The serodiagnosis of human infections with Yersinia enterocolitica and Yersinia pseudotuberculosis

The techniques of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting were evaluated for the serodiagnosis of human infections with Yersinia enterocolitica and Yersinia pseudotuberculosis. Lipopolysaccharide (LPS) was prepared from strains comprising four serogroups of Y. enterocolitica and five serogroups of Y. pseudotuberculosis, tested against 200 sera submitted to the Laboratory of Enteric Pathogens for routine serodiagnosis, and shown to contain antibodies to Yersinia LPS by agglutination. Forty four sera were found to contain antibodies that bound to one of the LPS preparations used in the immunoassay. Thirty five of the sera contained antibodies to the LPS of Y. enterocolitica O3, whilst three contained antibodies to the LPS of Y. enterocolitica O5, 27 and Y. enterocolitica O9 LPS respectively. Two sera had antibodies to the LPS of Y. pseudotuberculosis II and a single serum contained antibodies to Y. pseudotuberculosis IV. The SDS-PAGE-immunoblotting procedure described proved to be a reliable procedure for the serodiagnosis of infections with Y. enterocolitica and Y. pseudotuberculosis.     

鼠疫耶尔森氏菌LcrV基因的克隆及序列分析

为了研究鼠疫耶尔森氏菌（Y.pestis）保护性抗原V蛋白,从基因库中查得Y.pestis LcrV基因DNA序列,针对序列设计合成了一对PCR扩增引物,以本所保存的Y.pestis菌种为模板进行基因扩增,结果获得长约980bp的DNA片段。将扩增产物回收纯化,克隆至pGEM-T载体,构建重组载体pGEN-T/ypV,经过PCR,酶切鉴定,并对pGEM-T/ypV中的V基因片段进行测序,分析测序结果与己知序列相同,表明获得了LcrV基因。     

Characterization of IS1541-like elements in Yersinia enterocolitica and Yersinia pseudotuberculosis

We characterized Yersinia enterocolitica and Yersinia pseudotuberculosis insertion sequences related to insertion sequence 1541, recently identified in Yersinia pestis. For each of the two species, two insertion sequence copies were cloned and sequenced. Genetic elements from Y. pseudotuberculosis were almost identical to insertion sequence 1541, whereas these from Y. enterocolitica were less related. Phylogenetic analysis of the putative transposases encoded by insertion sequences from the three pathogenic members of the genus Yersinia showed that they clustered with those encoded by Escherichia coli and Salmonella enterica elements belonging to the insertion sequence 200/insertion sequence 605 group. Insertion sequences originating from Y. pestis and Y. pseudotuberculosis constitute a monophyletic lineage distinct from that of Y. enterocolitica.     

松鼠猴感染假结核耶尔森菌的诊治

2013 年2 月18 - 20 日,四川省雅安市碧峰峡野生动物园内6 只松鼠猴突然接连死亡,同时约有100 只松鼠猴也出现了不同症状,给动物园造成了严重的损失。为寻找传染病因和治疗防控措施,首先采用尸体剖解和病理切片对其进行了组织学检查,然后采用细菌分离培养和鉴定确定了病原菌,通过动物回归试验和耐药性试验发现其致病性和流行性特征,并采取紧急治疗和防疫措施及时控制了疫情。结果表明,死猴尸体内多处器官发生淤血坏死,组织病变严重并可见大量细菌。经鉴定,分离到的病原细菌为假结核耶尔森菌,该菌与鼠疫耶尔森菌同源性极高,且具有强烈的致病性、传染性和一定的耐药性,对丁胺卡拉霉素较为敏感。这是我国首次发现并报道的松鼠猴感染假结核耶尔森菌病例,本文对其诊断与治疗、流行病学研究和病理模型建立提供了一定的基础参考资料。     

Plasmid content in Yersinia pestis strains of different origin

Plasmid content in 242 Yersinia pestis strains from various natural plague foci of the U.S.S.R. and other countries was studied. Of these strains, 172 (71%) were shown to carry three plasmids described previously of about 6, 45-50 and 60 MDa, respectively. Twenty strains (8%) from different foci harboured additional cryptic plasmids, most often of about 20 mDa in size. Plasmid pPst displayed considerable constancy of its molecular mass. On the contrary, size variations of pCad (45-49 MDa) and, especially, pFra (60-190 MDa) were found. Molecular mass of these plasmids correlated with the host strain origin.     

鼠疫菌毒力调控研究

鼠疫菌通过一系列转录调控子(如CRP、PhoP、RovA和Fur)控制着一些关键毒力因子(如Pla、强毒力岛、III型分泌系统等)的基因表达。鼠疫菌可感应宿主体内信号刺激,紧密调控毒力因子的表达。在这个紧密调控过程中,调控子、毒力相关基因构成了一个动态网络。鼠疫菌在从假结核菌祖先演化的进程中,基因表达调控网络的重塑在鼠疫菌毒力进化过程中发挥着不可取代的作用。     

鼠疫菌噬菌体效价噬菌斑测定法的建立

目的建立鼠疫菌噬菌体噬菌斑效价测定方法。方法通过分析细菌接种浓度、孵育吸附时间及培养温度等参数,建立鼠疫菌噬菌体效价测定方法,并分析其精密性;建立鼠疫活疫苗鉴别及纯菌检查用噬菌体效价质量标准。结果经优化后确定细菌接种浓度为7×108/mL,不需孵育吸附,培养温度为29℃,所建立的检测方法精密性较好,用于鼠疫活疫苗鉴别及纯菌检查用噬菌体效价质量标准应不低于1×106PFU/mL。结论建立了鼠疫菌噬菌体噬菌斑效价测定方法,为鼠疫菌噬菌体及疫苗质量控制奠定了基础。     

A cloned chromosomal DNA fragment which differentiates Yersinia pestis from Yersinia pseudotuberculosis and Yersinia enterocolitica

Chromosomal DNA from reference Yersinia strains was digested individually with 9 restriction endonucleases. DNA fragments were separated and analyzed by electrophoresis through agarose gels. The clearest fragment patterns were obtained when EcoRI was employed. The Y. pestis fragment pattern obtained after the use of this enzyme showed the presence of a unique DNA fragment with molecular mass 1400 bp. This DNA fragment was cloned, purified, labeled with 32P and then used to probe EcoRI digests of all three Yersinia species. A strong hybridization signal was obtained with Y. pestis strain. No such signal was found with Y. pseudotuberculosis or Y. enterocolitica. These results indicate that the DNA fragment is species specific and could be used as a diagnostic DNA probe for Y. pestis.     

鼠疫耶尔森氏菌基因表达的环境调节

鼠疫耶尔森氏菌是烈性传染病鼠疫的病原菌,该菌在媒介(跳蚤)和宿主(哺乳动物)之间的循环过程中,基因表达适应环境谱的变化。本介绍鼠疫耶尔森氏菌适应环境信号如不同温度、离子浓度、pH等条件下的基因表达调控研究现状。     

巴尔通体细胞脂肪酸成分分析

【目的】分析影响巴尔通体脂肪酸成分的主要因素,建立适合巴尔通体脂肪酸成分分析的标准化方法,探讨脂肪酸图谱应用于巴尔通体分类鉴定的可能性。【方法】应用气相色谱技术分析不同培养条件下巴尔通体脂肪酸的组成与含量的变化;应用已构建的标准化方法获取10株巴尔通体标准菌株和9株来自不同地区的汉赛巴尔通体猫分离株脂肪酸图谱;应用SPSS16.0统计软件对获得的数据资料进行聚类分析。【结果】培养基、温度和传代次数主要影响巴尔通体脂肪酸的微量成分;10株巴尔通体标准菌株的成分相似,但也存在构成和含量上的差异;在所测巴尔通体中,检出可分辨脂肪酸成分有20种,共有成分为7种,其中C18:1ω7c、C18:0和C16:0累积含量达80%以上;猫分离株被准确鉴定为汉赛巴尔通体。【结论】巴尔通体脂肪酸成分受培养基、温度等培养条件影响,在脂肪酸提取方法标化后,可用于汉赛巴尔通体种水平分类鉴定。     

鼠疫耶尔森氏菌基因转录调控的研究进展

细菌基因转录调控是多种调控机制中研究最为广泛的一种模式。复杂而精细的基因转录调控网络有助于细菌应答外界环境压力,在病原菌致病与传播中均发挥着关键作用。本文以鼠疫耶尔森氏菌基因转录调控的相关研究进展为基础展开论述,重点阐述细菌的转录调控机制、转录调控的研究策略及鼠疫菌致病与传播中转录调控的作用,以期为深入研究鼠疫菌致病与传播中的基因转录调控分子机制提供新思路。     

IS1OO周围序列多态性分析技术的建立及其在鼠疫耶尔森氏菌分型中的应用

建立鼠疫耶尔森氏菌IS1000周围序列多态性（ISCP）分析技术,并探讨其在鼠疫耶尔森氏菌分型中的应用,根据鼠疫杆菌CO92株IS100的基因序列在其两端设计两条向外延伸的引物进行PCR扩增,电泳,获得的指纹图用RAPD,PHYLIP和Treeview软件分析,建立的ISCP分析技术稳定,可靠,利用该技术分析17个生态型的271株鼠疫耶尔森氏菌,扩增结果表明,指纹图有一定的差异,经RAPD,PHYLIP和Treeview分析可分为3个类型,IS100在鼠疫耶尔森氏菌染色体中虽然分布较广,但其周围序列变异较小,在遗传上较稳定,可作为鼠疫耶尔森氏菌的基因标志,研究该菌的分型与进化。     

鼠疫菌OxyR调控子蛋白对dps的转录调控机制

[目的]利用分子生物学实验研究鼠疫菌调控子OxyR对dps的转录调控机制.[方法]提取鼠疫菌野生株(WT)和oxyR突变株(ΔoxyR)的总RNA,采用引物延伸实验研究dps的转录起始位点,并根据产物的丰度判断OxyR对dps的调控关系.进一步采用实时定量RT-PCR的方法验证OxyR对dps的调控关系.PCR扩增dps的整个启动子区DNA序列,并纯化His-OxyR蛋白,通过凝胶阻滞实验(EMSA)验证OxyR对dps启动子区是否具有直接的相互作用.利用大肠杆菌OxyR识别基序,预测鼠疫菌OxyR对dps启动子区的结合位点,从而得出鼠疫菌OxyR对dps的转录调控机制.[结果]鼠疫菌dps有一个转录起始位点G(-40)(翻译起始位点为+1),其转录表达受OxyR的激活;体外实验及生物信息学预测结果表明OxyR能结合到dps启动子区-111到-78之间的碱基上.[结论]OxyR能直接结合到dps启动子区而激活其转录表达.     

重组鼠疫耶尔森菌LcrV抗原原液性质研究

目的进行重组鼠疫耶尔森菌LcrV抗原原液二聚体含量及性质研究,确定LcrV原液的相关质控标准。方法在不同缓冲体系(0.85%NaCl(NS)、20 mmol/L PBS),不同蛋白浓度(2.0、1.5、1.0、0.5、0.1 mg/mL)及不同保存温度(4℃、-20℃、-70℃)条件下保存LcrV抗原,采用SDS-PAGE和HPSEC方法定期检测LcrV二聚体含量及纯度。将连续三批检定合格的LcrV抗原原液进行质谱相对分子质量测定、等电点测定、N末端氨基酸序列测定、圆二色(CD)谱、HPLC肽谱及氨基酸组成分析,研究LcrV抗原的相关性质。结果随着保存时间的延长LcrV抗原二聚体含量增加,低温保存时二聚体不易大量形成。在-20℃和-70℃条件下,NS保存的LcrV抗原比PBS体系保存稳定,而在4℃条件下NS保存的LcrV抗原容易降解。LcrV抗原高浓度保存容易发生聚合。LcrV抗原在低质量浓度(0.1 mg/mL)保存时免疫学活性明显下降。质谱检测到LcrV单体和二聚体共同存在,且与理论相对分子质量一致。LcrV原液检测等电点范围为4.6～6.3。N末端测序、CD谱、HPLC肽谱图及氨基酸组成分析与理论结果一致。结论 LcrV抗原原液保存条件确定为:NS体系,蛋白质量浓度1.0～2.0 mg/mL,-20℃以下冻存。制备的LcrV抗原各项检测结果与理论结果一致,抗原性质稳定。     

Adhesive properties of the purified plasminogen activator Pla of Yersinia pestis

The beta-barrel outer membrane protease Pla from Yersinia pestis is an important virulence factor in plague and enables initiation of the bubonic plague. Pla is a multifunctional protease whose expression also enhances bacterial adherence to extracellular matrix. It has remained uncertain whether the increase in cellular adhesiveness results from modification of the bacterial surface by Pla, or whether the Pla molecule is an adhesin. Pla was purified as a His6-fusion protein from Escherichia coli and reconstituted with lipopolysaccharide to an enzymatically active form. Purified His6-Pla was coated onto fluorescent micro-particles (FMPs) that expressed plasminogen activity. Pla-coated FMPs also bound to laminin and to reconstituted basement membrane (Matrigel) immobilized on permanox slides, whereas only poor activity was seen with lipopolysaccharide-coated FMPs or bovine serum albumin-coated FMPs. The results show that the Pla molecule has intrinsic adhesive properties and that purified transmembrane proteins coated onto FMPs can be used for functional assays.     

Isolation and characterization of recombinant OmpF-like porin from the Yersinia pseudotuberculosis outer membrane

The encoding sequence of the pore-forming OmpF-like protein from the Yersinia pseudotuberculosis outer membrane was cloned and expressed in Escherichia coli cells. Conditions for isolation and refolding of recombinant monomer and porin trimer were selected. Their spatial structures were characterized by the intrinsic protein fluorescence and CD spectroscopy. It was shown that recombinant porins are similar in the composition of secondary structure elements to isolated porins, but have a considerably less compact tertiary structure. The pore-forming activities of the recombinant proteins are similar to those of Y. pseudotuberculosis native porins.     

鼠疫耶尔森氏菌rV270抗原的克隆、表达及其免疫保护作用评价

目的：为研制鼠疫亚单位疫苗,克隆、表达并纯化去除产生免疫抑制作用序列后的鼠疫耶尔森氏菌LcrV抗原（rV270）。方法：依据已知的LcrV的核苷酸序列,避开其产生免疫抑制作用的区段设计引物,扩增rV270基因并克隆到pET24a载体中,在大肠杆菌BL21中表达His-rV270融合蛋白：表达产物先后经Co^2＋亲和层析和Sephacryl S-200HR凝胶柱纯化,并在纯化过程中应用凝血酶切除His标塔;氢氧化铝佐剂吸附重组抗原后免疫BALB／c小鼠,初次免疫后第21天加强免疫1次,第5周使用104CFU鼠疫菌141强毒株攻毒,测定其免疫保护作用。结果：rV270以可溶性方式表达;应用Co^2＋亲和层析柱和Sephacryl S-200HR凝胶柱结合凝血蛋白酶切除His标签的方法可得到不含标签的较高纯度的重组蛋白;攻毒实验中实验组小鼠全部存活,而对照组全部死亡。结论：获得了具有良好免疫保护作用的rV270蛋白,可用于鼠疫亚单位疫苗的研究。     

Structural modification of a base disaccharide alters antiadhesion properties towards Yersinia pestis

Incubation in the presence of structurally modified disaccharides altered the in vitro attachment of Yersinia pestis GB to three human respiratory epithelial cell lines. Each disaccharide resulted in decreased attachment to the alveolar epithelial (A549) cell line. The best inhibitor of attachment for each cell line was the benzylated derivative of Galbeta1-4GalNAc. Highly negatively charged saccharides were efficient inhibitors, particularly for the bronchial epithelial (BEAS2-B) cell line. The data indicate that targeted modification of receptor ligands could offer a novel therapeutic preventing Y. pestis attachment to host cells.     

Development of a double-antibody sandwich ELISA for sensitive detection of Yersinia pestis

We developed a biotin–streptavidin-based sandwich ELISA for the sensitive and specific detection of Yersinia pestis. In this assay, the F1 capsular protein and Y. pestis were captured by anti-F1 mouse monoclonal antibody followed by detection with biotinylated-anti-F1 rabbit polyclonal antibody and HRP-conjugated streptavidin. The developed F1 ELISA could detect not only the F1 protein up to 29 and 17 pg/ml but also Y. pestis up to 177.8 and 129.2 CFU/ml in PBS buffer and human serum, respectively. In addition, the F1 ELISA did not show any cross-reactivity with various proteins and bacterial strains.     

鼠疫耶尔森菌F1抗原的性质研究

鼠疫菌F1抗原是鼠疫亚单位新疫苗最重要的候选抗原,对其性质的充分认识,将有助于抗原制造工艺和新疫苗的开发。F1抗原的性质研究包括:微观结构,一级核苷酸、氨基酸序列,二级结构,高分子聚集形态,以及F1抗原的理化性质。