Study Of PCV2 Nucleotide Vaccine And Bivalent Recombinant PRV Genetic Engineering Vaccine

Porcine circovirus type 2 (PCV2) could cause PMWS (Post-weaning pigsmultisystemic wasting syndrome), PDNS (porcine dermatitis and nephropathy syndrome),PNP (proliferative and necrotizing pneumonia), CT (congenital tremors), PRDC (porcinerespiratory disease complex) and reproductive disturbance. PMWS was first reported in1990s in Canada. From then on PCV2 had widely spread in the world and maintain highinfection rate in herd. PCV2 could cause progressive weight lose and high wasting, in theother hand the virus proliferates in the immune system of piglet, and cause disorder ofimmune function. The virus has caused great economic lose in the pig industry. PCV2was first found in 2000 in our country, and now the infection rate of PCV2 in our countrywas almost 50%. Vaccine is the powerful tool for the prevention of the PCV2. But lowvirus yield in cell make it difficult to develop traditional vaccine such as inactive andactive vaccine. Thus the new genetic engineering vaccine would play important roles inthe precaution of PCV2. In this study we have expressed the major capsid protein ofPCV2 in eukarya cells and constructed and assessed the immunoigenicity of DNAvaccine, suicide DNA vaccine and recombinant PRV vaccine.1. Expression and localization of the ORF2 and BVP22 genePCV2 ORF2 gene and BHV-1 VP22 gene are acquired by PCR, and then they arecloned into pMD18-T vector resulted in pTORF2 and pTBVP22, pTORF2 was digestedby Hindâ…¢and Bglâ…¡, meanwhile pEGFP-N1 was digested by Hindâ…¢and BamHâ… , thenORF2 gene is cloned into pEGFP-N1 and conformed that ORF2 and EGFP gene are inthe same reading frame, the plasmid is named pNORF2. The pNBVP22 was constructedby the similar method. The three plasmids: pEGFP-N1, pNORF2, and pNBVP22 aretransfected into Hela cell line by Lipofectamine2000 transfection kit. 18 h aftertransfected a fluorescent microscope is employed for observation. EGFP expression canbe observed in the three groups of cells. But the localization of the fluorescent inpNORF2 transfected cells were only in the nucleus, and the fluorescent in pNBVP22were mainly in the nucleus, but fluorescent can be observed in cytoplasm. These resultsindicate that the ORF2 gene and BVP22 gene that we have cloned could expressefficiency in the eukarya cells.2. Construction and immunogenicity of the nucleotide vaccine of PCV2 The ORF2 and BVP22 gene were amplified by PCR. The cloned ORF2 gene wasinserted into eukaryotic expression vector pCDNA3.1(+) and constructed nucleotidevaccine pCORF2. Then the nucleotide vaccine which contains BVP22 and ORF2 genewere constructed, BVP22 gene which has the function of protein transductionrespectively located the upstream and downstream of ORF2 gene, and the two genes werein the same reading frame. All the above vaccines were intramuscular injected BALB/cmice for twice, two weeks interval. 2 and 6 weeks after the first immune injection, serawere collected and the humoral antibodies were tested by ELISA. The result of ELISAshowed that pCORF2 could stimulate immunoreaction by two immunizations, but themice could not generate high antibodies. Mice immuned by pCORF2BVP22 andpCBVP22ORF2 could generated higher antibodies and all mice immuned bypCBVP22ORF2 were detected seroconvert by twice immunizations. These results showthat the immunogenicity of pCORF2 was low but BVP22 could enhance theimmunoreaction of ORF2 in vivo.3. Construction and immunogenicity of the suicide DNA vaccine of PCV2ORF2 gene was cloned into suicide vaccine vector pSCA1 and pSCHAME2a, resultin pSORF2 and pMORF2. And then the BVP22 gene was inserted into the upstream ofORF2 gene resulted pSBVP22ORF2 and pMBVP22ORF2. 6-weeks old mice in fivegroups were muscular injected with pSCA1, pSORF2, pMORF2, pSBVP22ORF2 andpMBVP22ORF2, and booster 2 weeks later. Sera were collected 2 and 6 weeks after thefirst immunize, humoral antibodies were tested by ELISA. From the ELISA result we cansee that the pSORF2, pMORF2, pSBVP22ORF2 and pMBVP22ORF2 can inducespecitific anti-ORF2 humoral antibodies by one immunization in two weeks. The enhanceeffect of BVP22 was not obviously in the suicide DNA vaccine.4. Generation of a recombination PRV Tk^-/gE^-/ORF2⁺ that can express PCV2ORF2 genePCV2 ORF2 gene was cloned into the PRV transfer plasmid pIECMV then resultedpIEORF2. The genome of PRV vaccinal strain Tk^-/gE^-/LacZ⁺ was digested by EcoR I,then co-transfected with transfer plasmid pIEORF2 into IBRS-2 cell line. Therecombination virus was acquired by three times plaque purifications. PCR and Southernblotting results showed that ORF2 gene has inserted into the expected location. Westernblotting and IFA results showed that the ORF2 gene can express in the recombinant PRV.TCID₅₀ of this recombination virus on different cell lines are IBRS-2 10^-7.1, CEF 10^-4.8,Marc-145 10^-6.8TCID₅₀/0.1mL. 5. Immunogenicity of the recombinant PRV Tk^-/gE^-/ORF2⁺ to PRV and PCV2 inpigletsFour-weeks-old piglets were immunized by the recombinant virus Tk^-/gE^-/ORF2⁺(n=5), Tk^-/gE^-/gI^-(n=5) and DMEM (n=3). Immunization was carried out based on amethod of prime-boost protocol, i.e. the piglets from the three groups received the firstintramuscular injection on one side of the neck, and a boost injection on the other side 3weeks later. Serum were collected on day 0, 21, 35, 49 after the first immunization andimmunogenicity of PRV Tk^-/gE^-/ORF2⁺ was tested by PRV enzyme linkedimmunosorbent assay (ELISA), PRV neutralizing assay, ORF2-ELISA. ORF2 specificlymphocyte proliferation response was tested on day 49 after the first immunozation. Theresults show that PRV Tk^-/gE^-/ORF2⁺ elicited significant humoral immune responses toboth PRV and PCV2, and the PCV2-specific lymphocyte proliferation response could bedetected on day 49 of this experiment. These findings suggest that the recombinant PRVTk^-/gE^-/ORF2⁺ may be a potential vaccine against both PCV2 and PRV.