Development Of PCV2 ORF₂ Gene Vaccine, PPV VP₂ Gene Vaccine And The Trivalent Genetic Engineering Vaccine Against Porcine Circovirus Type 2, Porcine Parvovirus And Pseudorabies

Aujeszky's disease (AD), Porcine Parvovirus disease and Porcine Circovirus type2-associated diseases (PCVD) are three important infectious diseases in swineworldwide caused by Pseudorabies virus (PRV), porcine parvovirus (PPV) andPorcine Circovirus type 2 (PCV2), respectively. At present, these three kinds ofinfectious diseases often co-infect or secondarily infect swine herds and causesignificant economic losses. Therefore, safe and effective vaccine is a key point inpreventing and controlling clinical diseases.Pseudorabies virus is an important live-viral vector. It is characterized by that thegenome is a large and linear DNA molecule of about 150kb into which foreign genescan be stably inserted.Furthermore, it is safe to human kinds. The gene-deletedpseudorabies virus(PRV) has been used as the vaccine for eradication program of PRand a live-viral vector to develop bivalent or multivalent genetic engineering vaccineagainst pseudorabies and other swine diseases. In this paper, these experiments werecarded out to develop ORF2 gene of porcine circovirus type 2 vaccine, VP2 gene ofporcine parvovirus vaccine and the multivalent genetic engineering vaccine againstporcine circovirus type 2, porcine parvovirus and pseudorabies.The main researchcontents are presented as the following:1. Expression of ORF2 gene of PCV2 and VP2 gene of PPV in IBRS-2 cellsThe pMD-ORF2* plasmid was digested with Hindâ…¢and Kpnâ… releasing ORF2gene fragment, while the pMD-VP2* plasmid was digested with BamHâ… and Kpnâ… releasing VP2 gene fragment.Then the fragments of ORF2 and VP2 were cloned intothe eukaryotic expression vector, and named pcDNA-ORF2 and pcDNA-VP2,respectively. Which were transfected into IBRS-2 cells with lipofectamine. After 36h,The above IBRS-2 cells were harvested for analysis. Western blotting indicated thatORF2 gene and VP2 gene were expressed in IBRS-2 cells.2. Studies on the immunogenieity and safety of PCV2-ORF2 gene vaccine andPPV-VP2 gene vaccine in miceBALB/c mice that was serologically negative to PCV,PPV and PRV wasimmunized with pcDNA-ORF2 and pcDNA-VP2 plasmids by intramuscular injection twice. Antibodies against PCV2 and PPV were tested respectively by indirect ELISA.The result showed that pcDNA-ORF2 and pcDNA-VP2 gene vaccines could inducemice to produce specific antibody against PCV2 and PPV. At 56d after the firstvaccination, lymphocyte proliferation activity was detected with MTT method, andthe nμmber of CD4⁺ and CD8⁺ T lymphocytes was also assayed by FluorescenceActivated Cell Sorter (FACS). The results of MTT showed that the spleniclymphocytes from mice vaccinated with pcDNA-ORF2 gene vaccines, pcDNA-VP2gene vaccines, PPV live vaccine, PCV live vaccine, PBS and pcDNA-3.1 (+) werestimulated by ConA, the lymphocyte stimulating index (SI) in treatment groups were1.57 and 1.68 respectively, which were significantly higher than those in controlgroups, PBS group (1.0) and pcDNA-3.1+group (1.04); but were lower than those inthese groups, PPV live vaccine group (1.76)and PCV live vaccine group (1.69). Inaddition, the detection results of T cells showed that peripheral blood lymphocytesCD4⁺ decreased while CD8⁺ increased. The above results indicated thatpcDNA-ORF2 and pcDNA-VP2 gene vaccines successfully induced hμmoral andcellular immune response in mice.To confirm the safety of pcDNA-ORF2 and pcDNA-VP2 gene vaccines, thegenomes of vaccinated mice were extracted for PCR amplification.The resultindicated that ORF2 and VP2 genes could not be integrated into the micechromosome, and the pcDNA-ORF2 and pcDNA-VP2 gene vaccines were safe.3. Construction of recombinant pseudorabies virus co-expressing ORF2 gene ofPCV2 and VP2 gene of PPVThe ORF2 gene of PCV2 and the VP2 gene of PPV were inserted into themultiple clone sites of universal transfer vector pPI-2.EGFP to construct the transferplasmid pPI-2.EGFP.ORF2.VP2.The transfer plasmid was co-transfected into ST cellswith the genome of SA215 constructing the recombinant pseudorabies virus.Therecombinant pseudorabies virus was identified by immunofluorescence assay, PCRtest and southern blotting and named SA215(D). Meanwhile, the fusion proteinexpressed by SA215 (D) was detected by SDS-PAGE and Western blotting. Theresult showed that the ORF2 gene of PCV2 and the VP2 gene of PPV weresuccessfully expressed in the recombinant pseudorabies virus.. Furthermore, the virusproliferation test showed TCID₅₀ of SA215 (D) in Vero, IBRS-2,ST and CEF cellshad no significant deviation compared with that of the parent SA215 and that theinsertion of foreign genes had no influence on the propagation of recombinant virus.4. Studies on the viral morphous and hereditary stability of SA215 (D)SA215 (D) was observed through scanning electron microscope. The resultshowed that there was not significant difference between virus particles of SA215 andSA215 (D). In addition, the hereditary stability of SA215 (D) was detected by PCR. The result indicated that ORF2 and VP2 genes had been stably inserted into thegenome of recombinant virus and the recombinant virus SA215 (D) was stable inheredity.5. Studies on the immunogenieity and safety of SA215(D) in miceBALB/c mice that was serologically negative to PCV, PPV and PRV wasinoculated with SA215(D) to evaluate its safety and immunogenicity. The results ofthe animal tests showed that SA215(D) is safe to mice. Furthermore, The vaccinatedmice could acquire protective immunity against lethal challenge of the virulentPRV(PRV Fa strain). Meanwhile, the tissues and organs from the mice vaccinated byFa, SA215 and SA215(D) were collected for pathological section.These resultsindicated the virulence of SA215(D) was significantly lower than that of PRV Fastrain, and equal to that of the parent strain SA215.The above results revealed that itwas a safe vaccine strain.Indirect ELISA test was performed to detect the antibody level. The resultshowed that the recombinant viruses could induce mice to produce specific antibodyin mice against PCV2, PPV and PRV in a(at) higher level. In addition, The result oflymphocyte proliferation assay showed the recombinant viruses could also elicitsplenic lymphocyte of mice to propagate. The detection result of T cells showed therecombinant viruses could improve the nμmber of T cells and strengthen the immuneability of mice. The above results manifested that SA215(D) could successfullyinduce mice to elicit humoral and cellular immune responses.The result of viralpermanent implanting showed SA215(D) could protect from the permanentimplanting of PRV Fa strain in mice.The above results revealed that the recombinant viruses could be suitablecandidate vaccine strain for developing a novel genetic vaccine to combat PCV2, PPVand PRV in the pig industry.