| By means of IFT, rabbit cross immunity test and ELISA we diagnosed the clinical case of Hog Cholera Virus (HCV).The MZ strain of HCV was isolated. Full-length fragments of envelope glycoprotein E2gene of MZ strain amplified with RT-PCR was cloned into PUC18 easy vector. The result showed that HCV E2 gene of MZ strain share 95%,94% with HCV SM strain, Brescia strain in nucleotide sequence respectively, and share 96%,94.2% identity in amino acids sequence respectively. A 1285bp cloned fragments encoding intact envelope glycoprotein E2 was obtained, including its signal peptide sequence (WLLLVTGA) and transmenbrane region (TMR).This study showed epitope was not variable obviously by analysising the hydrophobicity and the varintion of amino acid.It could be high immunogenicity.To construct recombinant virus of PRV and HCV,E2 gene fragment from recombinant plasmid digested with BamHI and Hindlll was cloned in the plasmid PP63LacZ which digested with Bell and EcoRV and transformed into strain DH5 a of E.coli, yielding plasmid PP63LacZE2.The recombinant plasmid PP63LacZE2 was indicated containing E2 gene by PCR and Dot-blot.By cotransfection of PRV Fa TK/gE-/gI-/LacZ (SA215) DNA and recombinant plasmid PP63LacZE2 DNA in Vero cells, 12 recombinant namly SA215(A)1 , SA215(A)2 et al were obtained with calcium phosphate transfection system.The results obtained from biotinlabeled probe of HCV E2,indicating positive. Through digestion with BamHI and Southern-blot acid hybridization of SA215(A)1 strain, this study showed the recombinant virus of PRV and HCV namly SA215(A) was successful. IFT, SDS-PAGE, electrophoresis and Western-blot confirmed HCV E2 gene was expressed in recombinant virus, about 51Kd fragment of envelope glycoprotein E2 gene. This study on the biological characteristics of SA215(A) strain showed this strain could replicated very well on vero cells, BHK21 cells et al. There were variability of cell culture at different cell lines. There was a great variation in inducing CPE between SA215 strain and SA215(A) strain: the time of SA215(A) inducing CPE on vero cell was prolonged, final plaque was smaller than parent virus SA215. SA215(A) wasneutralized by positive sera of PRV, SA215(A) was of serum properties of parent virus . No-morphdogical difference between SA215 and SA215(A) and obviously fall of reproductive capacity of SA215(A) under electron microscope. Through DOT-blot hybridiztion the result indicated SA215(A) and SA215 were of the same distribution on animal ,simply planting the following trigeminal ganglion .21-day-old SPF pigs inoculated intramuscularly SA215(A) strain could be protected from being affected at day 28 after oronasal challenge-exposure with 106 PF.U high-dose virulent PRV Fa strain and at day 42 after i.m. challenge-exposure with 1 ml high-dose virulent HCV SM strain ,when control groups were tenable ,the result showed SA215(A) was high efficacy .2121-day-old SPF pigs vaccinated i.m. with 5 *104, 1* 10s, 5 * 105, 1* 106P.F.U. SA215(A) strain ,challenge-exposure under the same conditions. The lowest immune dosage was determined 1*105 P.F.U.We made the production check technology were of recombinant live vaccine of PRV and HCV,SA215(A) strain was used as virus stock .production and test select chicken embryonic fibre cell and vero cell, repecticly three lot of live vaccine were prepared (01001,01002 and 01003) .The result indicated three lot of vaccines were up to standard . The titer of the vaccine was stable at different lot. The titer of a field dose was usually #105P.F.U. It was not significantly reduced after keeping the vaccine at -20@ and 4-8 @ for one year, but it was obviously reduced at room temperature for short-term, therefore the length of protection was determined at 4-8@ for one year. The safety was tested in newborn piglets,21-day-old post-weaning piglets and pregnant sows injected different dosage of.The fact that no any reverse reaction indicated the high safety of the vaccine. Through titers of antibodies test from injected animal of vaccine in d... |
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