| Actinobacillus pleuropneumonia (APP) is the pathogen of porcine contagious pleuropneumonia (PCP). It can cause serious infectious respiratory disease in pigs, bringing serious economic losses to the pig industry worldwide. According to capsular polysaccharide and lipopolysaccharide antigen, APP could be divided into 15 serovars, the prevalence and virulence of each serovar were different. Serovar 1,2,3 and 7 are prevalent in China. A. pleuropneumoniae major virulence factors include capsular polysaccharides, lipopolysaccharides, outer membrane proteins, transferrin binding proteins, RTX toxins, adhesion factors, urease, and proteases. According to the functions of these virulence factors, researchers have developed a number of candidate vaccines to prevent and control PCP. However, the vaccines used for clinic are inactivated and subunit vaccines, which displayed limited immune protections and could not provide cross-protections against different serovars. Therefore, it is urgent to develop new vaccines to prevent PCP. Investigations on molecular pathogenesis are the bases of design and development of new vaccines. Hence, it is essential to identify the genes associated with infection of APP. In this study, we constructed genome expression library of APP JL-03 (serovar 3), prepared the rehabilitation serum of swine infected with APP, and then used the immunological techniques to screen the in vivo induced genes of APP JL-03.14 antigens were identified and the immunogenicities of three of the genes were further investigated. The main results are summarized in four aspects:Construction of APP JL-03 genomic expression libraries: APP JL-03 genomic DNA was extracted and digested with Sau3AI. The 0.5-2 kb fragments were purified, cloned into the prokaryotic expression vector pET-28a/b/c and transformed into E. coli BL21(DE3) respectively. The resulted library sizes were 2.50×104 cfu/μL for pET-28a, 1.91×104 cfu/μL for pET-28b and 2.12×104 cfu/μL for pET-28c. The positive ratios of recombinant plasmids in the libraries were all higher than 60%, hence they could be used to do further studies.Preparation of antibody probes for library screening: Healthy pigs were infected with 5×105 cfu, the non-lethal dose of APP, then the recovery serum were collected. Somatic antigens and secretory antigens were adsorbed using nitrocellulose membrane for 4 times. Adsorption was reduced from 1.23 to 0.16 detected by ELISA. The results after the 3rd absorption and 4th absorption showed no difference. The antibody probes could be used for library screening.Screening in vivo induced antigens (IVIAs):The expression of proteins in the genome expression library were induced by IPTG. Then, 11 clones were identified using western-blot. The positive clones were sequenced and analyzed. The results demonstrated that these clones contained 14 open reading frames (ORFs).Validation of the immunogenicity of three IVIAs: Three conserved genes (pkyA,purR3, mutT2) were selected from the 14 ORFs identified to be cloned, expressed and immunogenicity of these genes were tested. pkyA, purR3, mutT2 were cloned into the expression vector pET-28a, expressed, purified and confirmed by western-blot. The results validated that these proteins were the antigens induced in vivo. Then, immunogenicity of recombinat PkyA, PurR3 and MutT2 were detected respectively on mice. Mice were immuned and chanllenged with 2×LD50 of APP JL-03 and APP 4074 (6×1015, 1×1010) respectively. PkyA, MutT2 showed better immune protections against APP JL-03 compared with PurR3. However, all the proteins showed weak protections against APP 4074. Relatively, PurR3 displayed better cross protection against APP serovar 1 and 3. |
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