Preparation And Immunogenicity Study Of Novel Duck Parvovirus Virus-like Particles

In recent years,duck short beak-dwarfism syndrome caused by the new duck parvovirus（NDPV）has been prevalent in Cherry Valley ducks and Muscovy ducks in some areas of China,seriously affecting the production efficiency of ducks.However,as an emerging disease,the disease still lacks effective means of prevention and treatment.At present,detection methods such as necropsy pathological changes and molecular biology can be used for accurate diagnosis of the disease.The sick ducks are no longer of breeding value,and vaccination is always the best strategy to control the disease.At present,there is no commercial vaccine against this disease.Some farms use gosling plague yolk antibody to prevent infection,which has a certain effect,but the effect is unstable.Therefore,it is of great significance to develop a safe and effective vaccine to prevent this disease.Virus-like particles（VLPs）are protein particles self-assembled by virus capsid proteins,which can mimic the structure of real virus particles,and because they do not contain infectious genetic material,they do not have the ability to replicate in the host body.It is safer than inactivated vaccines and attenuated vaccines.In this experiment,the recombinant VP2 protein was successfully expressed in a soluble form by using the E.coli expression system,which can be self-assembled into NDPV VLPs in vitro,and its immunogenicity was verified by animal immunization experiments,which provided a reference for the development of new subunit vaccines for NDPV.Divided into the following sections:1.The NDPV duck positive material was homogenized and filtered to remove bacteria,and the virus was isolated by inoculating 9-day-old Cherry Valley duck embryos via the allantoic cavity.5 generations of blind transmission were performed,and the embryos showed regular mortality,generalized The viral DNA extracted from the allantoic fluid of the dead duck embryos was identified by PCR and sequencing,and the results were consistent with the NDPV QH-L01 strain isolated and identified in our laboratory.The virus was isolated and cultured using duck embryo fibroblasts,and the infected cells showed lesions,while the TCID₅₀ result was determined as 10^-5.77/0.1 ml.Bioinformatics analysis of the VP2 gene:homology analysis showed 80.2%～99.9%sequence homology with the reference waterfowl minivirus VP2 gene;secondary structure analysis showed that the VP2protein is rich in irregularly coiled,α-helix and elongated chains;hydrophobicity predicts that the protein is a hydrophilic protein;contains glycosylation sites and B-cell antigens epitopes.2.Artificially synthesize the full-length gene of NDPV VP2 protein,insert the VP2 gene fragments into the expression vectors p ET30a and p Cold TF respectively to construct recombinant plasmids p ET30a-VP2 and p Cold TF-VP2,and then transfer them into DH5αcompetent cells respectively,and select positive colonies for PCR,enzyme Cutting and sequencing identification;the constructed plasmids were respectively transformed into expression bacteria BL21（DE3）to induce expression with IPTG,and the expression results were analyzed by SDS-PAGE electrophoresis for solubility.The expression product of p ET30a vector is mainly insoluble inclusion body protein,and the expression product of p Cold TF vector is mainly soluble protein.Select the p Cold TF vector with better soluble expression effect for subsequent experiments,and optimize the expression conditions of prokaryotic expression,including induction temperature,IPTG concentration,and induction time.The final optimal expression condition is:The best induction temperature is 16°C,the best IPTG induction concentration is 0.2 mmol/L,and the best induction time is 12 h.3.The expressed soluble VP2 protein was purified by nickel column affinity chromatography,and Thrombin enzyme was used to remove the TF tag to obtain a single VP2 protein.Western Blot was used to verify its reactogenicity,and a specific protein with a size of about 65～70k Da appeared.The morphology of VLPs was observed by transmission electron microscopy,and the particle size of VLPs was observed by dynamic light scattering.The results showed that the VP2 protein assembled by in vitro dialysis could self-assemble into particles with a size of about 18～25nm and a shape similar to that of Natural NDPV-like VLPs.4.Prepare VLPs oil adjuvant vaccine to immunize mice,and use ELISA method to detect the level of NDPV-specific antibodies in mice.The results show that all immunized groups can induce the body to produce high levels of specific antibodies;trace virus neutralization experiments detect neutralization Antibody level,Neutralizing antibodies play an important role in preventing viral infection,the average neutralizing antibody titer of the low-dose20μg VLPs immunization group was 1:91,the average neutralizing antibody titer of the high-dose 40μg VLPs immunization group was 1:98,which was slightly higher than that of the low-dose group,and the PBS control group had no neutralization Antibody production;lymphocyte proliferation experiments showed that spleen lymphocytes of mice immunized with VLPs could proliferate rapidly after being stimulated by specific antigens,and the levels of cytokines IFN-γand IL-4 in culture supernatant of spleen lymphocytes were higher than The PBS group indicated that VLPs could induce the body to produce cellular immune response.In summary,this study successfully constructed the prokaryotic expression vector of NDPV VP2,and realized the soluble expression of the target protein in E.coli through a solubilizing tag.The purified VP2 protein can spontaneously assemble into virus-like particles in vitro.After immunization with VLPs,It can induce the body to produce humoral immunity and cellular immunity,providing a candidate method for the subsequent development of efficient and safe NDPV new subunit vaccines.