Research On The Recombinant Virus Live Vaccine Of Pseudorabies Virus Coexpressing Porcine Parvovirus VP2 Gene And A Marker Enhanced Green Fluorescence Protein Gene

In the research, we successfully isolated one porcine parvovirus (PPV) strain from the embryonic resorption, fetal mummification, abortion and stillbirth which could reproduce on swine kidney primary cell, PK-15, ST and IBRS-2 cell lines, and produce prominence CPE: the infected cells appeared gathering and amalgamation. The isolated strain could agglutinate the RBC of guinea pig, and could be inhibited by the positive PPV serum. Bright green fluorescence was in the infected cells using immune fluorescence assay. Viron was 23nm in diameter and without envelope under electron microscope. According to these assays, we isolated a porcine parvovirus (PPV) strain, named PPV-SC1.A pair of primers of VP2 gene was designed according to PPV NADL-2 genomic sequence reported by Ana I.Ranz. After amplification from PPV-SCl RF-DNA, the fragment which was 2.0kb long was inserted into pUC19 vector, named pPVP2. And the sequence was determinated. The whole VP2 gene was 1740bp long and coded 579 amino acids. Multiple sequence alignment showed that there was little difference between PPV-SC1 VP2 and the others. Codons bias analysis showed that PPV-SC1 VP2 biased in some codons, especially those codons whose third was A, which was the same as PPV-SCl NS1. Hydropathicity analysis showed that there existed hydropathicity regions in 77-82, 291-304, 362-373, 400-411, 465-477 sites of the PPV-SCl VP2. There were no transmembrane helices in PPV-SCl VP2 by swiss TMPRED. There maybe existantigenicity sites in 60-68, 81-88> 266-275^ 351-357^ 398-404 of the PPV-SC1 VP2.After digestion with ApaL I and Mlu I , the reporting gene EGFP from plasmid pEGFP-Cl was inserted into the Stu I site downstream of initiation codon ATG of PRV gl gene, the transfer vector pPI-2.EGFP was constructed. And a pair of primers of VP2 gene was redesigned according to PPV-SC1 genomic sequence and the multi-cloning sites(MCS) of pPI-2.EGFP, Kpn I and BamH I sites were respectively introduced on the upper stream and lower stream of the primers. The VP2 gene obtained by PCR was inserted into pPI-2.EGFP vector, and the expression vector pPI-2.EGFP.VP2 was then constructed. Assayed using the methods of Dot-blots hybridization, restriction enzymes digestion, fluorescence observation and ELISA, the results suggested that the construction of pPI-2.EGFP.VP2 was successful. The successful construction of pPI-2.EGFP.VP2 would lay a foundation on the development of a genetically engineered PRV bivalent marker virus.PRV SA215 DNA and recombinant plasmid pPI-2.EGFP.VP2 DNA were co-transfected into Vero cells with calcium phosphate, and several recombinant virus strains were screened out with fluorescence microscopy assay and Dot-blots hybridization. The recombinant virus named SA215(B) DNA was analyzed with BamH I restriction enzyme digestion, Southern blotting. SDS-PAGE and Western blotting, the results confirmed that PPV VP2 gene had been inserted into the PRV SA215 DNA and expressed about 93kDa EGFP-PPV VP2 fusion proteins in recombinant virus. The results suggested that the construction of PRV-PPV recombinant virus was successful.Researches on the culture characteristics of SA215(B) showed the strain could replicate very well on Vero cells, BHK21 cells, IBRS-2 cells, PK-15 cells, ST cells, MDBK cells, Marcl45 cells and Chicken Embryo Fibroblasts, et al., while there were some culture variability on different cell lines. The time of SA215(B) inducing CPE on most cell lines (excluding ST cells) prolonged, and the plaque was smaller than parent virus SA215. SA215(B) still holded the serum properties of parent virus, which could be neutralized by positive PRV serum. Concerning the morphology of SA215(B) under electron microscope, there was not only the same viron as SA215, but also the analogous porcine parvovirus virus-like particles (PPV-VLPs) formed by VP2 gene expressing andself-assembling. The result of tissue Dot-blot hybridization indicated SA215(B) and SA215 were of the same distribution on swine, simply planting on the underside of trigeminal ganglion. The result of 37-day-old healthy pigs challenged with SA215(B)(PFU 107) showed SA215(B) was safe to pigs. The immunoassay result of 37-day-old healthy pigs inoculated intramuscularly with SA215(B)(PFU 106) showed the anti-PPV HI antibody was up to 1: 24 and the anti-PRV PD5<> was up to 1: 2' \ These results suggested that SA215(B) could be chosen as the PRV-PPV recombinant vaccine strain.