Reverse Genetics Modification Of Classical Swine Fever Virus Vaccine Strain

Classical swine fever(CSF),also known as pig cholera,commonly known as "rotten intestinal blast",is a high infectious disease of pigs,lethality rate of up to 90% or more,is one of the major infectious diseases to the pig industry.The outbreak of swine fever can lead to serious economic losses,the World Organization for Animal Health has classified it as a class A infectious disease,it has received the attention of various countries and has become a hotspot in various countries.The swine fever virus is an envelope virus with a size of about 40-60 nm and its genome size is about 12.3 kb,which is a single stranded stranded RNA that contains a large open reading frame(ORF),a 5’ untranslated region(5’UTR)and a 3’ untranslated region(3’UTR).The open reading frame can encoding about 3900 amino acid’s polyproteins and then producing 12 mature proteins under the action of signal peptidase,in the order of Npro-C-Erns-E1-E2-p7-NS2-NS3-NS4A-NS4B-NS5A-NS5 B.Among them,C,Erns,E1 and E2 were four structural proteins.Npro,P7,NS2,NS3,NS4 A,NA4B,NS5 A and NS5 B were 8 nonstructural proteins.In the first part of this study,p MC18-CMV-HM-CSFV was constructed by inserting CMV promoter and hammerhead ribozyme according to the infected swine fever virus’ s c DNA,which was constructed in the laboratory,and used for the rescue of classical swine fever virus.In order to verify the function of hammerhead ribozyme,we constructed four plasmids p GL2.0-CMV,p GL2.0-CMV-HM,p GL2.0-CMV-HM’ and p GL2.0-CMV-HM-5’UTR.The results showed that the hammerhead ribozyme can play about 62% of the shear effect by Dual-Luciferase Reporter Assay System and real-time quantitative quantitative PCR detection system.After the QPCR assay,it was found that the transformed plasmid p MC18-CMV-HM-CSFV was successfully rescued by transfection into the cells.This study laid the foundation for the development of swine fever vaccine.The second part of this study investigated the relationship between the core protein and the 5’UTR.The previous study in the laboratory found that the protein C of the classical swine fever virus had a modulating effect on the 3’UTR..In order to determine whether there is an interaction between the C protein and the 5’UTR,dual-luciferase expression plasmid p MIR-5’UTR-RLUC and p MIR-RLUC were constructed in this experiment.The detection of p MIR-5’UTR-RLUC into the cells by Dual-Luciferase Reporter Assay System revealed that the 5’UTR could play a corresponding role.The eukaryotic expression vector p CMV-C of classical swine fever virus C protein was co-transfected into PK-15 cells with 5’UTR eukaryotic expression vector p MIR-5’UTR-RLUC,and then detected by western blotting,the Real-time Quantitative PCR Detect ing System and Dual-Lucifer ase Reporter Assay System showed that there was no interaction between the C protein and the 5’UTR.This part of the study laid the foundation for exploring the effect of classical swine fever virus’ protein on 5’UTR function.