Expression Of Nonstructural Protein3of Japanese Encephal Itis Virus And Study The Effect Of Japanese Encephalitis Virus N S3Protein On Its Invitro Replication

Using the swine Japanese encephalitis virus (Japanese encephalitis virus, JEV)-Sichuan Ya’an isolate strain YA as material to do the following research:Molecular cloning, prokaryotic expression of NS3gene, preparing polyclonal antibodies and st udy of the effect on the recombinant NS3protein of Japanese encephalitis virus pr oliferation,and the main results are as follows:1. The expression plasmid construction, prokaryotic expression of NS3gene and preparation of polyclonal antibodyThis study using RT-PCR technology, amplified the whole ORF of NS3gene f rom the total RNA of swine Japanese encephalitis virus’s YA strain cultured in vitr o,and subcloned it into PMD19-T vector,and constructed the ecombinant plasmid P MD19-T-JEV NS3. Verified by double-enzyme cleavage(EcoR I、 Sal I) and genes equence analysis,and identified to be correct.Linked the NS3fragments into the pro karyotic expression vector pET-32a(+) double-enzyme cleavaged by EcoR I and Sal Ⅰ.Transformed recombinant plasmid pET-32a-NS3into the host strain Rosetta(DE3), fusion protein of NS3about88kD mass expressed successfully after IPTG inductio n.The optimum expression conditions are:When the final IPTG concentration is0.3mmol/L,and induced for4h at the temperature of37,most of the recombinant pr otein were inclusion body.Purified the protein by the Ni2+affinity chromatography, and renatured it by removing the urea. Prepared the rabbit anti NS3polyclonal anti body with the purified recombinant protein, the titer of agar diffusion test was1:8.2. The effect on the proliferation of JEV by the recombinant NS3proteinInoculated four bottles of JEV as normal, then added the NS3prokaryotic expr ession protein in the concentration of0.00mg/ml、0.42mg/ml、0.84mg/ml and1.26m g/ml,and paralleled with the one bottle inoculated with JEV and one bottle of BH K-21.Collected extracellular virus at different time points (0h,2h,4h,6h,8h,12h,24h,36h,48h and72h).Constructed recombination eukaryon expression plasmid pCI-neo-NS3and transfected it into BHK-21cell. Western-blot test results sh owed that BHK-21cells transfected with the recombinant plasmid can express a lar ge number of NS3protein. After inoculated with JEV,collected intracellular and ext racellular virus at different time points(0h、2h、4h、6h、8h、10h、12h、16h、20h and24h).Using the fluorescence quantitative method to detect the content ch anges in the content of JEV virus particles both intracellular and extracellular at ea ch time point,and analyze the data by using SPSS statistical analysis software to c ompare the proliferation of JEV.The results showed that:after the normally inoculat ed group infected by JEV, the virus content grew exponentially in10h, and reache d its maximum in18h (42982.49±1148.66copies/ml), then proliperated steadily; As the vrius adsorbed, proliperated and excreted,after6h in the supernatant, the vir us content grew exponentially, and reached its maximum in48h (85062454.36±1159786.29copies/ml). Statistical methods of analysis showed that the virus content of int racellular and supernatant in the group infected the eukaryotic expression vector wa s significantly higher than the control groups (P<0.01), which showed that the NS3eukaryotic expression protein could significantly accelerate the Japanese encephalit is viral replication;However, on account of the impact of external factors, the virus content of supernatant in the group which was added the prokaryotic expressed NS3protein was significantly lower than the control groups(P<0.01),which showed that it didn’t accord to the theoretical results.