Purification Of Porcine Circovirus Type 2 Antigen With Gram-positive Bacterial Enhancer Matrix

The immunization of porcine circovirus-associated disease(PCVAD)has important economic value.Currently,all commercial vaccines against PCV2 are made of virus culture with PK15 cell and oil adjuvant in China,while accompanied by great side effects clinically.Side effects are generally caused by the low-purity PCV2 vaccine antigens.Moreover,free nucleic acid of PCV2 may lead to immune suppression.Therefore,the study of concentration and purification for PCV2 antigen has significantly practical value,enhancing the quality of domestic vaccines.Gram-positive bacterial enhancer matrix surface display system,composed of GEM particles and protein anchors,is a novel technology of antigen preparation developed in recent years.GEM particles are nonviable spherical cell wall peptidoglycan skeleton,obtained by boiling Lactococcus lactis MG 1363 with hot acid and removing all the nucleic acid and protein of bacteria.PA protein anchors are a domain structure of N-acetylglucosaminidase,which can non-covalently bind to GEM particles.Studies have shown that exogenous protein fused with PA can anchor on the surface of GEM particles.Thus,GEM technology can be applied to the concentration and purification for PCV2 antigen by the means of designing and expressing the fusion proteins of PCV2 specific nanobody and PA.This research successfully established the GEM purification method of PCV2 virus and Cap protein VLP,and then GEM-PCV2 vaccine was made and its immunogenicity was preliminarily evaluated,which lays the foundation for the development of high potency PCV2 vaccine.The main contents are as follows:1.Preparation of the basic components of GEM-PCV2 purification systemPreparation of GEM particles:Lactococcus lactis MG 1363 were grown in fresh GM17 broth at 30? overnight.The cells were collected by centrifugation and resuspended in 0.1M HCl.The cell suspension was placed in boiling water for 30 min and then washed three times with PBS,and finally stored at-80 ? until use.The transmission electron microscopic(TEM)images clearly showed that the GEM particles stayed the original shape and size,with smooth surface,and all the intracellular contents were removed.Preparation of adaptor fusion protein:the N-terminal of adaptor fusion protein is PCV2 specific nanobody and the C-terminal is protein anchor.After the double digestion of recombinant plasmid pZQ-Cap-PA and pZQ-Nb stored in the laboratory,the recombinant protein anchors-nanobody prokaryotic expression plasmid pZQ-PA-nb was constructed by the connection of aboved two plasmids.Then the obtained pZQ-PA-nb was transformed into BL21 competent cells to induce expression.The result of SDS-PAGE revealed that the purpose protein,known as adaptor fusion protein of approximately 39 kDa,was expressed as insoluble inclusion bodies,and the most refolding adaptor fusion proteins existed in the supernatant.Preparation of GEM-adaptor protein complexes:1 U GEM particles were resuspended in 200 ?L adaptor fusion protein for 30 min at room temperature and the precipitation were collected by centrifugation.The results of TEM showed that a large number of small flocs appeared in the surfaces of GEM particlesafter the binding of GEM particles and adaptor fusion protein.2.Construction of PCV2-GEM purification methodThe mixture of precipitation GEM-Pb and PCV2 virus(NJ strain)was incubated at 37 ? for 1 h,and then centrifuged at 6000 rpm for 5 min.The supernatants and precipitations were separately collected for SDS-PAGE,electrophoresis analysis,TEM,western blotting,indirect immunofluorescence assay and PCR.All above identification confirmed that GEM-Pb could specifically bind to PCV2 virus and no virus remained in the supernatant after centrifugation,and only PCV2 virus,adaptor fusion protein and GEM particles leaf in the precipitation,which indicated that the precipitation GEM-PCV2 had a good reactogenicity as the original PCV2 virus and the concentrating and purifying PCV2 virus method based on GEM technology was constructed successfully.3.Detection of immune protective efficacy for GEM-PCV2 vaccineGEM-PCV2 was used as antigen to produce experimental vaccine,and then administrated piglets.2-3 weeks old piglets which were PCV2 antigen and antibody double negative were divided into 6 groups of 5 animals each,GEM-PCV2 single dose group,GEM-PCV2 fivefold dose group and four control groups.After immunization 21 days,the blood serum was separated and the PCV2-specific antibody levels were examined by PCV2 ELISA antibody detection kit.All immunized piglets were administrated with PCV2(NJ strains,105.0 TCID50/mL)intranasally and monitored in the next 25 days' changes,and then weighed,slaughtered and dissected.The results showed that,the PCV2 specific seropositivity of GEM-PCV2 fivefold dose group was 3/5,GEM-PCV2 single dose group was 1/5,and the other groups were disqualification.After administration 25 days,the body temperature and mental state of GEM-PCV2 fivefold dose group were normal with sharply rising serum antibody level,while that of Ingelvac CircoFLEX(?)commercial vaccine control group declined.The average daily gain of GEM-PCV2 fivefold dose group was lower than that of Ingelvac CircoFLEX(?)commercial vaccine control group.Meanwhile,the lung anatomy of GEM-PCV2 fivefold dose group showed no lesion and PCR test results were negative.Therefore,GEM-PCV2 fivefold dose group is promising for further research.4.Construction of purification methods for PCV2-VLP-GEMPreparation of PCV2-VLP:The recombinant capsid protein was expressed by E coli expression system CVC1102.Most capsid proteins existed in the soluble form with high expression level The results of western-blotting identified that the protein had good reactogenicity,and the recombinant Cap protein in vitro self-assembly formed large numbers of VLP observed by TEM.Construction of purification method:the mixture of GEM-Pb and Cap proteins were incubated at 37 ? for 1 h and then centrifuged at 6000 rpm for 5 min The supernatants and precipitations were separately collected for identification.The results of SDS-PAGE electrophoresis and western blotting showed that GEM-Pb and Cap protein VLP could bind specifically and effectively,and the majority of other proteins could be removed by centrifugation once;meanwhile the results of indirect immunofluorescence assay revealed that the PCV2-VLP-GEM presented a clear bright spot.Thus,this antigen concentration and purification technology is successful and lays a solid foundation for developing a new generation of PCV2 genetic engineering vaccine.