| The influenza virus is a family of orthomyxoviridae,which kills hundreds of thousands of people every year in the world.Influenza virus is divided into three types:type A,type B and type C.Among them,type A influenza virus has variability and is often closely linked with a worldwide pandemic.Vaccination can prevent and control influenza outbreaks.Compared with traditional vaccines and DNA vaccines,mRNA vaccines have the advantages of high safety,strong immunogenicity,rapid design and synthesis in vitro,and large-scale production.Due to the presence of a large number of RNases in vivo and in vitro,nRNA is unstable and can easily cause degradation.Appropriate delivery techniques can improve the stability of nRNA vaccines and effectively enhance the immune response elicited by vaccines.In this paper,we designed the influenza virus H1N1-FM1 strain HA mRNA vaccine,obtained HA mRNA vaccine through a suitable in vitro transcription system.We designed and prepared DEC205 scfv/tp fusion protein,studied effects of the HA mRNA vaccine-fusion protein complexes on immune effects in mice.1.mRNA vaccine design,construction,and in vitro expression assayThe design of mRNA vaccine target gene is based on the HA gene of H1N1-FM1 strain and sent to the company for synthesis.The broth returned by the company was amplified.The plasmid extracted from the broth was digested with Xbal and Pmel,and the HA mRNA vaccine was obtained by in vitro transcriptase cleavage.The HA mRNA vaccine was transfected into 293T cells by Lipofectamine2000.Western blot results showed that the HA mRNA vaccine was successfully expressed in cells.2.Study on in vivo delivery system of HA mRNA-DEC205 scfv/tp fusion proteinDendritic cells(dendritic cells,DCs)are the most important antigen-presenting cells in the body and have the ability to initiate immune responses.Research shows that targeting DCs surface membrane protein molecule DEC205 can enhance the ability of DCs to ingest,process,and present antigens.Protamine is a positive charge basic protein that can form complexes with negatively charged HA mRNA to prevent the degradation of mRNA.The DEC205 scfv(single chain antibody fragment,scfv)fusion protein can form complexes with mRNA to increase mRNA stability and targeted cell uptake capacity.The truncated protamine(truncated protamine,tp)is derived from protamine,has a large number of basic amino acid sequences and possesses the same ability to bind nucleic acids as protamine.Based on these characteristics,we design and synthesize DEC205 scfv/tp DNA fragment,ligated into the expression vector,transformed into E.coli for expression and extracted DEC205 scfv/tp plasmid,and expressed and purified the protein by eukaryotic expression system.According to the results of Coomassie brilliant blue staining,western blot and LC-MS,the purified protein was DEC205 scfv/tp fusion protein.In the HA mRNA-fusion protein complex immunopotency test,animals were divided into five groups:blank group,control group,protamine-HA mRNA(mass ratio 1:2)group,fusion protein-HA mRNA(mass ratio 1:1)group,fusion protein-HA mRNA(mass ratio 1:2)group.The mice were immunized with 10 ug of HA mRNA by intradermal injection.The mice were immunized at 0,14 and 28 days respectively.After the third immunization,the mice were infected with a lethal dose of the H1N1-FM1 strain virus on the 14th day.We observed changes in body weight and survival in mice and evaluated the immune effects of HA mRNA.The results showed that the fusion protein-HA mRNA vaccine(1:1)group had 60%survival rate;the fusion protein-HA mRNA vaccine(1:2)group had 40%survival rate.The results showed that the fusion protein mediated HA mRNA vaccine delivery had a certain degree of immunoprotection,and which increased as the dose of the fusion protein increased. |
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