A Preliminary Study On GD Gene MRNA Vaccine Based On Pseudorabies Virus Envelope Glycoprotein

In recent years,breakthroughs have been made in related technologies in the field of RNA molecules.mRNA vaccines have achieved certain research results in many infectious diseases such as influenza virus,Ebola virus,and Zika virus.mRNA vaccines deliver mRNA to cells and express proteins.,so that the body gets immune protection.mRNA as a vaccine molecule has an outstanding advantage over DNA as a vaccine molecule: it does not require any nuclear localization signal,transcription greatly reduces the risk of mutation and there is no possibility of integration into the genome.Pseudorabies in pigs is one of the most important infectious diseases in pig breeding industry.Vaccine immunity is still the most important way to prevent and control pseudorabies.However,since 2012,pseudorabies virus variants have become more prevalent in the country,causing huge economic losses in the pig industry.In this study,based on the molecular and functional properties of envelope glycoprotein gD,it is of great potential value to innovatively study the PRV envelope glycoprotein gD mRNA vaccine as a vaccine candidate for the prevention and treatment of PRV.The pseudorabies virus XJ strain is a virus template,and the gD gene of the clone envelope glycoprotein is amplified.The gD gene of the envelope glycoprotein is amplified to construct an mRNA cloning vector and an eukaryotic expression vector,and the in vitro transcription of mRNA and the eukaryotic expression are realized.The expression vectors were individually studied for immunogenicity.The main research content is:1.Amplification and Plasmid Construction of the Envelope Glycoprotein gD GeneTwo pairs of specific primers were designed and compared using the biological software Premier5.0,Oligo6.0 and BLAST,and the related research of this experiment.The first pair of primers introduced the BamHI and XhoI restriction sites at 5? and 3?,respectively.Two pairs of primers introduced BamHI,T7 promoter and XhoI cleavage site respectively at 5' and 3'.The DNA extracted from Vero cell cultured and expanded PRV-XJ virus was used as a template to amplify two segmentsof the target fragment and were then ligated with pMD19-T cloning vector and PVAX eukaryotic expression vector to construct the cloned recombinant vector pMD19-gD and eukaryotic expression.The vector PVAX-gD was double-digested and submitted to the company for sequencing and identification,which was exactly the same as the expected target gene sequence.2.In vitro transcription of pMD19-gD cloning vector,immunogenicity of mRNA vaccine and PVAX-gD eukaryotic expression vector.After pMD19-gD digestion,a linearized gD gene fragment of the envelope glycoprotein containing the T7 promoter was recovered,mRNA was obtained after addition of an in vitro transcription system,and the envelope glycoprotein gD gene mRNA and the eukaryotic expression plasmid PVAX-gD were transfected into BHK21 The cells were enriched after 40 hours and verified by western blot.There were clear specific bands.The envelope glycoprotein gD gene mRNA liposome was coated to prepare mRNA vaccine,mRNA vaccine and PVAX-gD eukaryotic expression vector for animal experiments.Five groups of BALB/c mice were immunized twice with thigh muscles,after the first immunization.At the 0th,2nd,4th,6th,and 8th weeks,the venous plexus blood was collected for ELISA antibody detection and virus neutralizing antibody assay respectively;the blood sample ELISA was used to detect cytokines IL-2 and IFN-? at the same time after the first immunization.Flow cytometry was performed to measure changes in T lymphocyte subsets.Analysis of the experimental data shows that both the mRNA vaccine and the PVAX-gD eukaryotic expression vector can induce mice to produce PRV gD-specific antibodies and virus-neutralizing antibodies.After the second immunization,specific antibody levels and virus-neutralizing antibody effects Prices have increased significantly.The proportion of CD4+/CD8+ cells in the experimental group increased significantly.The proportion of CD4+ in the experimental group reached 50%,which was 18%higher than that in the negative group.The proportion of CD8+ in the experimentalgroup reached 16%,which was increased by more than 3% compared with the negative group.In IL-2,the concentrations of mRNA+protective agent group and PVAX-gD group were 1.5 times higher than that of negative mice,while the mRNA group was slightly lower;the concentration of IFN-? mRNA+protective group and PVAX-gD group were negative mice.The 2-fold,mRNA group was nearly 2 times that of the negative mice.The challenge protection experiment further proved that the envelope glycoprotein gD gene mRNA vaccine can resist the virus attack,and the same effect as the DNA vaccine can induce good humoral and cellular immune responses..