| In 2016,American research scholars detected a novel circovirus in a sow that was clinically manifested as Porcine dermatitis nephritis syndrome(PDNS)and named it Porcine Circovirus 3(PCV3).In addition to affecting sow papular dermatitis,nephrotic syndrome and abortion,PCV3 can also cause poor growth of pigs.Besides,PCV3 is easily co-infected with PCV2,PRRSV and PEDV,which aggravates the incidence of pigs and seriously affects the world pig industry.The report of PCV3 in China can be traced back to1996.The disease has been widely spread in more than 20 provinces in China since 2016,and the antigen positive rate is 0%-26.32%.There is no effective treatment method,no effective cell line for amplifying PCV3 and no cross-protection of PCV3 and PCV2,so it is extremely urgent to carry out PCV3 genetic engineering vaccine(especially VLPs vaccine).At present,the genetic engineering vaccine of circovirus is mainly Cap VLPs produced by baculovirus and prokaryotic expression systems.In this paper,based on the more mature baculovirus expression system,PCV3 Cap protein was expressed,and the assembled VLPs were purified.The immunogenic agar expansion assay of VLPs was established,and the PCV3 VLPs produced by baculovirus were proved to be highly immunogenic.The research results are as follows:(1)Construction and identification of recombinant vector pBac-cap3-6HisIn this study,the PCV3 cap gene was cloned into the multiple cloning site of pBac-KB3-B2-6His to obtain the recombinant vector pBac-cap3-6His.This was co-transfected with linearized Bacmid into Sf9 cells to package recombinant baculovirus.The recombinant baculovirus was extracted and identified by genomic DNA,Western blot and electron microscopic observation.The results of gene identification indicated that the recombinant baculovirus genome contained the target gene.Western blot analysis showed that the PCV3 Cap protein could be successfully expressed.Electron microscopy showed VLPs with a diameter of about 16-18 nm,indicating that the PCV3 Cap protein was successfully assembled into VLPs.(2)Purification and electron microscopic observation of PCV3 Cap proteinThe supernatant collected from the lysed cells of the previous step was centrifuged by CsCl density gradient to purify the PCV3 VLPs.The purified VLPs were also identified by Western blot and electron microscopy.Western blot analysis showed that the purified Cap protein was highly soluble in the cytoplasm.High-purity VLPs with a diameter of 16-18 nm were observed under transmission electron microscopy.(3)Establishment of agar expansion method and detection of immunogenicity by agar diffusion methodWith reference to the PCV2 agar expansion assay,we used the PCV3 sample expressed in the prokaryotic expression of our laboratory as the detection antigen;after the PCV3 VLPs emulsified with 206 adjuvant,the rabbit was immunized subcutaneously,and the immunization was performed 3 times at intervals of 2 weeks.Serum collected after secondary immunization and third immunizations as antibodies;the prokaryotic expression of PCV3 was used as a control,and the PCV3 VLPs agar expansion method was established.The test results showed that after the second immunization,the plunging titer can reach 1:16,and after the third immunization,it can reach 1:64.This proves that it has good immunogenicity.In summary,this paper successfully expressed the PCV3 Cap protein by using the baculovirus insect expression system,and the PCV3 Cap protein can self-assemble into VLPs.After ultra-ion purification,the VLPs have uniform size,high purity and good immunogenicity.These results provide ideas and ways for the development of the PCV3 vaccine. |
