Immune Response Induced By Recombinant Adenoviruses Expressing ORF2 And ORF1-2 Fusion Gene Of PCV2

PCV belong to circoviridae circovirus, it was no peplos but single strand circo-DNA virus. It was the smallest animal virus up to now. According to the antigenicity and genetic constitution, PCV has two types, one was PCV1 and the other was PCV2. PCV1 has no pathogenicity, but it generally resided in swine and part of the porcine source continuous cell line; PCV2 has pathogenicity and it could cause post-weating multisystemic wasting syndrome(PMWS).The PCV2 genome contain 11 open reading frame(ORF). Among the total, ORF1 encoding virus replication protein and ORF2 encoding the major virus constitutive protein which has the major antigen neutralizing epitope. On the base of the previous investigate, the adenovirus was cultivated again and PCR,enzyme digest,Western-blot identification was done. Balb/c mice were immunized with Ad-ORF2,Ad-ORF1-2,Ad-ORF1+Ad-ORF2,Ad-C and PBS via muscle rout. The mice were immunized again in the second and fourth week after the first immunization. 5 mice were executed in the second week after the third immunization and lymphocyte were isolated from spleen in free germ. The splenocytes proliferation,lymphocyte subgroup,cytotoxic response of NK cells and CTL were deter mined. Also, the specific antibody of PCV2 was deter mined.1. The PCR,enzyme digest,and sequencing identification confirm that the target gene has correct insert position and has no variation. Western-blot identification and fluorescence microscope survey show that the recombinant adenoviruses could express the target protein, and the target protein could be recognized by the positive serum of the PCV2 infected pigs.2. The serum antibody was arising with the increase of the immunity times, and the highest serum antibody was appear two weeks after the third immunity, but the antibody begin to fall four weeks after the third immunity; Comparing to the control group , the population of the Th and Tc subpopulation of the experiment groups increased remarkably, but it has no significant deviation between the experiment groups(P>0.05); Comparing to the control groups, the splenocytes proliferation response to the ConA of the experiment groups increased remarkably, but it has no significant deviation between the experiment groups(P>0.05); The results of the cytotoxic response of NK cells indicates that cytotoxic response of NK cells the of the experiment groups had significant deviation compare to the control groups. The Ad-ORF2 group and Ad-ORF1 + Ad-ORF2 group has significant deviation compare to the Ad-ORF1+2 group(P<0.01), but there has no significant deviation between the Ad-ORF2 group and Ad-ORF1+Ad-ORF2group(P>0.05); The cytotoxic response of CTL of the Ad-ORF1+Ad-ORF2 group was the highestand the Ad-ORF2 group was the lowest among the experiment groups,but all the experiment groups has significant deviation compare to the control groups.In summary, the results of this experiment indicates that the recombiant adenoviruses of Ad-ORF2 group,Ad-ORF1-2 group and Ad-ORF1+Ad-ORF2 group could induce strong immune response of PCV2, including humoral immunoresponse and cellullar immunologic response. This study laid a foundation for the development of PCV2 genetic engineeringr vaccine.