Dissertation > Agricultural Sciences > Livestock, animal medicine,hunting,silkworm,bee > Animal Medicine ( Veterinary Medicine) > Livestock, poultry, wildlife diseases > Livestock > Pig

Development of PCV2 DNA Vaccine and Establishment of Indirect ELISA Method

Author ShiYunTong
Tutor ShaoLiangPing;ZhouLunJiang
School Fujian Agriculture and Forestry University
Course Clinical Veterinary Medicine
Keywords Porcine circovirus type Ⅱ PcDNA3.1 DNA vaccines Immune effect The prokaryotic expression Indirect ELISA
CLC S858.28
Type Master's thesis
Year 2010
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Objective: based on the recombinant plasmid pMD18-T-ORF2 build FZ0502 strain ORF2 DNA of porcine circovirus type Ⅱ (Porcine circovirus PCV2) DNA vaccine and evaluate its immune efficacy and safety. Methods: application constructed recombinant plasmid pMD18-T-ORF2 eukaryotic expression vector PcDNA3.1 under the same conditions double digested by T4 ligase, transformation competent bacteria DH5a by cultured at 37 ℃ and picked growth by shaking bacteria colonies large number of extracted plasmid; application BamH, and xhoI double digestion technology, identification screening positive recombinant plasmid; application endotoxin extracted plasmid extraction kit positive recombinant plasmid after liposome Lipofectamine 2000-mediated transfection single layer of Vero cells, 37 ℃ in the C02 incubator maintaining culture 4d extracted Vero cell total RNA by RT-PCR method identified the the ORF2 gene's expression situation; positive recombinant plasmid leg intramuscular immune Balb / c mice (100μg / only), at the same time set the of PBS and PcDNA3.1 empty vector control interval of 2 weeks were given a booster. Blood collection 14d and 28d respectively in the first free, was measured by ELISA serum antibody; taken after the first free mice 56d of the heart, liver, spleen, lung, kidney and brain parenchymal organ, using PCR to detect the DNA vaccine security. 'The result: pMD18-T-ORF2 and eukaryotic vectors PcDNA3.1 through double digestion connected transforming competent bacteria DH5α and cloned plasmid by double digestion, and the results show the 582bp target band positive plasmid named PcDNA3.1 -ORF2. Lipofectamine 2000 mediated After liposome monolayer of Vero cells, transfection, extracted Vero total cellular RNA, RT-PCR was identified ORF2 gene expression results show that the purpose strip appear 582bp position. PCV2 antibodies, nucleic acid vaccine induced PcDNA3.1-ORF2 PCV2 antibody indirect ELISA immunized mice was significantly higher than that the PCV2 antibody of PBS and PcDNA3.1 induced; the first free 56d, the acquisition of the organs of mice was amplified by PCR method , are not amplified PCV2-ORF2 object strip. Conclusion: We successfully constructed PcDNA3.1-ORF2 DNA vaccine by transient expression in Vero cells in vitro, and can induce a strong immune response; safety tests showed that the DNA vaccine is not integrated into the mouse chromosome is safe. Objective: To take advantage of the ORF2 gene prokaryotic expression vector PGEX-6P-1 expression discard the signal peptide, PCV2-Cap protein expression coated microtiter plates, establish a specific, rapid, sensitive indirect ELISA method. METHODS: Cultured E. coli BL21 containing recombinant plasmid PGEX-6P-ORF2 and induced by IPTG sonication and purification of inclusion bodies; diluted inclusion bodies and secondary antibodies, antigens optimum coating concentration is determined by titration and rabbit anti-pig IgG diluted concentration; tested serum-fold dilution, measured OD490 values, to determine the optimal serum dilution; Korea JBT PCV2 kit with the establishment of an indirect ELISA method detected 20 negative serum plus three times the standard deviation of the average OD490 value of detection as the yin and yang of the threshold; establish indirect ELISA method to detect porcine reproductive and respiratory syndrome virus, classical swine fever virus, porcine pseudorabies virus and porcine parvovirus positive serum, at the same time as PCV2-positive serum and negative serum control, validation indirect ELISA specificity; 16 were detected three times PCV2 negative and serum detected once a week, and by comparing the results of the analysis of variance differences; the PCV2 kit JBT with South Korea at the same time were 40 copies of yin and yang serum test, compare results, and calculate the coincidence rate; test and evaluate the test results and 204 serum samples were collected in Fujian Province, Fuzhou, Ningde, Xiamen, Putian region. Results: by reaction conditions fumble indirect ELISA method, determine the best package for the indirect ELISA method the concentration of antigen and rabbit anti-swine IgG 1.5μg / well and 1:3000 dilution; optimal serum dilution of 1 : 400; The positive standard threshold should be greater than 0.427; porcine reproductive and respiratory syndrome virus, classical swine fever virus, porcine pseudorabies virus, porcine parvovirus, PCV2 positive serum and PCV2 negative serum were measured, only PCV2 positive serum positive results; repeated test results show no significant difference between trials; Korea JBT PCV2 kit compare both negative and positive serologic test results, positive serum was 95%, in line with the negative serum was 90%, with a total in line with the rate of 92.5%; collected 204 of the farms in Fujian Province serologic test results show that with the establishment of the indirect ELISA method of PCV2 positive rate of 76.5%. Conclusion: 'established indirect ELISA method PCV2 positive serum specificity, reproducibility and good stability, can be used for serological diagnosis and epidemiological investigation of PCV2 antibody.

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