| Porcine parvovirus(PPV)and pseudorabies virus(PRV)are two important pathogen which effects on pig industry and both of them lead to reproductive failure in sows,such as delay to oestrus,abortion,embryonic death,mummification,weakling piglet.In this study,we present the generation of a recombinant PRV strain(r PRVSMX-VP2)which could express the VP2 protein of PPV clinical isolate.The biological characteristic and immune efficacy of the recombinant virus was studied,to lay the foundation for the bivalent vaccine,simplify the vaccination schedule and improve productivity.The main research is as follows:1.The screening of the PPV and the clone of VP2 geneIn this study,eight strains of PPV preserved were inoculated to ST cells and a significant CPE appeared.The multiple sequence alignment and phylogenetic trees of VP2 amino acid sequences were constructed.The amino acid homology of VP2 protein was high in different isolates,and only the individual amino acid sites were different.The variation sites of PPV were different in countries,however,the similarity of isolated strains in China was higher.The mutations of VP2 genes in these strains may also be related to their location.Four main groups or clades were formed in the VP2 amino acid phylogenetic trees.Our isolates are mainly in Group I except the HL strain.For the practical application in Hubei province,the VP2 gene of PPV-JB,which was isolated from Wuhan of Hubei province,was cloned and expressed in PRV live vector as antigen.2.Construction of the recombinant virus r PRVSMX-VP2The transfer plasmid pc DNA3.1-LR-VP2 was constructed based on pc DNA3.1(+).The recombinant virus r PRVSMX-VP2 was generated following the co-transfection with pc DNA3.1-LR-VP2 and genomic DNA of r PRVSMX.Non-fluorescent plaques were selected under fluorescence microscopy.After 5 rounds of plaque purification,the recombinant virus was validated by PCR amplification of VP2 gene and EGFP gene and sequencing of VP2 gene.3.The characters of r PRVSMX-VP2Western blotting and IFA were used to confirm the VP2 protein expression in PK-15 cells inoculated with r PRVSMX-VP2.In western blot,a specific 64-k Da band corresponding to the expected size of VP2 proteins was detected in r PRVSMX-VP2 infected PK-15 cells,but not in r PRVSMX infected PK-15 cells and nature PK-15 cell.IFA result manifested that distinct fluorescent signals were observed in the cells infected with r PRVSMX-VP2,but not in those infected with r PRVSMX.The results demonstrated that the VP2 protein was able to be expressed in r PRVSMX-VP2 infected cells.The one-step growth curve confirmed that the insertion of VP2 gene didn't affect the replication of r PRVSMX.The titers of r PRVSMX-VP2 increased gradually and reached the peak at 32 h post infection with titers of 108.2.4.Immunogenicity study of r PRVSMX-VP2 in miceSafety test in mice showed that r PRVSMX-VP2 is safe to mice with a max titer of107.0TCID50.The recombinant virus was injected in mice with 106.0TCID50,and then boost at 21 d post the first immunization(dpi).These results demonstrated that immunization of r PRVSMX-VP2 could elicit robust anti-PRV immune response,which has no difference with r PRVSMX.At 40 dpi,the Hemagglutination Inhibition(HI)antibody elicited by r PRVSMX-VP2 could reach to 27,which was lower than that of PPV vaccine group.5.Immunogenicity study of r PRVSMX-VP2 in growing pigsThe immunogenicity in pigs was similar to that of mice.r PRVSMX-VP2 was injected in growing pigs with 107.0TCID50.After the first immunization,the level of PRV-specific neutralizing antibodies of r PRVSMX-VP2 group and r PRVSMX group had a rapid growth,which can reach to 1:23.27±6.92 and 1:24.87±5.20.r PRVSMX-VP2 group and r PRVSMX group keep the equivalent g B ELISA antibodies at 35 dpi and 56 dpi.PPV-specific antibody elicited by r PRVSMX-VP2 was lower than PPV vaccine group,and the HI antibody r PRVSMX-VP2 group can reach to 1:192±73.9,which was significantly lower than that of PPV vaccine group(1:320±128). |
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