猪胸膜肺炎放线杆菌溶血毒素的克隆表达及其对小鼠免疫保护作用

将猪胸膜肺炎放线杆菌血清3型分离株的ApxⅡA、ApxⅢA、ApxⅣA基因和血清5型分离株的ApxⅠA基因分别克隆到原核表达载体pGEX-5X-3,并在大肠杆菌BL21中进行表达。SDS-PAGE结果表明重组菌表达的最佳条件为诱导时间2小时和IPTG终浓度1mmol/L。通过硫酸铵盐析和SephadexG-200凝胶过滤层析纯化表达产物。Westernblot检测结果显示表达产物具有免疫活性。按照不同组合将表达产物与弗氏佐剂等比例混合,制备3种亚单位疫苗。并在30日龄和45日龄免疫小白鼠,在60日龄分别用血清1、3、5、7和10型胸膜肺炎放线杆菌攻毒。血清1、5和7型胸膜肺炎放线杆菌攻毒后,3种亚单位疫苗分别提供58.4%、66.6%和91.7%的保护率。试验结果表明重组蛋白具有免疫保护作用,且含有四种融合蛋白的亚单位疫苗免疫保护效果最佳。     

胸膜肺炎放线杆菌RTX毒素与宿主的相互作用

摘要：胸膜肺炎放线杆菌引起猪传染性胸膜肺炎,给养猪业造成严重的经济损失。RTX毒素是胸膜肺炎放线杆菌主要的毒力因子,在该病原的感染与免疫中发挥“双刃剑”的作用。本文综述了近十多年来国内外在胸膜肺炎放线杆菌RTX毒素的研究进展,提出了毒素与宿主互作研究的必要性和技术可行性,认为毒素与宿主相互作用研究将诠释此病原的分子致病机理。     

构建胸膜肺炎放线杆菌apxIC基因插入突变株

目的：构建胸膜肺炎放线杆菌（APP）apxIC基因插入突变菌株,以鉴定ApxⅠ毒素的生物学特性。方法：根据apxⅠ核酸序列（U05042）设计1对引物,用于自APP血清10型参考菌株（D13039）基因组DNA中扩增apxIC基因及其上下游约2．8kb的基因片段,经克隆测序后在apxIC基因下游xbI酶切位点处插入约0．9kb的氯霉素（Chl）抗性基因表达盒,构建用于转化的转移载体pUIC-Chl^r,将转移载体DNA经电转化导入APP血清10型参考菌株中进行同源重组,以获得突变菌株。结果：在含有氯霉素的培养基中经筛选获得2株丧失溶血活性的突变菌株（D13039C-Chl^r）;利用PCR和Southern blot对突变菌株鉴定,显示氯霉素抗性基因已被插入细菌基因组中。结论：利用电转化和同源重组技术构建成功APP apxIC基因插入突变菌株,为分析ApxⅠ毒素的生物学特性,进而研制APP基因工程减毒活疫苗奠定了基础。     

猪传染性胸膜肺炎放线杆菌flic基因的原核表达与生物学活性分析

以猪传染性胸膜肺炎放线杆菌血清Ⅰ型菌株基因组为模板,根据其鞭毛区与其它细菌高度同源性设计引物,利用PCR方法扩增鞭毛蛋白基因flic片段,将其亚克隆到pMD-18T载体中并进行PCR和酶切鉴定,再将其亚克隆与载体pGEX-kG分别双酶切和连接,构建重组表达载体pGEX-flic,并将其转化大肠杆菌工程菌BL21进行原核表达.结果表明,目的基因片段长度为528 bp,在大肠杆菌BL21中获得成功表达,且表达蛋白能与猪抗APP血清发生反应.为进一步研究细菌鞭毛的抗原性及其他功能提供参考.     

复合PCR鉴定胸膜肺炎放线杆菌方法的建立及初步应用

根据胸膜肺炎放线杆菌(Actinobacillus pleuropneumoniae,App)apxIVA毒素基因序列和16SrRNA序列分别设计了一对特异性引物P1P4和一对通用引物S7S10,建立了检测App全部15个血清型的复合PCR方法。对App的15个血清型国际参考株和国内的11个App菌株进行检测,都能得到363bp和692bp的两个扩增片段。而放线杆菌等13株参考菌株只能得到692bp的扩增片段。该方法能将15个血清型的App菌株鉴定到种。检测的灵敏度达9pgDNA1300CFU。用建立的方法检测临床分离的302株可疑菌株,阳性4株,与其它鉴定方法相符。结果表明复合PCR可用于App菌株的鉴定。     

猪胸膜肺炎放线杆菌通过Adh诱导猪肺泡巨噬细胞氧化应激

【背景】猪胸膜肺炎放线杆菌(Actinobacillus pleuropneumoniae, APP)可导致严重的肺脏炎性损伤。细胞氧化应激与肺脏炎性损伤密切相关,但是APP感染和细胞氧化应激之间的关系仍不清楚。【目的】明确APP感染和细胞氧化应激之间的关系并对其机制进行初步探索,从而为进一步阐明APP致病机理奠定基础。【方法】APP野生菌5b (5b WT)和Adh基因缺失菌(5bΔAdh)分别感染猪肺泡巨噬细胞(porcine alveolar macrophage, PAM)后,荧光显微镜观察细胞内活性氧(reactive oxygen species, ROS)水平,RT-qPCR和Western blotting检测氧化应激相关分子的表达变化,利用NLRP3炎性体抑制剂MCC950处理细胞,分别感染5b WT和5bΔAdh后,生化反应法检测细胞内乳酸和丙酮酸含量,流式细胞术检测细胞内ROS水平及线粒体膜电位变化情况;采用tandem mass tag(TMT)标记定量蛋白质组学测序分析细胞蛋白表达变化并检测线粒体DNA(mitochondrial DNA,mtDNA)的释放情况。【结果】免疫荧光和流式细胞术检测结果均显示,5bWT组ROS水平明显高于5bΔAdh组,但经MCC950处理后可以逆转这一上升趋势;生化指标检测结果表明,相较于5bΔAdh组,5bWT组的丙酮酸和乳酸的含量下降更显著,而且经过MCC950抑制NLRP3活性后,细菌感染组的丙酮酸和乳酸的含量均有显著回升,与对照组无显著差异;5bWT和5bΔAdh感染后,猪肺泡巨噬细胞(porcine alveolar macrophage, PAM)中氧化应激关键转录因子核转录因子红系2相关因子2 (nuclear factor-erythroid 2-related factor 2, Nrf2)和GCLM均呈现出下降的趋势;Western blotting结果表明5b WT和5bΔAdh组烟酰胺腺嘌呤二核苷酸磷酸氧化酶2(nicotinamide adenine dinucleotide oxidase 2, NOX2)含量增高,并且5b WT组含量升高更明显;蛋白质组学分析表明共有15个蛋白的表达出现显著变化,GO分析发现这些差异蛋白主要参与ATP代谢、嘌呤代谢、炎症等重要生物学过程,KEGG分析发现这些蛋白与炎症调节、能量转运和氧化磷酸化等相关的信号通路密切相关;重要的是5b WT和5bΔAdh感染PAM后,线粒体受损细胞比例分别为69.6%和58.5%,均高于对照组,mtDNA也大量释放到细胞质中。【结论】APP可以通过Adh引起PAM氧化应激,并且NLRP3炎性体活化参与细胞氧化应激的形成。     

猪胸膜肺炎放线杆菌毒素ⅢA基因的克隆、序列分析及原核表达

因胸膜肺炎放线杆菌的致病性主要是由毒素决定的,故参照猪胸膜肺炎放线杆血清2型菌株的序列（GenBank L12145）设计了一对特异性引物,用PCR的方法扩增apxⅢA基因并得到了长3 466bp的片段,然后将其克隆到pMD18T中,经酶切鉴定和序列分析表明克隆是成功的;再将apxⅢA插入到原核表达载体pET28b后,转化BL21(DE3),在IPTG诱导下获得高效表达,经Western blotting检测证实表达产物有活性。以表达产物包被ELISA板,建立了特异、敏感的ELISA诊断方法。     

胸膜肺炎放线杆菌apxIC基因插入失活突变株构建及免疫原性分析

胸膜肺炎放线杆菌是引起猪传染性胸膜肺炎(APP)的呼吸道病原菌,其分泌的Apx毒素是最重要的毒力因子之一。为构建APP突变弱毒菌株,在apxIC基因下游XhoI酶切位点处插入氯霉素抗性基因(Chlr)制备转移载体,通过电转化导入APP血清10型参考菌株(D13039)进行同源重组,筛选获得apxIC基因插入突变菌株D13039C-Chlr。该突变菌株特性鉴定结果表明其溶血活性完全丧失,可正常增殖和分泌ApxI毒素,连续10次传代后基因组中插入的Chlr基因可稳定遗传,利用5个剂量(2×108CFU~2×106CFU)对每组3只小鼠腹腔攻毒结果显示突变菌株毒力较母源菌株降低至少100倍以上,将突变菌株作为弱毒活疫苗经滴鼻途径免疫仔猪后利用APP血清1型(4074)和血清10型(D13039)菌株攻毒进行免疫原性鉴定,结果显示血清1型攻毒后非免疫组4头仔猪全部死亡而免疫组4头中死亡2头,非免疫组肺损伤指数(34.4)显著高于免疫组(17.5),血清10型攻毒后非免疫组肺损伤指数(17.5)也高于免疫组(10.5),同时鼻拭子和肺组织样品的细菌重分离数及PCR检测阳性数非免疫组也明显高于免疫组,表明突变菌株作为弱毒活疫苗对仔猪具有一定的交叉免疫保护力。该突变菌株的构建为鉴定ApxI毒素活性及研制具有交叉保护活性的APP弱毒活疫苗奠定了基础。     

胸膜肺炎放线杆菌基因分型方法的建立及其临床应用

根据胸膜肺炎放线杆菌各血清型之间外毒素(Apx),外膜脂蛋白(OmlA),转铁蛋白B(TbpB)的基因差异,分别对各血清型进行PCR扩增,得到不同的特异性片段,可区分开生物Ⅰ型13个标准菌株血清型中的8个血清型。临床检测结果与血清学分型一致,将此分型系统用于临床送检的126份肺脏和42份扁桃体的病原学检测,可直接检测出胸膜肺炎放线杆菌感染。此方法还可以将一些尚未定型的菌株进行归类,弥补了血清学方法的不足,为细菌的流行病学调查提供了可靠的技术手段。     

猪胸膜肺炎放线杆菌转铁结合蛋白基因（rbp8）分析及建模

为了深入研究猪胸膜肺炎放线杆菌（Actinobacillus pleuropneumonie,App）转铁结合蛋白基因（Transferrin BindingProtein8,脚）的生物学特性,采用生物信息学方法,对GenBank中的5株App的TbpB的核酸及其氨基酸序列进行比对,选取其中的中国湖北分离株（JL03）对其分子结构、理化性质及功能域、蛋白质二级和三级结构等重要参数进行了预测和分析,并在三级结构的基础上进行了同源建模。结果表明,不同APP菌株之间核酸序列相似性较大,而氨基酸序列存在较大差异,二级结构以延伸链和随机卷曲为主要构件,其空间结构与脑膜炎双球菌GNAl870蛋白相似性较高,以此为模板成功构建了三维结构分子模型,为TbpB基因功能的深入研究提供了线索和参考依据。     

The CAMP effect of Actinobacillus pleuropneumoniae is caused by Apx toxins

Abstract Actinobacillus pleuropneumoniae shows synergistic haemolysis when cocultured with Staphylococcus aureus on blood agar plates. This CAMP effect has been attributed to a discrete CAMP factor, but also to the A. pleuropneumoniae -RTX-toxins I, II, and III. We examined the CAMP effect of recombinant Escherichia coli strains that secreted each of these toxins, and of A. pleuropneumoniae mutant strains that were devoid of one or more these toxins. We found that the E. coli strains were CAMP positive, whereas the A. pleuropneumoniae strain devoid of functional toxin genes was CAMP negative. This demonstrated that the CAMP effect of A. pleuropneumoniae is caused by the toxins and that no CAMP factor per se exists.     

Antigenic secreted proteins from Haemophilus paragallinarum. A 110-kDa putative RTX protein

Haemophilus paragallinarum is the causal agent of infectious coryza, an economically important disease for the poultry industry. This bacterium secreted proteins of 25-110 kDa during its growth in brain heart infusion, tryptic soy broth, or Luria-Bertani glucose phosphate media, all lacking serum. Some of these proteins were recognized by sera from chickens experimentally infected with H. paragallinarum. A 110-kDa protein was recognized by a serum pool from convalescent-phase pigs naturally infected with Actinobacillus pleuropneumoniae, and also by a rabbit polyclonal serum against Apx I as well as a rabbit serum against Mannheimia haemolytica leukotoxin, suggesting the presence of an RTX-like protein in H. paragallinarum. H. paragallinarum secreted proteins could be important immunogens in the control of infectious coryza.     

Actinobacillus pleuropneumoniae serotype 7 siderophore receptor FhuA is not required for virulence

A ferrichrome receptor, FhuA, was identified in Actinobacillus pleuropneumoniae serotype 7. An isogenic mutant with a deletion in the ferrichrome uptake receptor gene (fhuA) was constructed and examined in an aerosol infection model. The disease caused by the mutant was indistinguishable from disease induced by A. pleuropneumoniae serotype 7 wild-type; an isogenic mutant lacking expression of the exbB gene that is required for the uptake of transferrin-bound iron retained the ability to utilize ferrichrome, thereby indicating that an energy-coupling mechanism involved in ferrichrome transport remains to be identified.     

The epitope structure in lipopolysaccharide recognized by the serotype 2-specific monoclonal antibody of Antinobacillus pleuropneumoniae

Abstract: The polysaccharide structure recognized by a monoclonal antibody specific to serotype 2 lipopolysaccharide of Actinobacillus pleuropneumoniae was investigated using an enzyme-linked immunosorbent assay inhibition test. Lipopolysaccharide obtained from serotype 2, strain SH-15, was hydrolysed with acetic acid to liberate the polysaccharide portion, and the polysaccharide mixture was fractionated by gel filtration. The longer polysaccharide, composed of O -antigenic polysaccharide and core, fully inhibited the binding of monoclonal antibodies to a whole cell antigen of strain SH-15, whereas the core oligosaccharide without O -polysaccharide did not. No inhibition was observed with the monosaccharides which were the components of serotype 2 LPS. Enzyme-linked immunosorbent assay inhibition ability of O -polysaccharide was completely lost only by O -deacetylation. These results demonstrate that the epitope of the serotype-specific monoclonal antibody resided in O -polysaccharide of LPS and that the O -acetyl group was essential for the epitope structure.     

Induction of protective immune responses against the challenge of Actinobacillus pleuropneumoniae by the oral administration of transgenic tobacco plant expressing ApxIIA toxin from the bacteria

Actinobacillus pleuropneumoniae is the causative agent of porcine pleuropneumonia. Among the virulence factors, ApxIIA, a bacterial exotoxin, is reportedly expressed in many serotypes and is considered as a candidate for the development of a vaccine against the bacterial infection. Previously, we isolated a field strain of A. pleuropneumoniae serotype 2 in Korea and characterized its exotoxins to develop an oral vaccine. In this study, we initially confirmed the immunogenicity of ApxIIA expressed in Escherichia coli. We then developed transgenic tobacco expressing ApxIIA and tested its efficacy to induce a protective immune response against A. pleuropneumoniae infection after oral administration of the plant powder. We observed that protective immune responses were induced in mice after oral administration of the plant powder once a week for 4 weeks. Immunoassays revealed that the levels of antigen-specific immunoglobulin G against ApxIIA increased in mice that were fed a powder made from the transgenic plant, but not in mice fed a powder made from wild-type tobacco. Additionally, mice fed the transgenic plant powder were protected from an injection of a lethal dose of A. pleuropneumoniae. These results support that the transgenic plant may be a suitable candidate for an oral vaccine that could be used effectively against A. pleuropneumoniae infection.