Porcine Parvovirus HN-2011Molecular Biological Characteristics And Expressed VP2Protein In Insect Baculovirus Cell System

Porcine parvovirus (PPV) is considered an important cause of reproductivefailure in swine, embryonic death and resorption, mummified foetuses and stillbirth, as aresult of prolonged farrowing intervals, are typical clinical signs of PPV-inducedreproductive failure; In addition, PPV has gained importance as an agent able to potentiatethe effects of porcine circovirus type2(PCV2) infection in the clinical course ofpostweaning multisystemic wasting syndrome(PMWS). In this study,phylogenetic analysisthe NS1and VP2gene of PPV HN-2011strain, VP2gene was expressed by in insectbaculovirus cell system and analysis of the antigenic characteristics. The purpose of thisstudy was to understand the genetic background of PPV HN-2011strain and compare theimmune effects of classical inactivated PPV vaccine with recombinant VP2subunit vaccine.1.Cloning, Sequencing and Bioinformatics Analysis of Genome of PPV HN-2011StrainFive pairs of primers which cover the whole genome were designed to arnplify thewhole genome fragments by PCR reaction. The positive recombinant plasmids were sent toTaKaRa Biotechnology (Da lian) CO., LTD. for sequencing．The results show that the lengthof HN-2011is4773nt. There is no insertion or deletion in the coding region. There is a127-bp repeat bp which located downstream the part of ORF2encoding the structuralproteins VP1and VP2. PPV HN-2011isolate from Henan China, together with sequencesretrieved from GenBank, consisting of the complete NS1and VP2genes were sequencedand analysed. Phylogenetic analysis NS1and VP2genes shows that the PPV isolates aredivided into four groups, The Chinese PPV isolates are located on Group A and B viruses,only one JY strain in Group C.The selective pressures on the PPV genome shows that theNS1and VP2genes are under negative selection and positive selection, respectively.Mapping polymorphic sites of protein data onto the three-dimensional (3D) structure of PPVVP2revealed that almost all substitutions were located on the external surface of the viralcapsid.2. Analysis of immunological characters and expressed of porcine parvovirus VP2gene byinsect baculovirus cell systemAmplification PPV VP2gene by PCR, and inserted into the pFastbacI plasmid andconstruction the recombine plasmid pFastbac-VP2. The recombinant plasmid was transfected into insect’s cells and obtained the recombinant baculovirus. VP2gene wasexpressed the by insect’s cells. The protein was identified by SDS-PAGE, IFA and HA.The VP2protein emulsified with MONTANIDE ISA206and then immune8weeksguinea pigs. The PPV antibody of guinea pig was detected by HI.We found that recombinantVP2subunit vaccine can be induced high level antibody reaction, and immune withrecombinant VP2subunit vaccine was faster than classical inactivated PPV vaccine.