【作者】 王秀然；

【导师】 王兴龙；

【作者基本信息】 吉林大学 ， 预防兽医学， 2014， 博士

【摘要】 布鲁氏菌病是一种人兽共患病，该病不但严重影响养殖业的发展，对人类健康也构成巨大威胁。人畜布鲁氏菌病难以防控的原因是多方面的，其中，其致病机理（包括胞内寄生机理）不清、免疫机理不清是主要原因。脂多糖（lipopolysaccharide，LPS）是布鲁氏菌主要的毒力因子，也是引起哺乳动物免疫的主要抗原之一。目前布鲁氏菌LPS合成的途径并不完全清晰，已经发现的与布鲁氏菌LPS合成相关的关键基因有wbkA、gmd、wzm、wzt、wbkB、wbkC等基因，其中wzm、wzt基因既是LPS合成的关键基因，又是ABC转运系统的主要组成部分。ABC transporter（ABC转运系统）在细菌中是一个主要的细胞转运机器，由一系列同源基因编码的操纵子组成。该操纵子由ATP结合蛋白，膜蛋白，亚基结合蛋白3部分组成，通常情况下，形成由6个跨膜片段组成的2个完整的膜蛋白。ABC转运系统是膜外生物大分子合成的主要运输通道之一，参与胞外多糖的合成。此外，ABC转运系统在细菌营养成分的运输，毒力分子的外排等过程中也起着重要的作用。然而ABC转运系统对细菌毒力、免疫原性及蛋白质表达的影响还不清楚。本研究通过构建致死质粒，利用基因重组的方法敲除LPS合成的关键基因wzm和wzt基因部分序列，构建LPS缺失的突变株，研究突变株感染小鼠后的脾菌数，评价其毒力，并对其血清抗体水平、细胞因子水平、淋巴细胞增殖与转化差异进行了比较，通过2D电泳比较其蛋白质组的差异，研究基因缺失对菌株毒力及免疫原性的影响，比较其缺失前后的蛋白质组的变化，分析基因缺失对菌体基因表达的影响，为进一步揭示布鲁氏菌的致病机理、免疫机制提供实验依据，也可为布鲁氏菌粗糙型疫苗的研制奠定基础。实验结果如下：1.本实验成功构建了wzm和wzt基因敲除的重组质粒，经PCR扩增和酶切鉴定，该质粒包含sacB基因反向筛选标记，wzmf/wztf和wzmr/wztr目的基因两臂片段。通过电转化将重组质粒转入布鲁氏菌B. abortus S19，经kanr抗性，5%蔗糖TSB琼脂固体培养基筛选，经过PCR扩增和测序鉴定突变位点正确，经测序证明Δwzm和Δwzt分别缺失了511bp和446bp。将鉴定的阳性突变株Δwzm和Δwzt进行特异性引物PCR鉴定结果显示为布鲁氏菌，两株突变株的LPS粗提物进行SDS-PAGE鉴定结果表明突变株LPS-O侧链缺失，吖啶橙凝集反应和感染血清的虎红平板试验证明突变株O抗原缺失，表现为粗糙型。2.通过感染小鼠试验发现，感染8周后，亲本株S19，突变株Δwzm和Δwzt的log脾菌数分别为4.11±0.31，2.86±0.52，2.52±0.41，突变株Δwzm和Δwzt较亲本株S19感染小鼠后的脾菌数显著下降，脾指数分别为1.30±0.23，0.67±0.12，0.62±0.52，与亲本株相比脾指数显著下降，说明wzm/wzt基因缺失会造成毒力下降。3.免疫小鼠后，通过淋巴细胞增殖、淋巴细胞转化实验发现，Δwzm和Δwzt突变株引起的淋巴细胞转化CD4+/CD8+比值较亲本株S19要高，但是CD3+淋巴细胞数较亲本株降低，淋巴细胞增殖指数也较亲本株下降显著。通过ELISA检测血清中IgG抗体水平结果显示，Δwzm和Δwzt突变株免疫小鼠产生的抗体水平较S19亲本株要高，而且抗体持续时间较长。对免疫后小鼠的脾细胞体外培养诱导产生IFN-γ、IL-2、IL-4、IL-10、TNF-、NO均表现不同程度的下降，Δwzt比Δwzm突变株诱导产生的IFN-γ无论是细胞水平，还是体液水平比亲本株都要低。4.研究表明wzm和wzt基因缺失均使2,3-二磷酸甘油酸依赖型磷酸甘油酸变位酶、甘油醛-3-磷酸脱氢酶、乙酰辅酶A羧化酶的羧基转移酶亚基、50S核糖体组成蛋白L9、磷酸甲基嘧啶激酶、ATP/GTP结合蛋白、ABC转运单位底物结合蛋白等8个代谢相关蛋白的表达下调。此外wzm基因缺失造成核苷二磷酸激酶、果糖-2-磷酸醛缩酶、3-羟基丁酰CoA脱氢酶、核糖体循环因子、硫胺素-磷酸焦磷酸化酶、硫代甘氨酸氧化酶等6蛋白表达量下降，同时引起噻唑合成酶、钼酸盐转运蛋白、双组分系统效应调节蛋白、孔蛋白Omp2b、6,7-二甲基-8-ribityllumazine合成酶等6个蛋白表达量上调。wzt基因缺失造成6-磷酸-葡萄糖酸内酯酶、电子转移黄素蛋白亚基、饥饿蛋白、超氧化物歧化酶、50S核糖体组成蛋白L25、NAD(P)H还原酶、蛋白输出蛋白secB、ATP依赖的Clp蛋白酶水解亚单位、双精氨酸转运途径的信号序列结构域蛋白等8个基因表达下调，同时引起TAXI家族TRAP转运受体、铁硫ATP酶SufC、辅酶Q-细胞色素C还原酶FeS亚基、延伸因子Ts等4个蛋白表达上调。上述研究结果表明，布鲁氏菌wzm和wzt基因缺失产生了粗糙型突变株，进一步说明wzm和wzt基因与细菌LPS-O侧链的合成有关，两个突变菌株对小鼠的毒力较亲本株降低，揭示wzm和wzt基因通过影响LPS合成从而影响细菌毒力。两个突变株均能诱导产生比亲本株高的抗体效价，但是细胞免疫水平下降，说明wzm和wzt基因缺失均增强细胞免疫的同时抑制了体液免疫。比较蛋白质组学研究发现，wzm和wzt基因缺失均影响多种蛋白质的表达以下调为主，他们影响蛋白质表达调控的机制有待于进一步研究。更多还原

【Abstract】 Brucellosis is a zoonosis, which not only seriously affects the development ofaquaculture, also poses human health a huge threat. Prevention and control of humanand animal brucellosis is difficult for many reasons, but pathogenesis (including themechanism of intracellular parasitism) and immune mechanism are unclear that is themain reason.Lipopolysaccharide (LPS) is one of the major Brucella virulence factors, and isone of the major antigens in mammals immunized. Currently Brucella LPSsynthesis pathway is not completely clear, and several critical LPS synthesis-relatedgenes have been found, wbkA, gmd, wzm, wzt, wbkB, wbkC et al., in which wzm andwzt genes are major components.ABC transporter, in bacterial cells, is a major transporter of the machine consistsof a series of homologous genes encoding an operon. The operon is substantiallycomposed of three parts, ATP-binding proteins, membrane proteins, protein subunits,and is typically formed two integral membrane proteins by six transmembranesegments. ABC transport system is one important transport corridors in outermembranes on biological macromolecules synthesized, involved in the synthesis ofextracellular polysaccharides. In addition, ABC transporters also play an importantrole in transporting nutrient and exporting some molecules. However, the effect of theABC transporter system on bacterial virulence, immunogenicity, physiologicalmetabolism needs to be proved.In this study, a plasmid was constructed for knockout wzm/wzt gene by allelicexchange methods, and LPS deletion mutants were constructed. The spleen index and CFU of infected mice spleen was evaluated. And the differences of the parent strainand mutants on antibody level, cytokine level, lymphocyte proliferation and proteindifferences by2D electrophoresis. The effect of gene mutation on virulence,immunogenicity, and gene expression was evaluated, in order to further reveal thepathogenesis and immune mechanism of Brucella, and lay the foundation for thedevelopment of the rough Brucella vaccine.The results showed:1. The lethal plasmid was constructed for wzm/wzt knockout, and amplified byPCR and digested by restriction enzymes, containing sacB gene as a reverse selectionmarker, wzmf/wztf and wzmr/wztr arms of target genes fragments. The allelicexchange plasmid was electro-transformed into vaccine strain B. abortus S19, andscreened on TSB agar solid medium (kanr) with5%sucrose, and then screened byPCR amplification. The mutant sites were confirmed correct, and511bp,446bp genewere deleted. The specific primers multiple-PCR identification results showed asBrucella. LPS crude extracts of two mutant strains were analyzed by SDS-PAGE, andthe results showed that the mutants LPS molecule distribution is missing. Acridineorange agglutination of mutants and the test of serum by Rose Bengal plateagglutination test proved O antigen deleted on bacteria as rough mutants.2. The results showed that the logCFU of infected spleen of S19, Δwzm andΔwzt strains were4.11±0.31,2.86±0.52, and2.52±0.41, and the spleen indexswere1.30±0.23,0.67±0.12, and0.62±0.52respectively. And the Δwzm and Δwztmutants were decreased significantly compared to the parental strain S19, thatindicated wzm/wzt gene deletion caused virulence decline.3. The lymphocyte proliferation and lymphocyte transformation were analyzedthat Δwzm and Δwzt mutants induced lymphocyte CD4+/CD8+ratio higher than theparental strain S19, but the number of CD3+lymphocytes and lymphocyteproliferation decreased compared with the parent strain. IgG antibody levels detectedby ELISAin serum showed immunized antibody levels of Δwzm and Δwzt mutants in mice were higher compared to the parental strain S19, and longer maintained durationof the antibody. Mice spleen lymphocyte cells of mutants cultured in vitro to produceIFN-γ, IL-2, IL-4, IL-10, TNF-, and NO showed different degrees of declinecompared with the parental strain S19. And Δwzt mutant induced lower production ofIFN-γ thanΔwzm mutant.4. Results showed that wzm/wzt gene deletion caused2,3-bisphosphoglycerate-dependent phosphogl-ycerate mutase, glyceraldehyde-3-phosphate dehydrogenase,Acetyl-coenzyme A carboxylase carboxyltransferase subunit alpha,50S ribosomalprotein L9, Phosphomethyl-pyrimidine kinase, ATP/GTP-binding protein, and ABCtransporter substrate-binding protein expression down-regulation. wzm deletioncaused the decline of Nucleoside diphosphate kinase, fructose-bisphosphate aldolase,glyceraldehyde-3-phosphate dehydrogenase, Ribosome recycling factor,Thiamine-phosphate pyrophosphorylase, Glycine oxidase ThiO proteins, while causedThiazole synthase, Molybdate ABC transporter, Two component transcriptionalregulator, porin omp2b,6,7-dimethyl-8ribityllumazine synthase protein up-regulated.wzt gene deletion caused6-phosphogluconolactonase, electron transfer flavoproteinsubunit alpha, DNA protection during starvation protein, Superoxidedismutase[Cu-Zn],50S ribosomal protein L25, Aldo-keto reductases, Protein-exportprotein secB, ATP-dependent Clp protease proteolytic subunit, Twin-argininetanslocation pathway signal sequence domain-containing protein down-regulated,while caused TRAP transporter solute receptor, FeS assembly ATPase SufC,ubiquinol-cytochrome c reductase, iron-sulfur subunit, elongation factor Tsup-regulated.These results indicated that Brucella wzm and wzt genes deletion caused roughmutants, and further described that wzm and wzt genes were involved in bacterialLPS-O side chain synthesis. The virulence of two mutant strains was decreased thanthe parental virulent strains that revealed wzm and wzt genes were related to bacterialvirulence. Two mutants could induce higher antibody titer than the parent, but decreased levels of cell-mediated immunity, which indicated wzm and wzt genesaffected the bacterial immunogenicity. Comparative proteomics study found that manyproteins expression are affected by wzm and wzt genesand mostly decreased, whichneed further study on the regulation of protein expression.更多还原