Expression And Immunogenicity Of The Recombinant Capsid Protein Of Porcine Circovirus Type2in Insect Cells

Porcine circovirus type2(PCV2) was considered to be the main pathogen resulting in post weaning multisystem wasting syndrome (PWMS) which contributed to the immune suppress of infected pigs. PCV2infection was associated with kinds of porcine diseases, including respiratory diseases, reproductive failure, porcine dermatitis and nephropathy syndrome (PDNS), enteritis which are often referred to as porcine circovirus-associated diseases (PCVAD).PCVAD was a great disaster for the major swine producing countries and was economically important. The capsid is the main immunogenic protein of PCV2which contains a series of epitopes. So the capsid protein has been the main target for development of PCV2-specific serodiagnostic methods and vaccines. In this study, recombinant baculoviruses and insect cell lines expressing the capsid protein of PCV2were constructed and immunogenicity of recombinant capsid from baculovirus expression system was analyzed. The contents of this paper are including:1. Construction and identification of recombinant baculovirus expressing Cap protein of PCV2The ORF2genes of PCV2were obtained and cloned into donor plasmid pFastBac. After identification by PCR, double enzymes digestion and sequencing analysis, the recombinant donor plasmid was transformed into competent DH10Bac cells, and then the recombinant bacmids were screened and identified by blue-white plaque assay and PCR. The correct recombinant bacmids were prepared and transfected into Sf9insect cells, and then recombinant baculoviruses were obtained and confirmed by SDS-PAGE, Western blot, indirect immunofluorescene assay, sandwich ELISA and electron microscope. Experiment results showed that Cap proteins of PCV2were expressed in Sf9cells successfully and one of them (rBcapkm) could self-assemble into virus-like particles (VLPs) which were morphologically similar to natural PCV2particles. The construction of recombinant baculoviruses expressing PCV2Cap would be useful for the development of subunit vaccine against PCV2.2. Construction and identification of insect cell line expressing Cap protein of PCV2The aim of this study was to construct a recombinant plasmid that including cap gene of PCV2and to establish the Sf21and HF cell line which could express cap protein stably. Cap gene without NLS was amplified and cloned into pMIB-V5/HisA.The recombinant pMIB-V5/HisA was confirmed by sequencing and double enzymes digestion analysis and then was transformed into sf21cells via liposomes. The positive anti-Blasticidin cells were screened, and identified by Western blot. The results showed that the recombinant pMIB-V5/HisA was constructed correctly.After transfection, the Sf21and HF cell clones expressing cap protein were obtained with Blasticidin pressue selection. Western blot analysis showed that the recombinant cap protein had good reactogenicity. All the above results indicated that insect cell lines expressing cap protein were established successfully and this may be a ideal method to produce sub-unit vaccine against PCV2and other virus diseases.3. Immunogenicity of recombinant Cap of PCV2expressed in baculovirus expression systemIn this study, the immunogenicity of the recombinant Cap protein of PCV2expressed by baculovirus was examed by animal immunization. The mice immunization experiment showed that two vaccines (CPH and CP) based on this recombinant Cap protein were able to induce significantly higher titers of antibodies against PCV2and strong lymphocyte proliferation responses. The results of pig experiment showed that pigs vaccinated with CPH and iPCV2developed anti-PCV2ELISA antibody and neutralizing antibody after immunization. At the same time, no humoral immune response could be detected in the control groups. After challenge, pigs vaccinated with CPH and iPCV2showed little clinical signs compared with the challenge group. The viral load of serum and lymph node, microscopic pathological lesions of lungs and lymph node of the immunized pigs were lighter than that of the challenge group. It indicated that the recombinant Cap protein expressed by baculovirus could induce strong immune responses and provide sufficient protection against PCV2infection in pigs. So this research may be useful for further study about PCV2vaccine.