Study On The Effect Of Inhibitor SP And Activator PMA Of PKC To DEV Proliferation

Duck enteritis virus(DEV),also known as duck plague virus(DPV),is one of the important pathogens encumber the healthy development of duck industry. Nucleocapsid protein(NP) of DEV and its host cell receptor as protein kinase C inhibitor protein(PKCI)were found in our previous studies. In the host cell,PKCI act as an inhibitor of protein kinase C(PKC),PKC is key molecule in the host cell phosphorylation pathway,participate in the process of variety virus proliferation. The process of phosphorylation pathway exists in the DEV infection has not been reported. To this end,this study used classical inhibitor SP and specific activator PMA of PKC research phosphorylation pathway in DEV infection,tried to provide some new clues and ideas for the mechanism of DEV infection.1.Establishment of real-time fluorescence quantitative PCR method for detecting the PKC gene of duck: Accorded to the GenBank PKC gene,specific primers were designed and synthesised,the target gene fragment was obtained by PCR from the m RNA of duck embryo fibroblast(DEF) cells,then recombinanted plasmid of GV pXT19-T-PKC,after that,the plasmid has been used as positive sample,real-time fluorescent quantitative PCR and standard curve were constructed. The reproducibility,specificity and sensitivity were verifiled.The results showed that:the linear relationship of standard curve was Y=-3.3340x+44.199,the correlation coefficient was 0.9954,the amplification efficiency was 99.19%,melting curve appeared only a single specific peak,and for the H5,H7,H9 subtype avian influenza virus,newcastle disease virus,and duck hepatitis virus,the fluorescence signal were not detected. These indicated the establishment of real-time fluorescent quantitative PCR method had excellent stability,specificity and sensitivity.2.The effect of DEV virulence by SP and PMA: The duck enteritis virus virulent strain of Guizhou was taken and inoculated duck embryo fibroblast cells,the virus TCID50 value were determined; The DEF cells were cultured monolater, and treated by SP and PMA(concentration is 100nomol/L) for 4h, respectively, added series of diluted DEV for incubation,observed and measured the change of TCID50 value;And added 0.01 MOI DEV for incubation,observed and measured the change of TCID50 value,the results:the TCID50 was3.16×10-9/0.1mL of duck enteritis virus virulent strain of Guizhou;The virus TCID50 value decreased with the increased of the concentration by SP treated,but the virus TCID50 value recovered with the concentration increased to 100nomol/L;The virus TCID50 value increased when the concentration of 20 to 50nomol/L by PMA treated,but the virus TCID50 value decreased when the concentration of 100 to 200nomol/L. These indicated by SP and PMA treated can affect the virulence and proliferation of DEV.3. The effect of transcription level of PKC,PKCI and DEV-NP gene by SP and PMA:Treated the DEF cells were cultured monolater by SP and PMA,and infected with DEV,then the cell cultures were colledted,the transcription level of PKC,PKCI and DEV-NP gene was detected and analysised by real-time fluorescent quantitative PCR,the results showed:the transcription level of PKC, PKCI and DEV-NP gene was 2.62×109copies/μL, 2.61×102copies/μL and 6.65×100copies/μL by SP treated;respectively,the transcription level was3.92×108copies/μL, 5.24×101copies/μL and 2.55×108copies/μL by PMA treated; and control group the transcription level was 8.11×106copies/μL, 5.83×101copies/μL and2.74×102copies/μL. These indicated that by SP treated can reduce the proliferation level of DEV,but it can improve by PMA treated.