| Porcine circovirus type 2(PCV2)is the primary pathogen causing porcine circovirus-associated disease(PCVAD).PCV2 infects the immune system of pigs and causes immunosuppression of pigs,which in turn triggers mixed infections or secondary infections with other pathogens,which seriously endangers the pig industry in my country.Cap protein is the only structural protein of PCV2.Studies have shown that 60 Cap proteins can be assembled into a virus-like particle(VLPs).VLPs have a structure similar to real viruses and contain no viral nucleic acid,so they have good immunogenicity and safety.It is one hotspot of new vaccine research and development.The immunogenicity difference between the whole virus inactivated vaccine and the VLPs vaccine is still unclear.In this study,PCV2-VLPs were prepared using 293T cell expression system,PCV2-VLPs and their parental viruses were purified by affinity chromatography,and BACB/c mice were used as animal models to compare the immunogenicity of PCV2-VLPs and PCV2.The above research has laid the foundation for the research and development of new vaccines.To construct a recombinant plasmid(p CAGGS-Flag-Cap)expressing PCV2-Cap protein,the genomic DNA of PCV2-LG strain and a pair of specific primers were used to amplify the PCV2-Cap protein gene with 702 bp,and the amplified gene was then cloned into the eukaryotic vector p CAGGS-Flag by double enzyme digestion method.After sequencing to ensure the correct cloning,the Flag tag located at the N-terminus was mutated to the C-terminus of Cap protein by two mutation PCR with two pairs of primers successively,and a recombinant plasmid with the tag(p CAGGS-Cap-Flag)was constructed.The recombinant plasmid was identified by double enzyme digestion and sequencing,and then transfected into 293T cells to identify the expression of the target gene.The result of restriction enzyme digestion showed that the electrophoretic band size of the constructed recombinant plasmid p CAGGS-Cap-Flag was in line with expectations.A large amount of green fluorescent signal was found in the p CAGGS-Cap-Flag-transfected 293T cells,indicating that the Cap protein was successfully expressed in 293T cells.In order to better compare the immunogenicity of PCV2-VLPs and PCV2,the above two antigens need to be purified.The recombinant plasmid p CAGGS-Cap-Flag was transiently transfected into 293T cells,and the expressed Cap protein was purified by FLAG?M Purification Kit.A total of 3.2 mg of virus-like particles were purified with a purity of 95%was purified from 120 m L of cell culture medium.Observation of the morphology of VLPs formed by the recombinant Cap protein by electron microscope showed that the purified recombinant Cap protein exists in the form of VLPs with a diameter of about 17 nm,which is consistent with natural PCV2 virus particles.The result of SDS-PAGE showed that the recombinant Cap protein was 28.7 k Da and the molecular weight was in line with expectations.Western-blot identification showed that the recombinant Cap protein could be recognized by PCV2specific monoclonal antibody and had good antigenic activity.The VLPs assembled by the recombinant Cap protein had the similar antigen activity as the purified natural PCV2 by capture ELISA.Using PCV2monoclonal antibody 3A5 coupled with CNBr-activated Sepharose TM 4B,an affinity chromatography method was established for the purification of PCV2.A total of 6.5 mg of purified PCV2 with a purity of97%was obtained from 120 m L of virus medium.Only PCV2 virus particles were detected by electron microscopy,and the titers of the purified virus was increased by 100 times.The purified VLPs and purified PCV2 antigen were adjusted to ELISA titers to 1280,and prepared respectively into vaccines for comparison of immunogenicity in mice.Twenty 6-week-old SPF mice were selected and randomly divided into 4 groups,including a PCV2-VLPs immunization group,a PCV2inactivated vaccine immunization group,a challenge control group,and a blank control group.The second immunization in the same dose and route were conducted at 14 days post the first immunization.The mice were challenged via the abdominal cavity at 2 weeks post the second immunization,.The mice were killed at 4 weeks post challenge,and their lung tissue samples were chosen for nucleic acid testing to evaluate the immune effect.The results showed that both vaccines could induce a good PCV2 antibody response,and the antibody titer of the VLPs immune group could reach up to 12800,which was slightly higher than that of the PCV2 inactivated vaccine immune group(6400).The results of the neutralization test revealed that the neutralizing ability of the serum of the VLPs vaccine immunized group was about 62.2%,which was significantly higher than that of the mouse serum of the PCV2 inactivated vaccine immunized group.However,the neutralizing activity of the mouse serum of the above two groups is extremely significantly lower than the neutralizing activity of the pig positive serum,which is close to 100%..The results of nucleic acid detection of immunized animals after PCV2 challenge showed that both the VLPs immunized group and the PCV2 inactivated vaccine immunized group could achieve 100%protection.In summary,the recombinant plasmid p CAGGS-Cap-Flag was constructed and transiently transfected it into 293T cells.PCV2-Cap protein was expressed in 293T cells and found in the form of VLPs.It was verified that the purified PCV2-VLPs have similar morphological structure and antigenic activity to natural PCV2.Both purified PCV2-VLPs and PCV2 were used to prepare vaccines to immunize mice for immune efficacy tests.The results confirmed that both the VLPs vaccine immunization group and the PCV2 inactivated vaccine immunization group can achieve 100%protection,and the level of antibodies induced by VLPs was slightly better than that of PCV2,indicating that VLPs could be used in the development of new PCV2 vaccines. |
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