Isolation, Identfication And DNA Vaccine Preliminary Research Of Actinobcillus Pleuropneumoniae

In this paper, a strain of G" coccobacillus was isolated from pigs infected with Actinobacillus pleuropneumoniae. The bacteria were confirmed be 1 serotype of APP by microscopic examination, biochemical assay, drug sensitivity test, PCR and serological identification. The APP isolates needs special high nutritional condition,and can not grow in the common nutrient agar plate, but erupt ganoid, hygric, subtransparent, edge concinnous, little intumescent center, needle tip to medium sized coenobium in the TSA plate containing NAD. The coenobium can become bigger by the increase of cultural frequency. CAMP test presents positive reaction. It is in line with the biochemical character of APP that can ferment amylaceum, amylomaltose, saccharobiose, xylose, fructose, carbamide, ONPG, manicol and nitrate reductase. Drug sensitivity tests revealed that the APP was sensitive to penicillins and quinolones, moderate sensitive to cephalosporins, but resistant to tetracycline, chloramphenicol, trimethoprim and aminoglycosides.Apx I A N-terminal gene fragment and OmlA gene of APP serotype 1 were amplified by PCR, and then they were cloned into pMD18-T Simple vector in order to obtain the recombinant plasmid pMD-Apx I A and pMD-OmlA. Transfer the Apx I A gene into eukaryotic expression vector pIRES to construct the recombinant plasmid pIRES-Apx I A. Transfer the OmlA gene into the recombinant plasmid pIRES-Apx I A to construct the other recombinant plasmid pIRES-Apx I A-OmlA. Then the eukaryotic expression recombinant plasmid pIRES-Apx I A-OmlA was transfected into BHK-21 cells with Xfect transfection kit. The expressed product was detected by SDS-PAGE and indirect immunofluorescence assay. The green fluorescence and interest protein strap can be observed which show that eukaryotic expression vector pIRES-Apx I A-OmlA was constructed successfully. And it can be expressed in mammalian cells.The Balb/c mice were immunized using pIRES-Apx l A-OmlA DNA (group A), pIRES-Apx 1 A DNA (group B), empty vector pIRES DNA (group C), the APP serotype 1 vaccine (group D) and physiologic saline (group E) three times in the same dose and two weeks interval. To compare the immunogenicity of all immunity class by the detections that the change of IgG1, IgG2a, IL-4, IFN-y contents in blood serum, spleen lymphocytic proliferation reaction and the T-lymphocyte subsets variance. The results showed that the IgGl, IgG2a, IL-4, IFN-y contents in the immunized mice's peripheral blood of group A, B and D were step up. They had highly significant difference to group C and D (P<0.01). Group A was highly significant higher than group D (p<0.01). After the third immunization, the IgGl, IL-4 contents of group D were significant step up; but the IgG2a, IFN-y contents were significant step down. The value of IgG2a/ IgGl in each group immunized mice's peripheral blood could obtain that group A and B form antibody IgG2a mainly, and the APP vaccine group forms antibody IgGl mainly. After the third immunization, mice in group A, B and D could induce a strong proliferation of spleen cells, the SI values of group A were highly significant higher than group B and D (p<0.01); group B were significant higher than group D(0.01＜P<0.05). The percentage of CD3+, CD4+, CD8+in group A and B were highly significant higher than group C and E (p<0.01); The percentage of CD3+, CD4+in group D were highly significant higher than group C and E (p<0.01), and highly significant lower than group A and B (p<0.01); The percentage of CD8+in group D were highly significant lower than group A and B (0.01<P<0.05), and highly significant higher than group C and E (p<0.01); Between group A and B, the percentage of CD3+had significant difference (0.01＜P＜O.05), the percentage of CD4+had highly significant difference(p＜O.O1), and the percentage of CD8+had not statistically significant difference (P>0.05).Together the results, it can obtain that the co-expression eukaryotic expression vector pIRES-Apx I A-OmlA was constructed successfully. It can induce a strong humoral immunity and cell immunity in mice, the immunogenicity of co-expression DNA vaccine is better than single gene. This research offers a foundation to the further research about the APP DNA vaccine. At the same time, this research confirms that the DNA vaccine can not only induce the humoral immunity but also the cell immunity; however, it produces the cellullar immunologic response of Thl type mainly. But the inactivated vaccine produces the humoral immunoresponse of Th2 type mainly.