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Pleuropneumoniae Actinobacillus (Actinobacillus pleuropneumoniae, APP) is a Gram-negative non-spore Brevibacterium, has found 15 serotypes. APP can play swine pleuropneumonia (Porcineinfectious pleuropneumonia). The disease with dyspnea, pulmonary hemorrhagic necrosis and fibrinous adhesions as the main feature, a higher mortality rate, easily mixed infection with other pathogens. APP is a multi-pathogen virulence factors, many of its virulence factors, have been found with the APP pathogenicity virulence factors include capsular polysaccharide (CP), lipopolysaccharide (LPS), outer membrane protein (OMP ), the outer membrane lipoprotein (OMLA), transferrin binding protein (TBP), hemolytic exotoxin (Apx), protease, penetration factor, adhesion factor, fimbriae, urease. Gene sequences in GenBank No. U86675 serotype 1 APP OMLA gene sequences were designed and synthesized primers using PCR technology to APP bacterial DNA as a template, amplified OMLA gene, the gene is connected to pMD18-T vector and sequencing, sequencing results showed an open reading frame with 1095 bp encoding 364 amino acids. The OMLA gene nucleotide sequence and deduced amino acid sequences with other reported OMLA gene homology, mutation analysis, the results show, OMLA nucleotide sequence of a type, nine-and 11-type homology of 100 %, and type 2, type 3, type 4 homology was 76.2%, 63.3%, 63.8%, and type 5, type 6, 7 type homology was 62.5%, 63.3% and 64.4%, and 8 type, 10 type and 12 type homology was 77.3%, 62.2% and 98.6%. OMLA gene encoding the amino acid sequence of type 1, type 9 and 11 was 100% homology with other serotypes homologies were 62.2 ~ 98.6%. In pMD18-OMLA as template, amplified the gene encoding the mature protein (1032bp), and inserted into pET-32a () expression plasmid was constructed pET32a ()-mOMLA Mature prokaryotic expression plasmid. Positive recombinant plasmid was transformed into E. coli BL21 (DE3), IPTG-induced expression of recombinant mature protein, SDS-PAGE, Western-blot confirmed the expression of a fusion protein of about 60kD and detect its solubility. Testing to determine OMLA recombinant mature protein optimum conditions for IPTG 0.4mmol / L, 37 ℃ induction culture 4h, the optimal expression conditions, the relative expression of recombinant mature protein content can reach 47.5%. Purified recombinant mature protein refolding recombinant protein respectively, recombinant protein and inactivated APP, APP and inactivated PBS emulsified with an equal amount of adjuvant immunized mice during indirect ELISA antibody titer was detected in the immune times After a 5 × LD 50 of APP serotype 1 strains of mice were challenged poison. The results showed that the recombinant protein has a immunogenicity can stimulate the body to produce high levels of antibodies (> 1:3200), after immunization of experimental animals infected with the same type of APP has a protective force.
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