| Pseudorabies (Pseudorabies, PR), also known as Aujeszky’s disease and its pathogen pseudorabies virus (Pseudorabies virus, PRV) is a member of the herpes virus family (Herpes viridae)α-herpes virus subfamily (Alpha herpes viridae). The PRV is a linear double-stranded DNA virus with the genome size about150Kb. And G+C content as high as73%. Pseudorabies can cause cattle, sheep and wild animal’s fever, itching outside (except pigs), encephalomyelitis and other symptoms.The pig is the natural host and the main source of infection of pseudorabies virus. Pseudorabies is one ot the major infectious c worldwide, causing tremendous economic losses to China’s pig industry. The previous study found that the capsule membrane glycoprotein gB.the most-conserved gene of the herpes virus,be belonging to the essential glycoprotein. is a major immunogen of the pseudorabies virus, can mediate the interaction between virus and cell. As the target antigen recognized by animal immune system, the gB glycoprotein plays an important role in the diagnosis and eradication of pseudorabies.In this study, the gB glycoprotein of pseudorabies virus was chosen as a research object, using DNAMAN and Oligo6.3software for the analysis of gB gene sequences of pseudorabies virus logged in Genebank. we designed and synthesized three pairs of specific primers respectively: one pair of primers without restriction sites, one pair of fluorescent specific primers and TaqMan probe, one pair of primers containing EcoRl and Xbal restriction sites.one: PRV virus DNA extraction using DNA/RNA extraction kit, a total length of about2.8Kb of the PRV-gB gene was obtained using PCR technology, and was cloned into pMD20-T vector, constructed the recombinant plasmid pMD20--gB successfully. By gene sequencing, comparison with the Genebank, and analysis of the biological characteristics, our laboratory isolated PRV strain was finally identified as Nanyarig strains (Namyangju).two: The recombinant plasmid pMD20-gB was chosen as a positive reference, we established a rapid quantitative PCR using fluorescent specific primers and TaqMan probe for the detection of pseudorabies virus, the detection technique is of high sensitivity, specificity, and reliability. By testing the positive reference, the test results show the established pseudorabies virus TaqMan quantitative PCR detection limit is about1.50×103copies/reaction: its sensitivity is1000times higher than that of the PCR method, and the repeatability is good. By testing of60clinical samples, the quantitative PCR not only detected positive samples tested by the PCR. but also found two more positive samples which are negative by PCR. the results further confirmed the established method is fast and sensitive, can be used in the detection of PRY infection and in the quarantine and inspection of imported and exported meat.three: In order fo obtain a large quantity of lower-cost gB diagnostic antigen, we use specific primers containing EcoRI and XbaI restriction sites, use recornbinant piasmid pMD20-gB as a template, using PCR technology, re-amplified gB gene sequence, and successfully constructed Pichia yeast’ expression vectors pPlCZaA-of gB containing gB main epitope genes, it showed that there is no gene mutations, and protein coding is correct by sequencing and DNAMAN software analysis. Pichia yeast X-33strains of competent cells was used to transform restructured expression piasmid pPlCZaA-of gB. incubating at30℃for about a week, we take a single colony for further liquid culture, the yeast genome was extracted for identification by PCR. the results indicate that this study successfully obtained recombinant bacteria expressing main epitope gene of PRV-gB. Protein expression was induced by1%methanol. the size of the target protein is about103KD by SDS-PAGE analysis. The relationship between the expression quantity of the target protein and the culture conditions was studyed, the optimized expression conditions are: the OD600value of the initial bacteria concentration for inducing is1.2.28.8℃shaker for agitation, add methanol to a final concentration of1%every24hrs. inducing expression for72hrs. and the protein expression approaching maximum. The recombinant protein concentration induced at optimal expression conditions after purification is up to8.715mg/mL.four: the concentrated and purified recombinant protein gB mentioned was used as antigen to coat and prepare ELISA plate for antibody detection, indirect EL1SA was set up for the detection of PRV-gB positive/negative sera. The results show that the recornbinant protein is of good reactionogenicity. which can be used as the detection antigen. The positive sera of classical swine fever, porcine parvovirus. porcine reproductive and respiratory syndrome were tested by the established gB-ELISA. the test results show that the gB protein has no cross reaction with those sera, the specificity is good, can be used for the detection of PRV-gB protein antibody. The gB antigen was used to immunize New Zealand white rabbits, the antibody titer is about1:8by the agar gel diffusion test and neutralization test. This experiment shows that the gB protein has good immunogenicity. Collecting blood from the rabbits which has reached a certain antibody titer level, the sera was pooled as gB antiserum. which will be used for the verification of the diagnostic test kits and for the laboratory quality control. |
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