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.     

Identification of Yersinia pestis with varied plasmid composition using monoclonal and polyclonal fluorescent immunoglobulins

Abstract The efficiency of serological identification of Yersinia pestis strains which contain different plasmids was assessed with polyclonal and monoclonal immunoglobulin preparations in the direct fluorescent antibody method. Plague polyclonal luminescent immunoglobulins recognize only those Y. pestis strains which contain pPst, pFra plasmids or both. Anticapsular plague monoclonal antibodies interact only with capsule-forming plague agent strains (pFra+) grown at 37°C. With plague monoclonal lipopolysaccharide antibodies one can identify all Y. pestis strains irrespective of their plasmid content and cultivation temperature. However, these antibodies cross-react with Yersinia pseudotuberculosis bacteria in 60% of cases. The problem of laboratory diagnosis of the plague organism, whatever its plasmid profile, can be solved through the development of a test kit involving two preparations such as plague lipopolysaccharide monoclonal luminescent antibodies and pseudotuberculosisspecific luminescent adsorbed immunoglobulins.     

鼠疫耶尔森氏菌质粒上重要毒力相关基因的克隆与表达

鼠疫耶尔森氏菌含有3种质粒pMT1、pPCP1和pCD1,这3种质粒编码鼠疫耶尔森氏菌的多种重要毒力因子。首先通过生物信息学技术选定了18种可能重要的毒力相关基因作为拟克隆和表达的目的基因。通过：PCR技术、TA克隆技术、双酶切技术获得目的片段。这些目的片段再分别克隆入原核表达载体pET32a中,构建了一系列重组表达质粒,其中12个重要的毒力相关基因在原核表达载体pET32a中有稳定的高效表达,表达量占细菌总蛋白的20％～40％。实验结果为进一步研究质粒编码的毒力因子的结构与功能,及其作为新型疫苗选择的可能性奠定了基础。     

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。     

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。     

Cellular fatty acids as chemical markers for differentiation of Yersinia pestis and Yersinia pseudotuberculosis

Aims:  Gas chromatography (GC) was utilized to investigate the cellular fatty acids (CFAs) composition of 141 Yersinia pestis isolates from different plague foci of China, and 20 Yersinia pseudotuberculosis strains as well.
Methods and Results:  The whole cell fatty acid methyl esters (FAMEs) were obtained by saponification, methylation and extraction followed with analysis using a standardized Microbial Identification System (MIS). Y. pestis and Y. pseudotuberculosis strains are quite similar in major CFA profiles, which include 16:0, 17:0 cyclo, 3-OH-14:0, 16:1ω7c and 18:1ω7c, accounting for more than 80% of the total CFAs.
Conclusions:  Yersinia pestis could be easily differentiated from Y. pseudotuberculosis by plotting the ratios of some CFA pairs, i.e.,14:0/18:0 vs 18:1ω7c/18:0, 3-OH-14:0/18:0 vs 18:1ω7c/18:0, 16:1ω7c/18:0 vs 18:1ω7c/18:0, 12:0/18:0 vs 18:1ω7c/18:0 and 12:0 ALDE/18:0 vs 16:1ω7c/18:0 fatty acids.
Significance and Impact of the Study:  In the present study, the normalized Sherlock MIS and Sherlock standard libraries were used to analyse the fatty acid composition of different strains of Y. pestis and Y. pseudotuberculosis . Meanwhile, ratios of certain CFA components were found to serve as chemical markers for differentiating the two closely related bacteria that are difficult to be differentiated by simply comparing CFA profiles based on other researches.     

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。     

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。     

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.     

鼠疫耶尔森氏菌VcrV基因在大肠杆菌中的高效表达

将测序后的鼠疫耶尔森氏菌（Yersinia pestis)LcrV基因重组质粒pGEM－T／ypV酶切,克隆于原核表达载体pBV220,构建成pBV/ypV表达质粒,转化大肠杆菌DH5α,进行PCR及酶切鉴定,筛选阳性克隆,进行温控诱导表达,SDS－PAGE检测表达产物,在相对分子质量38000处有－表达条带,经薄层扫描分析目的蛋白带占全菌蛋白的38．4％以上,主要以可溶形式存在。     

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.     

鼠疫溶菌疫苗免疫小鼠的体液免疫应答

为选择以F1抗原为主要有效成分的鼠疫溶菌疫苗(Whole cell lysate of Yersinia pestis vaccine,WCLY)的免疫程序,设计了这组试验。在37℃培养鼠疫EV菌,通过超声波裂解法制备鼠疫溶菌疫苗。设计(0,2周)、(0,4周)、(0,2,4周)三种免疫程序,以每剂总蛋白量7.9μg、31.5μg和126.0μg三个剂量皮下接种NIH小鼠。分别在第一针免疫后2、4、8、12周采集血清,通过间接ELISA检测抗鼠疫菌F1抗原和总抗原抗体。结果显示:免疫后血清抗体上升很快,2周内即可测出;无论哪种免疫程序,至12周时抗体滴度仍保持高水平;加强免疫后,抗体水平在4周或8周达到较高,可与活疫苗免疫者相比;溶菌疫苗的接种剂量为7.9μg时,动物只出现轻度不良反应。提示鼠疫溶菌疫苗需要两剂免疫,最短可间隔2周,接种剂量应不超过7.9μg,疫苗中应富含F1抗原。     

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。     

鼠疫耶尔森氏菌环二鸟苷酸代谢及生物被膜形成调控研究进展

鼠疫耶尔森氏菌(Yersinia pestis,以下简称"鼠疫菌")是烈性传染病鼠疫的病原菌,以鼠蚤作为传播媒介。鼠疫菌在其传播媒介鼠蚤的前胃中形成生物被膜从而促进其在宿主间传播。鼠疫菌生物被膜的形成受第二信使分子环二鸟苷酸(c-di-GMP)的正向调控。鼠疫菌中c-di-GMP由二鸟苷酸环化酶(DGC)HmsT和HmsD合成,由磷酸二酯酶(PDE)HmsP降解。文中主要介绍影响鼠疫菌环二鸟苷酸代谢及生物被膜形成的调控因子,并对其作用机制进行讨论和总结。     

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.     

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。     

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。