Kinetic Study Of PCV2d VLPs In Mouse Lymph Nodes

The application of virus-like Particles started from the development of human vaccine,attributed to its safety and effectiveness.After decades of research,VLPs has long been the research object of various vaccine development platforms.The scope of VLP technology is reflected not only in the development of vaccines,but also in drug delivery,materials and biocatalysis applications.VLPs provides several key advantages for drug delivery:1.Efficient drug delivery rate;2.Uniform self-assembly;3.Artificial modification can activate the function;4.Varied in size and shape;5.Low toxicity and good biocompatibility.In this study,108 samples of diseased pigs from 9 places including ChangSha,YueYang and ZhuZhou in HuNan province were tested to isolate the strains with strong antigenicity.Through DNA extraction and PCR detection of PCV2 cap gene on these 108 samples,the results showed that 57 samples were positive for PCV2,and the positive rate of PCV2 was 52.7%.Cap genes were sequenced in 17 of 57pcv2-positive samples,and 3 samples?17.6%?of PCV2a,5 samples?38.4%?of PCV2b,and 7 samples?53.8%?of PCV2d were obtained.2 samples were not sequenced successfully.Virus isolation was carried out on 5 and 7 copies of PCV2b,and a PCV2b?KU317482?and a PCV2d?MH718995?without other pathogen infection were obtained.The virus titer reached 10^5.5TCID50/ml and 10^5.66TCID50/ml,respectively.Then,the cap gene of the popular PCV2d was used to link pET100 to construct the expression vector pET100-H,and the PCV2 cap protein was expressed by the Escherichia coli expression system.After purified by the Ni column,the PCV2 VLPs was verified by SDS-PAGE,and the assembly product was verified by transmission electron microscopy,and the particle diameter was about 17nm.PCV2 VLPs was identified by exclusion chromatography and indirect immunofluorescence.Finally,the dynamics of PCV2 VLPs in popliteal lymph nodes of mice were investigated.After subcutaneous injection of PCV2 VLPs into the plantar region,mice were sacrificed at the specified time point,popliteal lymph nodes and inguinal lymph nodes were taken for frozen sections,and the dynamics of VLPs in lymph nodes were studied by immunofluorescence.The results showed that after immunization,PCV2d VLPs was divided into two parts and entered popliteal lymph nodes successively.The first part of VLPs was detected at capsule in the popliteal lymph nodes 5 minutes after immunization.most of the antigen entered the medulla area through the trabecula,and a small part of the antigen was transported into the follicles by interacting with non-specific B cells.With the passage of time,VLPs was palpated from the lymph nodes into the cortex and co-located with B cells.At 12h,the first part of VLPs signal was weakened.With the passage of time,VLPs entered the cortical area from the subcapsular sinus and co-located with B cells.At 12h,the signal of the first part of VLPs was weakened.In the second part,VLPs entered the lymph nodes between 12h and 24h.Most of the antigens were recognized by B cells and transported to the B-T cell boundary.A small part of the antigens were transported to the medulla area via trabecula.The formation of germinal center was observed for the first time on the 4th day after immunization.After 3 days of proliferation,a complete germinal center was finally formed,and a large number of VLPs signals were found in the light zone of the germinal center.The reaction of Germinal center stopped after2 weeks,and PCV2 VLPs was still present in the popliteal cortical region at day 28.No PCV2d vlps signal was detected in the inguinal lymph nodes.The results of this study provide an experimental basis for the research of the vaccine based on PCV2d VLPs and its drug carrier.