The Research On PCV2 DNA Vaccine Of PVAX1-ORF2-ISS And Combined Immunization With Porcine Cytokine Genes

Porcine circovirus virus(PCV2) is recognized as the essential infectious agent of postweaning multi-systemic wasting syndrome(PMWS),porcine dermatitis and nephropathy syndrome(PDNS),porcine respiratory disease complex(PRDC), proliferative and necrotizing pneumonia(PNP) and congenital temorall(CT-All).It is epidemic in many countries in the world and is also a major infectious disease in intensive pig farms of China.It caused a great economic loss to world's pig industry. Therefore,there are many scientists and other people engaging pig raising paying much more attention to PCV2.However,there has not effective commercial vaccine in China and only a few researches for PCV2 vaccine in foreign countries up to now.Thus,it is very important to develop novel PCV2 DNA vaccine and apply cytokine molecular adjuvant to improve immune effects of vaccine.This research was carried out to construct novel PCV2 DNA vaccine,prepared vaccine with CNP complex and did combined immunization with porcine cytokine molecular adjuvants.The aim of this study was to improve the immunity of PCV2 DNA vaccine and evaluate the effects of porcine cytokine molecular adjuvants,which supply theoretical basis and experimental data for prevention of PCV2.The main content of the research is as follows.1.The isolation and identification of PCV2 Sichuan strain.Using ELISA to detected blood serum antibody of 987 pigs from 9 farms revealed that the PCV2 positive rate was 74.3%.104 specimen of dead pigs from 9 pig farms were identified as PCV2 by PCR and the virus were isolated.The results showed that the PCV2 positive rate of the specimen was 77.9%;A PCV2 whn strain was isolated,presented inclusion body in cell plasm and cell nuclear of PK15 cell,and the virion whose diameter was 12±2nm, presented crystal lattice arrangement by electron microscope;The complete genome of the virus was sequenced and the full-length was 1767nt,which showed 92.7-97.5% genetic homology with the isolates of China and 94.2-96%with European,American and South Africa isolates.We are successfully isolated and identificatied PCV2 in Sichuan province and got accession number in Genbank,which offered new materials to research of PCV2.2.The construction of pVAXI-ORF2-ISS DNA vaccine and the preparation of the DNA vaccine-chitosan-nanoparticle(CNP) complex.The pVAX1-ORF2-ISS DNA vaccine was constructed through the following steps.ORF2 of PCV2 and 11 CpG motif as ISS sequence were inserted into pVX1 eukaryotic expression vector.Then identified by enzyme digestion analysis.The results showed that the two fragments were rightly inserted into vector.CNP was used to coating pVAX1-ORF2-ISS plasmid.The transmission electron microscope and glue blocking analysis indicated that vaccine plasmids combined with CNP and pVAX1-ORF2-ISS DNA vaccine were successfully constructed and prepared.We had successfully constructed a kind of pVAX1-ORF2-ISS novel DNA vaccine with pVAX1 vector and ISS,which was reported at the first time.3.The identification of porcine cytokine molecular IL-4,IL-6 and IL-6/4 and the preparation of CNP as molecular adjuvant.The porcine cytokine molecular IL-4,IL-6 and IL-6/4 eukaryotic expression plasmid offered by ministry of education key laboratory for Sichuan university biological resource and ecological environment.Enzyme digestion analysis was applied to identify the three kinds of plasmid and the results showed that the length of target fragments accorded with expectations.CNP was used to coating IL-4, IL-6 and IL-6/4 plasmid.The transmission electron microscope and glue blocking analysis indicated that all the plasmids combined with CNP and cytokine molecular adjuvant were successfully prepared.We successfully prepared IL-4,IL-6 and IL-6/4 eukaryotic expression vector,which was reported at the first time.4.The combined immunization experiment on pigs using PCV2 pVAX1-ORF2-ISS DNA vaccine and porcine cytokine molecular adjuvant.By testing piglets in every experimental group of specific antibody titers and the quantity of lymphocytes CD³⁺, CD⁴⁺ and CD⁸⁺,analysis and evaluation were achieved about the pVAX1-ORF2-ISS DNA vaccine and porcine cytokine molecular adjuvant.Moreover,safety experiment was performed using white mice.The results showed that pVAX1-ORF2-ISS DNA vaccine could induce the generation of specific antibody and cellular immunity responses,and the duration of immunity could last over 60 days.The effects of pVAX1-ORF2-ISS and pVAX1-ORF2 DNA vaccines were also compared.The result proved that pVAX1-ORF2-ISS was better than pVAX1-ORF2 DNA vaccine(P＜0.05) and the difference was extremely significant(P＜0.01) when compared with empty vector and blank control groups.Also,the three cytokine molecular adjuvant could extremely significantly improve the immune effects of pVAX1-ORF2-ISS DNA vaccine(P＜0.05). IL-6 was the best,followed by IL-6/4 and IL-4.DNA vaccine safety experiment indicated that the pVAX1-ORF2-ISS DNA vaceine was safe and reliable.White mice were common used to study PCV2 DNA vaccine of pVAX1-ORF2-ISS and combined immunization with porcine cytokine molecular adjuvant,and it was the first time using pigs as animal model,which could get more practical experimental data.This research was carried out to construct novel PCV2 DNA vaccine,prepared vaccine with CNP complex and did combined immunization with porcine cytokine molecular adjuvants.It had improved the immunity of PCV2 DNA vaccine and evaluate the effects of porcine cytokine molecular adjuvants,which supply theoretical basis and experimental data for the research of novel,effective and safe vaccine of PCV2.