Construction And Immunogenicity Of Recombinant Adenovirus Expressing Coding Gene Of VP2 Of Porcine Parvovirus

Porcine parvovirus(PPV) has been implicated as one of the etiological agents of porcine reproductive failure disease,the disease caused by PPV is widespread in many countries and has a serious ecomonic impact on the swine industry worldwide.The VP₂ protein is the major objective gene for PPV genetic engineering vaccine,because it is the major capsid protein of PPV and possess favourable immunogenicity.In the study,the VP₂ gene was cloned into the adenovirus expressing vector to generate the recombinant plasmid rAd-VP₂ and expressed in 293 cells,and its immunogenicity was studied with piglets.The study laid the foundation for development of the recombinant vaccine against PPV.1.In this study,a recombinant replication-defective human adenovirus serotype 5(Ad-5) carrying VP2 protein gene was generated by homologous recombination in E.coli strain BJ5183 cells and subsequent transfection of HEK293-A cells.The DNA fragment was coloned by PCR and then was inserted into the multiple cloning site of the transfer vector pShuttle-CMV.The positive recombinant pShuttle-CMV vector was linearized by restriction enzyme Pmeâ… .To obtain homologous recombination,the mixture of linearied pShuttle-CMV-VP2 and adenovirus backbone vector pAdEasy-1 were co-transformed into E.coli strain BJ5183 cells by electroportion.The recombinant adenovirus pAd-vp2 was confirmed by Pacâ… digestion,and then transfected HEK293A cells with Transfast Transfection Reagent Kit.The cell CPE could be observed within one week after transfection.The expression of VP2 of PPV was identified by RT-PCR,indirect immunofluorescent assay(IFA) and the recombinant virus was analysised by Western blot analysis to decide the size of the recombinant protein.The virus titer was 10^-12 TCID₅₀ /ml.Then the virus was passaged by 10 times in HEK293A and the titer was still stable.2.The fragment of VP2 was amplified with a primer pair by PCR.The PCR product was linked with pMD18-T vector,cloned into expression vector pET-32a and transformed into E.coli BL21 for the prokaryotic expression.The interest gene was induced to express in E.coli with IPTG.The expressing product were examined by SDS-PAGE and western-blotting.Results showed that the structural protein VP2 gene of PPV can be expressed successfully in E.coli.Molecular weight of the fusion protein was 45kD and can be recognized by the positive serum of swine that was infected with PPV.and almost of the recombinant protein exists as inclusion body.The expressed protein was purified by His-Bind purification Kit.17μgmL^-1 protein was used as coated antigen to establish an indirect ELISA assay for the detection of antibody in the serum of swine infected with PPV.3.Eight piglets(30-day-old) were randomly divided in four groups and inoculated with recombinant adenovirus and control groups inoculated with PPV vaccine and PBS.At days of 7,14,21,28,42,56 after immunization,the serum samples were colleted for detection of antibody to PPV by indirect enzyme-linked immunosorbent assay and hemagglutination inhibition assay.The result showed that the level of antibody began to rise after 7days of immunization and achieved maximum after two weeks and could continued several weeks.We can conclude that the expression of the vp2 gene of PPV in recombinant adenovirus was stable and could induce PPV special antibodies in piglets.It might play a significant role in developing recombinant adenovirus vaccines of PPV.