Isolation And Identification Of Actinobacillus Pleuropneumoniae And Analysis Of Immunogenicity Of Six Outer Membrane Proteins

Swine pleuropneumonia is caused by actinobacillus pleuropneumoniae of Pasteurellaceae Actinobacillus which is a gram-negative coccobacillus characterized local lung lesion, extensive hemorrhage and pleuritis, which is a severe contagious and mortality respiratory disease. Currently, A.pleuropneumoniae contains15serotypes except several unclassified strains, inactivated vaccine is an effective measure to control and prevent it, while it has weak cross-protection among serotypes, so inactivated vaccine provides a limited protection. Six outer membrane proteins of A.pleuropneumoniae including omlA, plpA, lolB, omp2, ompAe and plpB are cloned and expressed by Prokaryotic and researched in their immunogenicity.A strain of A.pleuropneumoniae was isolated from swine in Hunan province. The strain was identified based on PCR identification, biochemical test, susceptibility test and pathogenicity experiment, and the result showed this strain was serotype1. According to the whole genome sequence of A.pleuropneumoniae in Genbank, six paris of primers were designed by DNAstar. OmpAe contained BamH Ⅰand Sal Ⅰ two restriction sites, and other five genes contained EcoR Ⅰ and Sal Ⅰ two restriction sites. The six genes were amplified by polymerase chain reaction (PCR) based on the template of A.pleuropneumonae. Digestion products were linked to expressing vector pET-28a(+), and then transmitted into DH5a. After positive and restriction identification, the recombinant plasmids were transmitted into BL21(DE3). SDS-PAGE analysis showed that the recombinant BL21(DE3) bacterium were highly expressed and the expressing products of omlA, plpA and lolB were expressed in the forms of soluble, while omp2, mpAe and plpB were inclusion bodies. OmlA, omp2and plpA showed good immunogenicity by Western blot.K.M female mice with a weight of25g were immunized with six purified proteins respectively with Freund’s adjuvant, after14days and28days of primary immunization to strengthen the immune, and the immunization dose was50μg each time. Mice were challenge with7LD50after the third immunization. The protection rates of omlA and omp2were17%,17%respectively, while control group and the other experimental groups were0%.According to the challenged result, LTB sequence was inserted into in front of omlA and omp2. All groups contained group Ⅰ (omlA+omp2), group Ⅱ (LTB-omp2), groupⅢ(LTB-omlA) and groupⅣ(saline). Immunization dose and method was the same as before, but the challenged dose was5LD50. The protection rates of group Ⅰ group Ⅱ, groupⅢ, groupⅣ were33%,33%,17%,0%respectively. The result showed that omlA and omp2provided a higher protective effect than immunized respectively. The protective effect of omp2was better than omlA when inserted into LTB sequence.