| Porcine pleuropneumonia,which was used to be called porcine contagious pleuropneumonia or haemophilus pleuropneumonia ,is a highly contagious, lethal disease of respiratory tract with high morbidity and mortality.Actinobacillus pleuropneumoniae(APP), the etiological agent of porcine pleuropneumonia , has been identified as two biotypes and 15 serotypes at present, and there is no efficient cross-protection between the serotypes. It is essential to research the differentially expressed genes and immune response related genes between the different APP serotypes for developing genetically engineering vaccine and constructing appropriate detecting or diagnostic methods. The experiments to determine the virulence and the cross-protection were conducted, two high virulence and no cross-protection strains, APP serotype 1 and 5 ,were selected as the research objects. By using improved Representational Difference Analysis (RDA),a differentially expressed gene cDNA library and a subtractive genomic DNA library had been constructed respectively. A portion of genes of the libraries were sequenced,comfirmed by Southern-blotting and The homology of these sequences was searched in the GenBank+ EMBL+DDBJ+PDB databases with BLAST program, the differentially expressed genes and genomic differential genes of APP serotype 1 and 5 were systematically analyzed. The differential genes of the libraries were expressed by using Ribosome Display technique , panned with positive serum of serotype specificity , the specific immune response related genes were amplified and recovered by RT-PCR. By screening the differentially expressed genes cDNA library ,there were 17 genes that were up-regulated in APP serotype 1(drived by serotype 5), among them, there were 8 genes that have a high degree about 93%-100% homology to the functional genes of Propionibacterium acnes (PA), and one gene has no significant similarity to any known gene, whereas 31 genes that were up-regulated in APP serotype 5(derived by serotype 1),with 10 genes that have 98% homology to the functional genes of Propionibacterium acnes, and 14 genes have no significant similarity to any known gene. The subtractive genomic DNA library were screened, 8 genes of APP serotype 1 and one unknown gene, 10 genes of APP serotype 5 and two unknown genes were obtained. 8 genes of the 10 differential genes of APP serotype 5 had 93-100% homology sequences with APP serotype 5b strain L20 .After three rounds panning and collecting, there were 6 differentially expressed immune response related genes including two unknown sequences , two genes with 90.5% homology with PA and two genes homology with the human's cDNA were selected out from the differentially expressed genes cDNA library of APP serotype 1 ,while there were 14 immune response related genes , five unknown sequences, 6 genes with over 95% homology with PA from the differentially expressed genes cDNA library of APP serotype 5 were obtained .when the subtractive genomic DNA libraries were screened ,we got 8 immune response related genes ,3 genes high homology with Pseudomonas alcaligenes , one unknown sequence from APP serotype 1, and 10 immune response related genes, 8 genes 93%-100% homology with APP serotype 5b strain L20,two unknown sequence from APP serotype 5.According to the analysis of the subtract gene libraries and the immune response related genes, we have got 48 differentially expressed genes between the APP serotype 1 and 5 . Out of the 48 differentially expressed genes, 18 genes were found in Propionibacterium acnes KPA171202 genome and they have a sequences homologies with 93%-100%,moreover, 8 genes of them are immune response genes. Especially, genes differentially expressed in serotype 1 and 5 were found co-exist in the genome of PA. The expression product can stimulate organism generate antibody, PA may share significant common antigen with APP serotypes 1 and 5 ,and the PA may have the cross-protection effect on APP serotype 1 and 5.In order to comfirm the presumption, the cross-immune test and the CP prevent APP infection test were conducted out. The results indicated that strong cross-protection effect had been worked between PA and APP, and the titer of Sero-response against PA by the anti- APP serotypes 1 and 5 positive serum is 1:8000-1:16000(the positive serum anti APP titer is 1:32000),the titer of Sero-response against APP by the anti-PA positive serum is 1:800-1600(the titer of anti PA positive serum is 1:3200), Vaccinated mice with the live PA, and challenged with the amount of 10 times of LD50 APP serotype 1and 5, the efficacy of the live PA against the APP serotype 1 is 95%, and 90% for APP serotype 5.At the fifth day from the challenge, all APP were cleared out of the body.In this study, we have obtained the differential expressed genes of APP serotype 1and 5 cultured in vitro, and its differential genes of genome and immune response related genes, we have demonstrated that there are 18 differential expressed genes of APP serotype 1and 5 have >90% homology with the PA , and there are a strong cross-protection effect between the APP and PA . Vaccinated with PA can protect the mice against the APP's infection, this study lays the foundation for establishing specific immune diagnostic methods and developing novel genetically engineering vaccine.
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