand HindⅢand those of gene sequencing showed that the eukaryotic expression vector FLAG-Cbl was successfully constructed.The results of Western blot confirmed that the expression of NP protein was decreased at 6 h after H9 N2 subtype AIV infection in the A549 cells transfected with the FLAG-Cbl recombinant vector.Furthermore,the mRNA levels of the NP,NS and HA genes were significantly decreased at 6 and 12 h in the A549 cells transfected with the FLAG-Cbl recombinant vector,suggesting that Cbl proteins inhibited the replication of the H9 N2 virus at the early stage.After knockdown of the endogenous Cbl proteins in the A549 cells with siRNA,the HA gene level of H9 N2 subtype AIV increased significantly at 12,24 and 36 h,which suggested that the decrease of the Cbl proteins might be beneficial for AIV replication.These results indicated that Cbl proteins might inhibit H9 N2 subtype virus amplification in the A549 cells at the early stage,which provided an important experimental basis for further study of the antiviral mechanism of Cbl proteins,and offered an important idea for anti-AIV drug developments.

关 键 词：Cbl蛋白 H9N2亚型AIV 重组蛋白表达 病毒基因表达 SIRNA 

分 类 号：S852.6[农业科学—基础兽医学]