鸡CMPK2的原核表达及其抗H9N2禽流感病毒的初步研究

Preliminary study on the expression of chicken CMPK2 and its resistance to H9N2 avian influenza virus

为研究鸡CMPK2基因的遗传进化特点和生物学功能,本研究以鸡成纤维细胞系(DF-1)为模板,通过RT-PCR扩增鸡CMPK2基因完整阅读框(ORF)(762 bp),利用MEGA7软件对CMPK2氨基酸序列进行同源性和遗传进化分析。并构建了重组质粒p3xFLAG-CMV-14-CMPK2和重组原核表达载体pET-28a-CMPK2,利用原核载体表达CMPK2目的蛋白后制备小鼠抗鸡CMPK2的多克隆抗体,并采用western blot检测其反应性。构建的CMPK2氨基酸序列进化树结果显示,鸡CMPK2与爬行类动物CMPK2遗传关系较近,而与哺乳动物CMPK2氨基酸序列的遗传距离相对较远。为研究鸡CMPK2是否具有抗病毒功能,利用H9N2禽流感病毒(AIV)感染DF-1细胞,感染后6 h、12 h时分别采用荧光定量PCR及western blot方法检测鸡CMPK2基因转录水平和蛋白表达情况,结果显示H9N2 AIV感染能够引起鸡CMPK2 mRNA转录水平和蛋白表达水平的明显上调。进一步利用DF-1细胞过表达CMPK2及利用特异性siRNA干扰CMPK2的表达后,分别采用荧光定量PCR及western blot检测鸡CMPK2蛋白对H9N2 AIV复制的影响。荧光定量PCR以及western blot结果显示,过表达鸡CMPK2蛋白可以抑制H9N2 AIV的增殖。而下调鸡CMPK2蛋白的表达后H9N2 AIV增殖水平显著上升(P<0.01)。上述结果首次证实,鸡CMPK2是H9N2 AIV复制的负调控因子,能够抑制AIV的复制。本研究丰富了AIV与抗病毒宿主因子的研究内容,也可为抗禽类病毒感染药物靶点的研究提供重要参考。

In order to study the genetic evolution characteristics and biological function of chicken CMPK2 gene,the chicken fibroblast cell line DF-1 was used as a template in this study to amplify the complete open reading frame(ORF)(762bp)of chicken CMPK2 gene by RT-PCR,and the amino acid homology and genetic evolution of CMPK2 were analyzed by MEGA7 software.The p3xFLAG-CMV-14-CMPK2 recombinant plasmid and pET-28a-CMPK2 prokaryotic expression vector were constructed,and the mouse anti-chicken CMPK2 polyclonal antibody was prepared by expressing the target protein of CMPK2 using pET-28a-CMPK2,and its reactivity was detected by western blot.The phylogenetic tree of the constructed the amino acid sequence of CMPK2 showed that the genetic relationship with reptiles CMPK2 is relatively close,while the sequence genetic distance from those of mammals is relatively far.In order to study whether chicken CMPK2 has antiviral function,the H9N2 AIV was used to infect DF-1 cells.Fluorescence quantitative PCR and western blot were used to detect chicken CMPK2 gene transcription and protein expression at 6 hours and 12 hours post infection.The results showed that H9N2 AIV infection can significantly upregulate chicken CMPK2 mRNA level and protein expression level.Further CMPK2 was overexpressed and interfered with specific siRNA in DF-1 cells,then fluorescence quantitative PCR and western blot were used to detect the effect of chicken CMPK2 protein on the replication of H9N2 AIV respectively.Results of quantitative PCR and western blot experiments showed that overexpression of chicken CMPK2 protein could inhibit the proliferation of H9N2 AIV,while the proliferation level of H9N2 AIV increased significantly(P<0.01)after downregulating the expression of chicken CMPK2 protein.The above results affirmed for the first time that chicken CMPK2 is a negative regulator of H9N2 AIV replication and can inhibit the replication of AIV.This study enriches the research on AIV and host antiviral factors,and also provides an important reference for finding antiviral drug targets against avian virus infection.