| Duck hepatitis A virus 1(DHAV-1)belongs to Picornaviridae,which is a common high deadly,infectious diseases,main infecting the 4 weeks duckling,has a great harm to the breeding industry.2C protein belongs to P2 in DHAV-1 coding regions,which plays a vital role in the process of the formation of RNA virus replication complex.For now,it's little study on DHAV-12C protein,referring to other small RNA virus family.In our study,we use a prokaryotic expression system to obtain protein 2C for the research of the subcellular localization of 2C protein in duck embryo fibroblast.Besides that we detect the function of the NTPase enzyme activity,including wild strain and site-directed mutants strains that play an essential role of the 2C NTPase activity.The results are as follows:1.The prokaryotic expression,preparation of polyclonal antibody and Subcellular localization of DHAV-1 2C proteinConnecting the correct amplification segments of 2C to pET32a,but only obtaining the insoluble protein in Rossta(DE3)of about 50 kDa protein size,purifying the 2C protein and preparing the anti-his mouse serum,used as the basis of subsequent subcellular localization function study materials.Firstly,the location condition of transient transfection with pEGFP-C2-2C protein,transfection after 24 h,Laser Confocal Microscope found that 2C protein distributed in the nuclear and cytoplasmic.Secondly,we carried on the natural infection situation of DHAV-1 virus,Laser Confocal Microscope found that 2C protein distributed outside the cytoplasmic before 4h,and started entering the nucleus after 6h,until going thoroughly into the nucleus after 20 h,and are no longer out of the nucleus.After that,we conducted the golgi apparatus and endoplasmic reticulum with 2C protein localization,indirect immunofluorescence test found that inoculation of DHAV-1 virus after 4h,the endoplasmic reticulum and the golgi apparatus colocalized with 2C protein.2.Detction the NTPase activity of DHAV-12C protein and its mutantsConsidering the insolubility of 2C protein,reference to previous studies,adding a maltose binding protein(MBP)at the N terminal of 2C to increase its soluble expression.Connecting the correct amplification segments of 2C to pET28a,obtaining the soluble protein in Rossta(DE3)induced by IPTG.The collected proteins are 75kDa,detction the NTPase activity after purfying the proteins.Carrying out the NTPase enzyme activity with purified 2C protein by Phosphate Colorimetric Assay Kit,but the dNTP enzyme hydrolysis efficiency is higher than the NTP enzyme hydrolysis efficiency,the hydrolysis results are as follows,dATP>dGTP>dCTP>dTTP>ATP>GTP>UTP>CTP.We also explore the biological characteristics of the enzyme activity(NTP),the results show that the NTP enzyme activity dependent on magnesium and effected by the common metal ions such as Ca2+,Mn2+ and Zn2+,the influence are as follows,Mg2+>Mn2+>Ca2+>Zn2+.The optimum condition is 2.5mM Mg2+,25mM NaCl,8 pH.Due to the NTP enzyme activity plays a very important role in viral replication,as guanidine hydrochloride is the commonly used inhibitors virus replication,we test the effect of guanidine hydrochloride on enzyme activity(NTP),indicating that 1mm NTP enzyme began to fell significantly,and when theconcentration is more than 50 mm,almost all the NTP enzyme activity are inhibitited.In order to determine the critical site of the NTP activity of 2C.we mutate the "GK" of the amino acid in 151 to 152,after predicting by bioinformatics.Testing enzyme activity on site-directed mutants with the optimum condition,indicating that single mutation amino acid mutations in "K" and at the same time amino acids "GK" will make the enzyme activity decreased significantly,but single mutation amino acid "G" makes higher enzyme activity. |
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