Isolation And Identification Of Sheep Pox Virus Jilin Strain And Construction And Immune Effect Evaluation Of CVLPs P32

Sheep pox is an acute,intense and highly contagious infectious disease caused by sheep pox virus(SPPV)with a high incidence.Infected sheep would have impaired ability in milk and hair production,have the abortion of pregnant ewes and death in severe cases,causing serious economic losses to the breeding industry and trade around the world.Furthermore,it is one of the animal diseases that must be reported by the Office International Des Epizooties(OIE)and a type of epidemic disease that is tightly controlled in China.Vaccination is the main method to prevent and control sheep pox,but traditional vaccines have many limitations.Inactivated vaccines have weak immunoreaction and is not able to provide cellular immune response.Although attenuated vaccines have good immune effects,but they may cause pockmarks on the body and pregnant ewes may have abortion after immunization.Therefore,the development of a kind of safe,efficient and new vaccine is essential for the prevention and control of sheep pox.Virus-like particles(VLPs)are nano-sized hollow particles assembled from the structural proteins of viruses,which are very similar to real viruses in size but do not contain infectious nucleic acids,so they are safe particles.VLPs have a unique spatial structure,high-density,repetitive and ordered epitopes are exhibited on the surface of the particles.VLPs are about 100 nm,and can be easily taken up by antigen-presenting cells,inducing highly effective immune responses.Moreover,VLPs allow foreign proteins insertion without affecting their assembling and is considered a good vaccine vector platform.In the present study,the Newcastle disease virus-like particles were used as a vector platform to construct cVLPs P32,by embedding the important protective antigen P32 of SPPV using an insect baculovirus expression system to the membrane of Newcastle disease VLPs by GPI anchoring strategy.BALB/c mice and sheep were used as experimental animals to evaluate the immune effect of the particle vaccine from both humoral and cellular immunity,providing safe and effective new vaccine candidates for the prevention and control of sheep pox.The main contents are as follows:1.Isolation and identification of SPPV in a sheep farm in Nong'an,Jilin Province.In December 2014,Sheep from a farm in Nong'an,Jilin Province,suddenly experienced elevated body temperature,mental dysfunction,difficult breathing,increased pulse,conjunctival flushing,etc.There was a lot of mucus secretion around the mouth and nostrils,ulcers were found in the mouth,red spots or pox rashes of different sizes were found in the fur parts such as the tail roots,breasts and labias,which meet the clinical symptoms of sheep pox.Further pathological examination showed that there are a lot of gray acne nodules on the surface of lungs,and lesions in heart,spleen and gastric mucosa were also observed.Pathological sections from the lung showed that there were eosinophilic virus inclusion bodies in some of the alveolar wall epithelial cells,hyperplasia and metaplasia of bronchial mucosal epithelial cells,and some of the mucosa were shed.The pathological sections from the spleen showed lymphocytes infiltration around the artery.Lung SPPV was isolated and a typical SPPV was observed by transmission electron microscopy.Furthermore,the OFTU cells were used to isolate the virus,and the sheep poxvirus RPO30,P32 and K1 genes were amplified and compared with the corresponding sequences of goat pox virus.The results showed that there was 21 bp deletion in RPO30 gene of Jilin isolate,and there was also deletions between 902-925 bp region in K1 gene.The phylogenetic tree analysis showed that although the P32 gene of Jilin isolate has a high homology with the sheep isolate(AY077834)published in GenBank database,but it has formed an independent phylogenetic branch.2.Construction of chimeric virus-like particle VLPs P32.Based on the vector platform of Newcastle disease VLPs and the GPI protein anchoring strategy,chimeric virus-like particles cVLPs P32 were constructed by the insect baculovirus expression system.The extracellular domain of P32 gene was cloned into pFastBac1-GPI vector,constructing the recombinant shuttle plasmid pFastBac1-GPI-P32,which was followed by transforming into DH10bac.After repeated screening of blue and white spots,recombinant plasmid rBmid-GPI-P32was constructed and transfected into adherent sf9 cells.The rBV-GPI-P32 was obtained from cells after three generations of blind passages.The rBV-GPI-P32 and the cryopreserved rBV-M were inoculated to a cell density of 2×10~6/mL sf9 cell suspension according to MOI=5(1:1).After incubation at 27°C for 72-96h,chimeric virus-like particles cVLPs P32 were purified by ultracentrifugation and sucrose density gradient.The results of transmission electron microscopy showed that hollow nano-particles of about 100 nm formed by typical Newcastle disease M protein were observed in cVLPs P32.The results of immunoblot analysis showed that P32 protein and Newcastle disease M protein were both correctly expressed,indicating that the assembling of cVLPs P32 was correct.3.Evaluation of immune effect of chimeric virus-like particles cVLPs P32.The immunogenicity of cVLPs P32 was evaluated using a mouse model,and the parental animals were also used to evaluate the immunological effects of cVLPs P32.Indirect ELISA was used to detect mouse serum antibodies.The results showed that chimeric virus-like particle cVLPs P32 could induce earlier humoral immunity than soluble GPI-P32 protein,and the antibody level was also significantly higher.However,5?g cVLPs P32 induced humoral immune response and 10?g cVLPs P32 is equivalent.Consistent with above phenomenon,flow cytometry analysis of mouse spleen T lymphocyte subtypes showed that cVLPs P32 has stronger ability to induce the differentiation from naive T lymphocytes into CD3~+CD4~+T and CD3~+CD8~+T lymphocytes than soluble P32protein.The immune effect of the parental animal,sheep,showed that the chimeric virus-like particle cVLPs P32 induced sheep to produce higher total IgG antibodies and specific antibodies against P32 protein.In summary,this study constructed a chimeric virus-like particle cVLPs P32that harness the Newcastle disease virus-like particles as a carrier to display the protective antigen protein P32 protein outside the capsule correctly.After immunization,it is able to induce high levels of cellular immune response and humoral immunity response,which lays a foundation for the prevention and control of sheep pox.