禽多杀性巴氏杆菌C48-3株外膜蛋白H的致病作用

目的:探讨禽多杀性巴氏杆菌外膜蛋白H(OmpH)的致病作用。方法:用大肠杆菌表达系统表达强毒株C48-3的重组蛋白OmpH,亲和层析法纯化N端带有6个组氨酸标签的重组OmpH,通过皮下注射兔制备抗OmpH抗血清,用生物学功能实验比较野生株C48-3、突变株ΔompH和互补株C48-3C的粘附能力、血清抵抗性和抗吞噬作用。结果:与野生株和互补株相比,突变株对CEF细胞的粘附能力显著降低,而抗OmpH抗体显著抑制野生株和互补株对CEF细胞的粘附,但该抗血清不影响突变株的粘附能力。野生株和互补株在鸡血清中的存活率显著高于突变株,但灭活鸡血清处理不影响它们的存活率。小鼠腹腔巨噬细胞对突变株的吞噬能力显著高于野生株和互补株,而抗OmpH抗体增强巨噬细胞对野生株和互补株的吞噬能力,但该抗体不影响巨噬细胞对突变株的吞噬能力。结论:OmpH是禽巴氏杆菌的致病因子,它在该菌对宿主的感染与致病过程中发挥重要的作用。     

禽多杀性巴氏杆菌C48-3株成熟黏附蛋白的表达及其免疫原性检测

目的：在大肠杆菌原核表达系统中高效表达禽多杀性巴氏杆菌成熟黏附蛋白Cpm39,并检测其免疫原性。方法：通过BamHⅠ和SalⅠ双酶切重组载体pMD18-cpm39获得cpm39基因片段,将该基因片段克隆至表达载体pMAL-p2X上,构建表达质粒pMAL-p2X-cpm39,转化至大肠杆菌BL21（DE3）,在IPTG诱导下表达融合蛋白,用禽多杀性巴氏杆菌C48-3株Cp39天然粘附蛋白的免疫血清经Western印迹检测其免疫原性。结果：SDS-PAGE结果显示表达的融合蛋白相对分子质量为78×103,与预期结果相符,而Western印迹结果表明诱导表达的融合蛋白MBP-Cpm39能与Cp39天然黏附蛋白抗体发生特异性反应。结论：构建了表达质粒pMAL-p2X-cpm39,获得了具有免疫原性的重组融合蛋白,为进一步研究禽多杀性巴氏杆菌成熟黏附蛋白的免疫保护功能奠定了基础。     

多杀性巴氏杆菌aroA基因缺失突变株的构建及鉴定

【目的】构建多杀性巴氏杆菌aroA基因缺失突变株,并验证其致病性。【方法】采用正向筛选同源重组技术构建多杀性巴氏杆菌aroA基因缺失突变株,利用PCR对突变株进行鉴定,分析其遗传稳定性、生长特性和致病性。【结果】成功构建多杀性巴氏杆菌aroA基因缺失突变株,连续传代20代,遗传稳定;突变株体外生长曲线表明,在前6h生长速度稍慢于亲本菌,随后两者生长速度一致。对小鼠的致病性试验表明:经腹腔注射aroA基因缺失突变株在1.0×106 CFU对小鼠无致死性,而亲本菌株在1.0×102 CFU对小鼠是致死性的。【结论】本研究获得多杀性巴氏杆菌aroA基因缺失突变株,对小鼠的致病性是减弱的。多杀性巴氏杆菌突变株的构建有助于研究其致病机理。     

禽多杀性巴氏杆菌粘附蛋白Cp39的交叉免疫保护作用

【目的】比较体内外增殖禽多杀性巴氏杆菌荚膜蛋白、天然粘附蛋白Cp39和重组粘附蛋白rCp39对小鼠的交叉保护作用。【方法】用NaCl提取法制备鸡胚尿囊液和DSA培养基增殖的C48-3株荚膜蛋白,并用电洗脱方法纯化Cp39蛋白,将rCp39蛋白以可溶形式表达在大肠杆菌BL21后,用Amylose Resin亲和层析柱纯化。分别以100μg剂量的鸡胚尿囊液增殖菌体荚膜蛋白、DSA培养基培养菌体荚膜蛋白、纯化的Cp39蛋白和rCp39蛋白通过皮下注射各试验组小鼠,生理盐水为对照组,第二次免疫后2周分别以A:1型菌C48-3株(6.7×102cfu)和A:3型菌C51-3株(1.1×103cfu)进行攻毒试验。采集免疫后小鼠血清,用ELISA法检测抗体水平,并计算免疫保护率,来评价4种抗原对小鼠的交叉保护效果。【结果】SDS-PAGE结果显示,体内外增殖禽多杀性巴氏杆菌荚膜蛋白的条带和分子量相似,且体内外表达的Cp39蛋白的分子量相同;ELISA结果表明Cp39免疫组小鼠和rCp39免疫组小鼠血清rCp39蛋白特异性抗体的水平显著高于其他两组(P0.05);保护试验表明,体外增殖菌体荚膜蛋白免疫组小鼠对同源C48-3株和异源C51-3株攻毒的保护率分别为100%和60%,鸡胚尿囊液增殖菌体荚膜蛋白免疫组小鼠、Cp39免疫组小鼠和rCp39免疫组小鼠对同源C48-3株和异源C51-3株攻毒的保护率分别为100%和80%。【结论】粘附蛋白Cp39是禽多杀性巴氏杆菌荚膜蛋白中的主要交叉保护抗原,可以作为禽霍乱亚单位疫苗。     

多杀性巴氏杆菌抗原的研究进展

多杀性巴氏杆菌在自然界分布广泛,是畜、禽、野生动物及人类的一种共患性病原,可以在同种和不同种的动物间传播,并引起多种畜禽巴氏杆菌病,本文就多杀性巴氏杆菌的抗原成分进行概述。     

兔多杀性巴氏杆菌C51-3株黏附蛋白的表达、纯化及其抗原性检测

应用PCR从兔多杀性巴氏杆菌C51-3株基因组DNA中扩增出编码36 kD黏附蛋白的cp36基因, 将其克隆到pMD18-T载体并对插入片段进行测序。以重组质粒pMD18-cp36为模板, 用PCR扩增得到编码信号肽除外的成熟黏附蛋白基因cpm36, 并克隆到原核表达质粒pQE30中, 得到重组质粒pQE30-cpm36, 转化大肠杆菌M15, 在IPTG诱导下表达融合蛋白CPM36, 经Ni2+-NTA亲和层析纯化。DNA测序结果表明cp36基因片段大小为1032 bp, 与已报道的16个血清型多杀性巴氏杆菌cp36基因的核苷酸序列比较, 同源性在76.9%~100%之间。SDS-PAGE结果显示, 表达分子量约为37 kD的带有6×His标签的CPM36蛋白, 与预期分子量相符。Western blotting结果表明, 抗重组蛋白抗体分别能与CPM36蛋白和多杀性巴氏杆菌36 kD蛋白发生特异性反应, 证明原核表达蛋白具有抗原性, 为进一步开展多杀性巴氏杆菌免疫保护性抗原的研究奠定了基础。     

禽巴氏杆菌C48-3株成熟黏附蛋白基因cpm39的克隆及序列分析

目的：对禽巴氏杆菌C48-3躺株编码成熟黏附蛋白的基因cpm39进行克隆和序列分析。方法：通过PCR从禽巴氏杆菌C448-3。基因组DNA中扩增出cpm39基因,克隆到pMD18-T载体中,转化大肠杆菌DH5d,并对目的基因进行核苷酸序列测定;用Clustal X和Mega 2．1软件将测定的序列与GenBank中已登录的16种血清型巴氏杆菌株核苷酸序列进行同源性分析。结果：测序结果表明cpm39基因大小为1002bp,与已知的16个血清型巴氏杆菌cpm39基因核苷酸序列的同源性为81．5％～100％。结论：克隆得到禽巴氏杆菌C。躺株编码成熟黏附蛋白的cpm39基因,该基因在不同血清型巴氏杆菌中具有很高的同源性,该蛋白可以作为研制预防巴氏杆菌病亚单位疫苗的候选抗原。     

抗多杀性巴氏杆菌植物乳杆菌细菌素的生物学特性研究

目的 针对多杀性巴氏杆菌耐药性不断增强的情况,寻找替抗产品。方法 使用MRS培养基分离酸菜中的乳酸菌菌株,采用16S rRNA基因测序鉴定分离菌株。对分离菌株进行发酵培养,研究其无菌发酵上清液对酶的敏感性、热稳定性、酸碱稳定性和其抑菌谱。结果 分离得到了1株优势乳酸菌YWH-4,经过16S rRNA基因测序鉴定该菌株为植物乳杆菌。植物乳杆菌YWH-4发酵上清液对胃蛋白酶具有高敏感性,推测其发酵上清液中具有抗菌活性物质细菌素。该细菌素具有良好的热稳定性,经100℃处理2 h后仍有较强抑菌活性;具有酸碱稳定性,在pH值3.0～5.0之间保持良好抑菌活性。结论 植物乳杆菌YWH-4所产细菌素对多杀性巴氏杆菌具有良好的抗菌活性。     

Cloning and characterization of the major outer membrane protein gene (ompH) of Pasteurella multocida X-73.

The major outer membrane protein (OmpH) of Pasteurella multocida X-73 was purified by selective extraction with detergents, followed by size exclusion chromatography. The planar lipid bilayer assay showed that OmpH has pore-forming function. The average single channel conductance in 1.0 M KCl was 0.62 nS. The gene (ompH) encoding OmpH has been isolated and sequenced by construction of a genomic library and PCR techniques. The coding region of this gene is 1,059 bp long. The predicted primary protein is composed of 353 amino acids, with a 20-amino-acid signal peptide. The mature protein is composed of 333 amino acids with a molecular mass of 36.665 kDa. The ompH gene encoding mature protein has been expressed in Escherichia coli by using a regulatable expression system. The ompH gene was distributed among 15 P. multocida serotypes and strain CU. Protection studies showed that OmpH was able to induce homologous protection in chickens. These findings demonstrate that OmpH is a protective outer membrane porin of strain X-73 and is conserved among P. multocida somatic serotypes.     

类鼻疽伯克霍尔德菌bopA基因敲除株的构建及生物学特征

【【背景】类鼻疽杆菌是一种能够引起人类疾病甚至死亡的胞内寄生菌,Ⅲ型分泌系统在该菌入侵上皮细胞、逃避宿主免疫以及毒力因子的分泌过程中发挥重要作用,其中bopA基因为TTSS-3基因编码的重要效应蛋白,在类鼻疽杆菌的免疫逃逸中发挥重要作用。【目的】构建类鼻疽杆菌bopA基因敲除菌株,并对其生物学特征进行初步研究。【方法】构建pK18mobSacB-ΔbopA自杀质粒,通过大肠杆菌S17-1λpair以接合的方式转入类鼻疽杆菌,利用同源重组敲除了bopA基因,并用蔗糖平板筛选出菌株,最后在细胞和动物水平检测敲除菌株的表型变化。【结果】构建了bopA敲除的类鼻疽菌株,并通过细胞和动物实验证实敲除bopA基因后,细菌的细胞侵袭和胞内存活以及体内定殖能力都显著降低。【结论】利用同源重组成功构建类鼻疽bopA基因敲除株,为深入研究该基因的作用靶点奠定了实验基础。     

高致病性2型猪链球菌plcR基因敲除株的构建及其相关生物学特性的研究

【目的】构建高致病性2型猪链球菌05ZYH33菌株plcR基因敲除株,通过比较突变株与野生株生物学特性的差异,研究plcR基因在2型猪链球菌致病过程中的作用。【方法】利用同源重组技术敲除plcR基因,多重交叉PCR及RT-PCR鉴定并测序验证。比较野生株与突变株基本生物学特性的差异,小鼠攻毒实验分析plcR基因缺失对细菌毒力的影响。【结果】经RT-PCR证实05SSU0241与05SSU0242共转录,通过多重交叉PCR及RT-PCR证实成功构建plcR基因缺失突变株,基本生物学特性显示突变株的生长速率、菌落形态、溶血活性均无显著改变,小鼠致病性试验结果显示,野生株攻毒的小鼠死亡率为70%,突变株攻毒的小鼠死亡率为40%,毒力较野生株显著降低。【结论】plcR基因作为2型猪链球菌有毒株基因组中特有的外源基因,在细菌致病过程中具有重要作用。     

Isopycnic characterization of lipopolysaccharides from Pasteurella multocida

In a novel application of an established procedure, isopycnic density gradient centrifugation procedures were used to analyze material obtained from the Westphal phenol extraction procedure of Pasteurella multocida cells. The initial phenol phase contained most of the lipopolysaccharides (LPS) and the major component had a buoyant density of 1.38 g/ml in CsCl density gradients. Repartitioning the phenol phase with an equal volume of water produced a second aqueous phase which contained most of the LPS. This LPS appeared as a single symmetrical band with a buoyant density of 1.40 g/ml. Buoyant density patterns obtained with schlieren optics in CsCl density gradients were useful in characterizing LPSs from P. multocida.     

Persistence of Pasteurella multocida in wetlands following avian cholera outbreaks

Avian cholera, caused by Pasteurella multocida, affects waterbirds across North America and occurs worldwide among various avian species. Once an epizootic begins, contamination of the wetland environment likely facilitates the transmission of P. multocida to susceptible birds. To evaluate the ability of P. multocida serotype-1, the most common serotype associated with avian cholera in waterfowl in western and central North America, to persist in wetlands and to identify environmental factors associated with its persistence, we collected water and sediment samples from 23 wetlands during winters and springs of 1996-99. These samples were collected during avian cholera outbreaks and for up to 13 wk following initial sampling. We recovered P. multocida from six wetlands that were sampled following the initial outbreaks, but no P. multocida was isolated later than 7 wk after the initial outbreak sampling. We found no significant relationship between the probability of recovery of P. multocida during resampling and the abundance of the bacterium recovered during initial sampling, the substrate from which isolates were collected, isolate virulence, or water quality conditions previously suggested to be related to the abundance or survival of P. multocida. Our results indicate that wetlands are unlikely to serve as a long-term reservoir for P. multocida because the bacterium does not persist in wetlands for long time periods following avian cholera outbreaks.     

Development of a toxA gene knock-out mutant of Pasteurella multocida and evaluation of its protective effects

Pasteurella multocida is an important veterinary and opportunistic human pathogen. In particular, strains of P. multocida serogroup D cause progressive atrophic rhinitis, and produce a potent, intracellular, mitogenic toxin known as P. multocida toxin (PMT), which is encoded by the toxA gene. To further investigate the toxigenic and pathogenic effects of PMT, a toxA-deleted mutant was developed by homologous gene recombination. When administrated to mice, the toxigenicity of the toxA mutant P. multocida was drastically reduced, suggesting that the PMT contributes the major part of the toxigenicity of P. multocida. Similar results were obtained in a subsequent experiment, while high mortalities were observed when toxA(+) P. multocida bacterial culture or culture lysate were administrated. Mice immunized with toxA(-) P. multocida were not protected (none survived) following challenge with toxA(+) P. multocida or bacterial culture lysate (toxin). These results suggest that the toxigenicity of P. multocida is mainly derived from PMT.