Development Of PCV2 DNA Vaccine And Establishment Of Indirect ELISA Method

Objective:To develop ORF2 DNA vaccine against PCV2 FZ0502 strain with recombinant plasmid pMD18-T-ORF2, to evaluate and determine the security and effect of immunization on mice vaccinated with the vaccine.Methods:The recombinant plasmid pMD18-T-ORF2 was constructed and eukaryotic vector PcDNA3.1 was double digested in the same condition. The plasmid was connected by the T4 DNA ligase, transfered to competent DH5αbacteria incubated at 37℃and the colonies were obtained, a large number of bacterial plasmids were extracted. The positive recombinant plasmids (DNA vaccine) were sieved through the double digestion with BamH and xhoI. Using no-endotoxin plasmid extraction kit to extract positive recombinant plasmid, which was mediated by liposome Lipofectamine 2000, then transfered to Vero cells growing in monolayer and Vero cells were cultured in CO2 incubator at 37℃for 4 days. Total RNA were extracted from Vero cells and ORF2 gene was identified by RT-PCR.12 Balb/c mice at the age of 6-8 weeks were selected and divided into treatment group (4 mice) injected intramuscularly with positive recombinant plasmids (100μg of each), control groupⅠ(4 mice) injected intramuscularly with PcDNA3.1 (100μg of each) and control groupⅡ(4 mice) injected intramuscularly with PBS (100μL of each).2 time injections were conducted in two weeks interval. On the 14th and the 28th day after the first vaccination, serums were collected to determine the effect of immunization by ELISA. On the 56th day after the first vaccination,, the genomes of parenchymal organ including heart and liver and spleen and lung and kidney of vaccinated mice were extracted for PCR amplification to confirm the safety of PcDNA3.1-ORF2 gene vaccines.Results:582bp band were screened and obtained by enzyme digestion, the positive plasmid was named PcDNA3.1-ORF2. Vero cell total RNA was extracted and the expression of ORF2 gene was identified by RT-PCR, and the 582bp target band were also screened and obtained. PCV2 antibody of immunized mice was detected by indirect ELISA method, showing that the antibody concentration of DNA vaccine (treatment group) was significantly higher than that of control groups. At the 56th day after the first vaccination, the organs of mice were collected and amplified, were the target band of PCV2-ORF2 was not amplified by PCR in the all collected organs.Conclusion:The PcDNA3.1-ORF2 DNA vaccine is constructed and developed successfully. Vero cells is transiently expressed in vitro. PcDNA3.1-ORF2 DNA vaccine can successfully induced immune response in mice and it is confirmed to be security. Objective:To develop a fast and sensitive indirect enzyme-linked immunosorbent assay(ELISA)for determination of Porcine Circovirus 2 antibody, with the expressed PCV2-Cap as protein-coated to coat ELISA plate and the prokaryotic expressed vector PGEX-6P-1 to express the discarded signal peptide ORF2 gene.Method:E.coli BL21 with plasmid PGEX-6P-ORF2 were incubated and induced by IPTG. The bacteria were crushed by ultrasonic and inclusion bodies were purified. Through dilution of the inclusion bodies and secondary antibodies, the optimal diluted packet antigen concentration and rabbit-anti-swine IgG concentration were determined with the matrix antigen titration method.The best diluted concentration of serum was determined by OD490 values in different dilutions of serum.20 negative serum samples were determined with the developed indirect ELISA and South Korea co-JBT kit and critical values of negative and positive were determined by the standard deviation of average OD490 values. The specialities of the developed indirect ELISA method was determined by examinations of positive serum of PRRSV, CSFV, PPV, PRV, and positive and negative serum of PCV2.16 serum samples of PCV2 positive and negative were examined 3 times in each week interval by the indirect ELISA to analyse the variance differences.40 positive and negative serum samples were detected at same time by the South Korea's PCV2 JBT kit and the developed indirect ELISA to compare their compliance rate.204 serum samples collected from farms in Fuzhou, Ningde, Xiamen, Putian regions in Fujian Province were determined by the developed indirect ELISA and the results were evaluated.Results:The optimum coated antigen concentration was 1.5μg for each hole, the optimum dilution of rabbit-anti-swine IgG was 1:3000, the optimal serum dilution was 1:400, and the critical value of positive standards was more than 0.472. The positive serum of PRRSV,CSFV,PRV,PPV,PCV2 were detected, only positive serum of PCV2 was showed positive result. The repeatability test showed no significant difference between South Korea's PCV2 JBT kit and the developed indirect ELISA,showing that the positive coincidence rate was 95%, the negative coincidence rate was 90%, and the average coincidence rate was 92.5%.204 swine serum samples from farms were detected by the developed indirect ELISA, the positive rate was 76.5%.Conclusion:The developed indirect ELISA method can only be used for determination of PCV2 positive serum. It has good specificity, repeatability and stability and can be used for the serodiagnosis and the epidemiological study of PCV2.