【作者】 谢芳；

【导师】 韩文瑜；

【作者基本信息】 吉林大学 ， 预防兽医学， 2010， 博士

【摘要】 胸膜肺炎放线杆菌(APP)是引起猪传染性胸膜肺炎疾病的病原菌,该病是世界范围内流行、严重危害现代化养猪业的重要传染病。APP具有众多血清型,其致病力和免疫原性各不相同,主要血清型间无免疫交叉保护,是制约APP致病机理和疫苗研究的瓶颈,严重影响了APP的诊断、检疫和预防。本研究选择毒力存在差异、而无免疫交叉保护的APP血清1型和3型菌株为对象,采用代表性差异分析法(RDA),分别构建了基因组DNA差减基因文库和cDNA差异表达基因文库,系统筛选1型和3型基因组差异基因和差异表达基因,经生物信息学分析后,从中选取自转运黏附素基因(差异基因)的功能区adh和蛋白酶clpP基因(差异表达基因)进行克隆与序列分析,并采用实时定量PCR分析clpP基因的相对表达,分别对adh和clpP基因进行原核表达,得到GST-Adh和GST-ClpP重组蛋白,测定其对昆明小鼠抗APP感染的免疫保护性,并利用凝血酶切除GST,纯化Adh蛋白,检测Adh对猪肺上皮细胞的黏附活性。结果表明,获得APP 1型8条差异基因片段,APP3型11条差异基因片段,经PCR鉴定发现这19条差异基因在15个血清型中分布不同;获得APP 1型22条上调表达基因,APP 3型20条上调表达基因,clpP基因在APP 1型的相对表达量为3型的3.22倍;成功获得融合蛋白GST-Adh和GST-ClpP,大小分别约为81kDa和48kDa,并以可溶性的形式获得表达;GST-Adh蛋白对APP血清5b型的保护率为7/10,对血清1型、3型和5a型无明显保护作用;Adh蛋白对猪肺上皮细胞具有黏附活性,该蛋白及其抗体能抑制APP对猪肺上皮细胞的黏附作用;GST-ClpP蛋白对APP血清1型的保护率为6/10,对血清3型无明显保护作用。本研究为今后APP不同血清型致病机理研究、新型多价疫苗研究以及特异性分子诊断技术的建立提供必要的基础。更多还原

【Abstract】 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 ApxⅠ, 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 ApxⅢ, 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 ApxⅠ, 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 ApxⅢ, 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.更多还原