| Actinobacillus pleuropneumoniae is the causative agent of porcine pleuropneumonia, an extremely contagious and often fatal respiratory disease responsible for major economic losses to the swine industry worldwide. This disease is one of the most infectious diseases that severely hamper the healthy development of the modern swine industry worldwide. The 15 currently known serotypes of A. pleuropneumoniae interfere accurate diagnosis of this disease and serotyping. All serotypes are capable of causing disease, although significant differences in virulence and the unclear pathogenic mechanisms. In addition, these serotypes show significant differences in immunogenicity, and the current inactivated vaccines and subunit vaccines can not provide cross-protection for a large number of serotypes that needs to find serotype-specific protective genes to develop genetic engineering multivalent vaccine. To overcome these challenges, it is urgent to screen and identify gene differences among different serotypes of A. pleuropneumoniae systematically at the molecular level. Therefore, in this study, A. pleuropneumoniae serotypes 1 and 3 were selected as study object because they show significant differences in pathogenicity and immunogenicity. Genomic differential genes and differentially expressed genes between A. pleuropneumoniae serotypes 1 and 3 were screened and identified systematically, and the function of autotransporter adhesin and ClpP protease were studied on. Genomic DNA of A. pleuropneumoniae serotypes 1 and 3 was extracted, and genomic differential genes were screened by using representational difference analysis (RDA). After three rounds of subtractive hybridization and PCR amplification, the RDA products were cloned into the pMD-18T vector to construct genomic subtractive libraries of serotypes 1 and 3. After sequencing, the nucleotide similarities of the differential DNA fragments were determined by BLASTN analysis in GenBank. Southern blotting analysis and PCR-based identification were performed to confirm the differential DNA sequences. 8 differential DNA sequences from the serotype 1 were identified, including the encoding genes of hemolysin Apxâ… , transferring binding protein, adhesin, ABC transporter protein and hypothetical protein. 11 differential DNA sequences from the serotype 3 were identified, including the encoding genes of hemolysin Apxâ…¢, hypothetical proteins, and proteins that participate in LPS synthesis, energy metabolism and carbohydrates synthesis. The distribution of these 19 sequences among the 15 serotypes was identified by using PCR, and each serotype showed a different distribution pattern.Specific primers for 5' end sequence of autotransporter adhesin gene were designed, and 5' end sequences ad1 and ad5a of autotransporter adhesin gene of A. pleuropneumoniae serotypes 1 and 5a were amplified, cloned and sequenced respectively. The result of sequence analysis found significant differences in nucleotide sequences between ad1 and ad5a, showing only 50.65% homology. The length of ad1 sequence is 3041bp, the BLASTN homology is 71%, the length of its ORF is 2244bp, encoding 748 amino acids. The length of ad5a sequence is 2147bp, the BLASTN homology is 99%, the length of its ORF is 2145bp, encoding 715 amino acids. The deduced amino acide sequences based on ad1 and ad5a genes and autotransporter adhesin sequences of A. pleuropneumoniae serotypes 5b and 7 reported in Genbank were analyzed by using SMART software. The result showed that autotransporter adhesin gene of serotype 5b was functional gene, but autotransporter adhesin genes of serotypes 1, 5a and 7 were pseudogenes. The results of bioinformatics analysis for autotransporter adhesin sequence of serotype 5b showed that the protein has highly antigenicity and contains 112 epitopes, N terminal 1-67aa is the signal peptide region, a large number of adhesion motifs were distributed in the N terminal 124-612aa. It is inferred that N terminal 68-612aa region of autotransporter adhesin was an important functional area responsible for adhesion. The encoding gene was named adh. The adh gene was cloned into the prokaryotic expression vector pGEX-4T-1 to construct the recombinant expression plasmid, and recombinant protein GST-Adh was successfully expressed in E.coli, with the molecular weight of approximately 81kDa. After GST affinity purification, single GST-Adh was obtained and the GST tag was removed with thrombin. Through observation by using laser scanning confocal microscope, Adh protein can adhere to lung epithelial cells. The results of adhesion inhibition tests revealed that the Adh protein and its antibody can inhibit the adhesion of A. pleuropneumoniae to lung epithelial cells. After immunization 4 weeks with GST-Adh, antibody titers of mice are up to 1:12800. The result of protective immunity test indicates that GST-Adh protein plays a certain role in immune protection against A. pleuropneumoniae serotype 5b infection, with survival rate of 7 / 10, but it fails to confer good protection against serotypes 1, 3, and 5a.RNA of A. pleuropneumoniae serotypes 1 and 3 was extracted, and double-stranded cDNA was prepared using the RNA. Differentially expressed genes were screened by using representational difference analysis (RDA). After three rounds of subtractive hybridization and PCR amplification, the RDA products were cloned into the pMD-18T vector to construct cDNA subtractive libraries of serotypes 1 and 3. After sequencing, the nucleotide similarities of the differentially expressed genes were determined by BLASTN analysis in GenBank. Northern blotting analysis was performed to confirm the differentially expressed genes. 22 differentially expressed genes from the serotype 1 were identified, including the encoding genes of hemolysin Apxâ… , transferring binding protein, adhesin, ABC transporter protein, protease, molecular chaperone, hypothetical protein, and proteins that participate in LPS synthesis, metabolism, bacterial transcription and nucleic acid metabolism. 20 differentially expressed genes from the serotype 3 were identified, including the encoding genes of hemolysin Apxâ…¢, lipoprotein, hypothetical proteins, and proteins that participate in LPS synthesis and metabolism.TaqMan probes and specific primers for protease gene clpP and housekeeping gene recF were designed, relative expression levels of clpP gene in A. pleuropneumoniae serotypes 1 and 3 were determined by using real-time quantitative PCR. The result showed that the relative expression level of clpP gene in A. pleuropneumoniae serotype 1 is 3.22 times as high as the expression level in A. pleuropneumoniae serotype 3. The clpP gene of A. pleuropneumoniae serotype 1 was amplified, cloned and sequenced. The result of sequence analysis showed that the length of nucleotide sequence is 591bp, encoding 196 amino acids. The clpP gene was cloned into the prokaryotic expression vector pGEX-4T-1 to construct the recombinant expression plasmid, and recombinant protein GST-ClpP was successfully expressed in E.coli, with the molecular weight of approximately 48kDa. After GST affinity purification, single GST-ClpP was obtained. After immunization 4 weeks with GST-ClpP, antibody titers of mice are up to 1:12800. The result of protective immunity test indicates that GST-ClpP protein plays a certain role in immune protection against A. pleuropneumoniae serotype 1 infection, with survival rate of 6 / 10, but it fails to confer good protection against serotype 3.In this study, genomic differential genes and differentially expressed genes between A. pleuropneumoniae serotypes 1 and 3 were successfully screened and identified. The recombinant protein for functional area of autotransporter adhesion has the adhesion activity to lung epithelial cell and plays a certain role in immune protection against A. pleuropneumoniae serotype 5b infection. The expression levels of the clpP gene in A. pleuropneumoniae serotypes 1 and 3 are different. The recombinant ClpP protein plays a certain role in immune protection against A. pleuropneumoniae serotype 1 infection. This study may contribute to future research on the pathogenic mechanisms of different serotypes, serotyping-based diagnosis methods and multivalent vaccines.
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