Design And Evaluation Of E2 Structural Based Nano-Vaccine Of Classical Swine Fever Virus

Classical swine fever?CSF?is a highly contacted and destructive infectious disease caused by classical swine fever virus?CSFV?,also known as classical swine fever or swine cholera.The mortality rate is as high as 80%to 90%,which is one of the most serious infectious diseases in the swine industry.The epidemic and outbreak of the disease has brought huge economic losses to the pig industry.At present,the main method for the prevention and treatment of swine fever is vaccination,in which the development of the Hog Cholera Lapinized Virus?HCLV?has played a positive role in the prevention and control of CSF in China and even in the world.However,the prevalence of swine fever in China is still complex.To control the epidemic and prevent the outbreak of this disease,it has become an inevitable trend to develop a new vaccine that is efficient and safe.The application of nanotechnology in vaccinology,especially in the past decade,has shown an exponential growth trend.Nanomaterials have been widely used as a delivery system to enhance antigen processing,or as an immunostimulatory adjuvant to activate or enhance immunity.Because of its small particle size,low toxicity and good biocompatibility,AuNPs can be used as vaccine vectors to present antigens to improve the immune effect of vaccines.It is an ideal material for preparing nano vaccines.The CSFV E2 protein is a major protective structural protein on the envelope of virion particles and is an ideal candidate antigen for the swine fever subunit vaccine.In this study,the E2 envelope glycoprotein of the CSFV was coupled to the AuNPs and the E2 protein was multiplexed on the surface of the AuNPs to prepare a novel nanovaccine E2-AuNPs.The immunization efficacy was evaluated by immunizing mice.A new idea was proposed for the development of a new type of vaccine,and a certain theoretical and practical foundation was laid.The overall implementation plan is mainly divided into the following sections:1.Preparation and identification of AuNPsAuNPs were prepared using the Turkevich method.1 mL of 1%hydrated tetrachloroauric acid?HAuCl₄??10 mg/mL?was added to 100 mL of double distilled water?ddH₂O?and allowed to boil under vigorous stirring.8 mL of 1%sodium citrate?Na₃C₆H₅O₇·2H₂O??10 mg/mL?was added to the boiling solution,and after 15 minutes of reaction,the color of the solution became red and the reaction was stopped.The shape,size and Zeta potential of the obtained AuNPs were characterized by transmission electron microscopy?TEM?and dynamic light scattering?DLS?.The results showed that the AuNPs had a particle size about 24.4 nm and a Zeta potential about-34.3 mV.AuNPs were monitored by ultraviolet-visible absorption?UV-Vis?scanning surface plasmon resonance and showed a maximum absorption peak at?max=523 nm.2.Expression,purification and identification of CSFV E2 proteinAccording to the amino acid sequence of the E2 protein of the HCLV in the NCBI database,the CSFV E2 protein coding sequence without nuclear localization signal and with no transmembrane region was codon optimized in the E.coli expression system and cloned into the pUC57 vector.The pUC57-E2 gene was used as a template to construct a recombinant prokaryotic expression plasmid pET-28a-E2 and induced by Isopropyl?-D-Thiogalactoside?IPTG?in E.coli BL21?DE3?.The result of SDS-PAGE and Western blot showed that there was a protein band at 43 kDa,which was consistent with the expected result.The target protein mainly existed in the form of inclusion bodies and could react with swine fever virus positive serum.After the E2 protein is denatured,purified and renatured,soluble E2 protein is obtained.The protein was detected by SDS-PAGE and Western blot.The results showed that there was a single protein band at 43 kDa and could be recognized by the anti-CSFV monoclonal antibody,indicating that the purified renatured protein was of high purity and activity.3.Preparation and physical characterization of nano-gold particle-E2 protein particle antigen?E2-AuNPs?1 mL of E2 protein was mixed with 1 mL of AuNPs?0.2 mg/mL?and stirred at 4°C.for1 h.The supernatant was collected by centrifugation?17000 g,30 min?for protein concentration determination.The precipitate was suspended in 10 mM Tris-HCl to prepare nano-gold particle-E2 protein particle antigen?E2-AuNPs?.Through TEM observation,a circle of protein corona was formed around the gold particles after coupling.The DLS measurement showed that the particle size of the gold particles after coupling increased from24.4 nm to 78.8 nm,and the Zeta potential increased from-34.3 mV to-26.1 mV.UV-Vis showed that after coupling,the maximum absorption wavelength of E2-AuNPs migrated from?max=523 nm to 530 nm.The above data all showed that the gold nanoparticles were successfully coupled with E2 protein and form a stable particle complex.4.Biological evaluation of E2-AuNPsThe E2 protein and E2-AuNPs were coated on 96-well plates in serial dilutions and subjected to biological evaluation by enzyme-linked immunosorbent assay?ELISA?,the results show that E2-AuNPs have almost the same antigenic activity as E2.Indirect immunofluorescence assay?IFA?was used to detect the uptake of E2 and E2-AuNPs by antigen presenting cells?APCs?,the results showed that E2-AuNPs group had more E2proteins internalized into APCs.And the E2 protein group and the cytotoxicity assay showed that the AuNPs did not produce cytotoxic effects on APCs.5.Immunological evaluation of swine fever virus CSFV E2 protein nano vaccine?E2-AuNPs?Six-week-old female BALB/c mice were randomly divided into 8 groups.Each mouse was injected subcutaneously with 100?L of vaccine.Each vaccine consisted of E2,E2-AuNPs,E2-CpG,E2-AuNPs-CpG,E2-Freund's adjuvant,E2-AuNPs-Freund's adjuvant,AuNPs and10 mM Tri-HCl as a negative control.Three weeks after the initial immunization,the animals were immunized again and the serum was harvested on the 0th,21st,and 42th days after immunization and detected by ELISA.After 49 days of immunization,mice were sacrificed to take their spleens.Lymphocytes were isolated from mouse for T-lymphocyte proliferation assay.The results showed that AuNPs could significantly increase the specific antibody level of E2 protein in the presence or absence of adjuvant;the E2-AuNPs group had significantly higher stimulation index?SI?than E2 protein group and AuNPs group.The significance of this study lies in the successful establishment of the system of refolding of CSFV recombinant E2 protein inclusion bodies,successful preparation of a novel nano vaccine of CSFV E2 protein,and evaluation of its immune effects.These studies indicate that AuNPs serve as an antigen carrier to enhance immune responses.Therefore,the combination of antigenic proteins and AuNPs will be a simple and effective strategy for the preparation of novel structural vaccines.