我国新发现的鼠疫自然疫源地鼠疫菌基因组序列测定及分析

目的 测定和分析我国新发现的鼠疫自然疫源地鼠疫菌株（耶尔森菌）的全基因组序列,探讨玉龙疫情菌株(D106004)与邻近两个疫源地剑川菌株(D182038)和西藏菌株(Z176003)的亲缘关系。方法 采用全基因鸟枪法及Solexa方法对3株鼠疫菌株进行全基因组测序,并进行比较基因组学分析。基因组间编码序列比较分析采用BLAST软件进行,基因组间重排分析采用MAUVE软件进行。结果 鼠疫菌株D106004、D 182038、Z176003均具有1个染色体和3个质粒,菌株间染色体、质粒特征基本相似;3株菌株间编码序列的蛋白相邻类的聚簇(COG)功能分类及插入序列数目比较差异无统计学意义(x2值分别为3.03、0.257,P均＞0.05)。菌株间编码序列、单核苷酸多态性(SNPs)和基因组重排结果显示,在3株菌株中,有2882个基因具有100％的同源性。其中D106004菌株预测的3636个基因中与D182038菌株一致的基因有2994个,90％以上相似的基因有240个;与Z176003菌株一致的基因有3113个,90％以上相似的基因有200个;D106004菌株与Z176003、D182038菌株的同义SNPs数为59、68个,非同义SNPs数为104、203个;D106004与Z176003菌株之间可分为11个重排片段,较之D106004与D182038菌株之间的16个重排片段数目明显减少。结论 3株鼠疫菌株基因之间具有高度同源性,D106004与Z176003菌株之间的亲缘关系较之与D182038菌株更为接近,玉龙疫源地菌株可能是由西藏疫源地菌株进化而来。     

鼠疫菌特异基因的克隆表达和抗原性分析

目的 在大肠埃希菌中克隆表达鼠疫菌特异基因(YPO1089.pst、ymt),分析重组蛋白的抗原性.方法 利用PCR技术扩增目的 基因,PCR产物经纯化、双酶切后,与质粒载体pET-30a(+)相连接并转化大肠埃希菌BL21(DE3)感受态细胞.经异丙基硫代-β-D-半乳糖苷(IPTG)诱导表达目标蛋白,并进行10%的变性聚丙烯酰胺凝胶电泳,蛋白免疫印迹分析其特异性,最后采用亲和层析法纯化诱导表达的融合蛋白.结果 成功构建了pET-YPO1089、pET-pst和pET-ymt 3个重组表达质粒,经优化诱导表达条件,重组的rYP01089、rPst和rYmt在大肠埃希菌中均得到了稳定高效地表达;免疫印迹结果表明重组的rPst可与鼠疫阳性血清发生特异性的免疫反应;目标蛋白经纯化后纯度可达95%以上.结论 鼠疫菌的特异性抗原能够在原核蛋白表达系统中获得高效表达,rPst具有敏感且特异的免疫学特性,实验结果为开发新型鼠疫诊断试剂奠定了基础.     

青藏铁路沿线鼠疫菌质粒的研究

目的 检测青藏铁路沿线鼠疫菌携带的质粒种类及相对分子质量.方法 采用碱裂解,酚一氯仿抽提法提取鼠疫菌质粒,经琼脂糖凝胶电泳进行检测并分析质粒的相对分子质量.结果 所检测的18株鼠疫菌具有6×106,45×106,52×106,65×106,92×106质粒,其中大质粒的变化范围在52×106～92×106.结论 青藏铁路沿线鼠疫菌除具有规范的质粒图谱外,鼠疫菌最大质粒的变化有一定的规律性,具有分类属性,对研究鼠疫自然疫源地空间结构和鼠疫菌的遗传学特性具有重要意义.     

鼠疫菌6 kb质粒核酸序列同源性比对分析

目的 分析云南省鼠疫菌6 kb(pYC)质粒核酸序列与GenBank中公开报道的多种细菌序列的同源性.方法 通过美国国立图书馆网站序列比对搜索工具(BLAST),对鼠疫菌pYC质粒全序列和开放式读码框架(ORFs)以及翻译蛋白进行核酸与核酸、蛋白与蛋白比对分析.结果 pYC质粒部分基因与宋内志贺菌质粒pKYM具有97.1%的同源性,与流感嗜血杆菌基因有92.1%的同源性,与伤寒沙门杆菌基因LT2和pIMVS1分别具有88.2%和87.2%的同源性,与大肠埃希菌0157:H7、K-12、ECOR31株局部基因分别具有81.4%、81.4%和84.7%的同源性.pYC质粒ORFs翻译蛋白ORF1与副鸡嗜血菌复制蛋白B具有47.2%的相似性,ORF4与大肠埃希菌推定蛋白具有52.7%的相似性,ORF5与假结核菌TriE蛋白具有48.3%的相似性,ORF6与大肠埃希菌PilxS/VirB5相似蛋白具有42.3%的相似性、与小肠结肠炎耶尔森菌TriD蛋白具有38.5%的相似性,ORFIO与伤寒沙门杆菌LT2细胞质蛋白具有83.1%的相似性.与大肠埃希菌0157:H7推定蛋白具有81.9%的相似性.ORF11与大肠埃希菌K12诱导损伤蛋白J具有81.4%的相似性.结论 pYC质粒DNA序列与肠杆菌科部分基因具有高度同源性,可能编码类似埃希菌属和耶尔森菌属推定蛋白、诱导损伤蛋白、TriD和TriE蛋白、Pilx5/VirB5相似蛋白.     

利用分子生物学技术对鼠疫耶尔森菌的分类鉴定

目的 对4株可疑菌株进行系列验证实验,以确定其是否为鼠疫耶尔森菌(以下简称鼠疫菌)自然分离株.方法 用鼠疫细菌学常规方法和分子生物学手段确定其生物学表型特征、特异性基因及基因组特征.结果 4株菌具备鼠疫菌的典型形态特征;能被鼠疫噬菌体完全裂解;主要生化特性为阿胶糖(+)、鼠李糖(-)、麦芽糖(+)、蜜二糖(-)、甘油(-)、脱氮(+),与鼠疫菌同源菌株菌苗株(EV76)一致;其毒力因子为F1(+),VW(+),Pgm(-),PstⅠ(+),与鼠疫菌菌苗株(EV)特征完全一致;109个/ml可疑菌对实验动物小白鼠无致死能力.具有鼠疫标识基因;差异片段(DFR)分型结果,4株菌的24个DFR扩增,DFR谱不同于任何鼠疫菌自然分离菌株基因组型.结论 尽管4菌株具备鼠疫菌自然分离株的一些表型特征,但基因组特征表明其不是鼠疫菌自然分离株,而是鼠疫菌同源菌株菌苗株(EV菌).     

鼠疫耶尔森菌亲缘性分型方法研究进展

狭义的亲缘性是指具有一定的血缘或者婚姻的社会关系,现在亲缘关系的范围正在扩火,而本文中亲缘性是指具有相同的遗传背景,相似的进化分支,结构或功能.现将研究鼠疫耶尔森菌亲缘性的方法综述如下.     

青海省鼠疫疫源地鼠疫耶尔森菌的基因型分布

目的研究青海省鼠疫耶尔森菌(鼠疫菌)基因型分布特征。方法对分离到的青海省148株鼠疫菌,根据已经证实的22个差异区段设计引物,每株鼠疫菌的每个基因差异区段都采用PCR技术进行验证。结果148株青海省鼠疫菌共包括8个基因型,即1、5、7、8、14型、新基因组型和Ype-ancestor型,其中以5型和8型为主。祁连山南麓和青海湖环湖地区的祁连、门源、刚察、海晏、共和、天峻等的菌株绝大多数属于基因型8型,占40．5％(60／148);位于青南高原的格尔木、玉树、扎多、治多、称多、囊谦和曲麻莱等的菌株,其基因型主要为5型,占31．8％(47／148)。青藏高原青海田鼠鼠疫疫源地鼠疫菌基因型全部为14型,占8．1％(12／148)。结论在青海省分离的鼠疫菌中,喜马托雅旱獭鼠疫疫源地鼠疫菌的基因型以5型和8型为主,青海田鼠疫源地鼠疫菌以基因型14型为主。     

鼠疫耶尔森氏菌16S-23SrRNA基因间区分析

目的　发展一种快速准确的鉴定鼠疫菌的方法。方法　 Ｐ Ｃ Ｒ 反应加限制性内切酶消化的方法。结果　对来自不同疫源地的 １０３ 株鼠疫菌 １６ Ｓ２３ Ｓｒ Ｒ Ｎ Ａ 基因间区进行扩增,均可扩出两条长为 １ １４６ｂｐ 及 １ ０９０ｂｐ 的片段,扩增产物用限制性内切酶 Ｔａｑ Ｉ, Ｍ ｓｐ Ｉ消化后的酶切图谱相同。而对照菌株小肠结肠炎耶尔森氏菌,鼠伤寒沙门氏菌,大肠杆菌,痢疾杆菌,霍乱弧菌的扩增产物及酶切图谱与鼠疫菌均不相同。结论　鼠疫菌 １６ Ｓ２３ Ｓｒ Ｒ Ｎ Ａ 基因间区高度同源,基本为两种类型,长度分别为 １ １４６ｂｐ 及 １ ０９０ｂｐ,其他菌株与其明显不同;该方法将有助于快速准确的鉴定鼠疫菌。     

鼠疫耶尔森菌外膜蛋白及其功能研究进展

鼠疫耶尔森菌和其他革兰阴性杆菌一样,其细胞结构由双层膜结构包绕,内层为内膜(亦称胞浆膜),外层是细胞与外环境的分界,称为外膜.     

鼠疫菌质粒上标识基因的筛选

目的 筛选鼠疫菌质粒上保守、稳定、特异的DNA标识序列.方法 选择32条源于鼠疫菌质粒上DNA序列,采用常规PCR技术,对云南家、野鼠两型共40株鼠疫菌、47株非鼠疫菌、鼠疫菌疫苗株EV76(阳性对照),进行了特异性验证试验和稳定性鉴定试验.结果 筛选出4对相对保守、稳定、特异的DNA标识序列:YPMT1.05c,YPMT1.03c,YPMT1.42及YPMT1.04c.结论 所筛选的4对鼠疫菌DNA标识序列,可用于鼠疫快速诊断.     

广西鼠疫耶尔森菌质粒图谱及分子流行病学意义

目的分析广西鼠疫耶尔森菌株质粒DNA种类,从分子水平探讨其流行病学意义。方法采用Kado改良法提取质粒,经琼脂糖凝胶电泳。结果从广西鼠疫自然疫源地分离到31株鼠疫耶尔森菌含有pYC、pPst/pPCP1、pYV/pCD1、pFra/pMT1和尚未明确其类型的111.36×106(相对分子质量,Mr)5种质粒。其中有29株由pYC、pPst/pPCP1、pYV/pCD1、pFra/pMT14种质粒组成,1株由pYC、pPst/pPCP1、pYV/pCD1、111.36×106(Mr)4种质粒组成,另一株由pYC、pPst/pPCP1、pYV/pCD13种质粒组成。结论广西鼠疫耶尔森菌株含有pYC、pPst/pPCP1、pYV/pCD1、pFra/pMT1和尚未定型的111.36×106(Mr)5种质粒,与云南省南部、东南部和贵州省鼠疫耶尔森菌株的质粒图谱相同。     

从动物和土壤中直接检测鼠疫耶尔森氏菌

目的　探讨用防污染的 PCR- EL ISA(U DPE)技术检测人为污染的土壤和感染的动物标本的可行性。方法　根据鼠疫耶尔森氏菌 Cafl和 Pla基因分别设计了 3条引物 ,组合成 3对引物 ,建立 U DPE检测技术。结果　利用该实验体系可以检出每克土壤中 10 0 0个鼠疫耶尔森氏菌的污染。比检测纯细菌的敏感性低 2 0 0倍 ,说明土壤中有抑制 PCR反应的因子。对 2 0只实验组动物 40份标本 (肝脏和脾脏 )的培养、U DPE和 PCR-电泳检测表明 ,3种方法的检出吻合率为 10 0 % ,均能从 40份标本中检出靶细菌 ,而用 3种方法检测对照组动物的 2 0份肝脏和脾脏均为阴性。结论　利用 UDPE技术可以被用于动物脏器中鼠疫耶尔森氏菌的检测     

中国鼠疫耶尔森菌染色体结构特征的研究

目的 了解中国鼠疫耶尔森菌不同分离株之间的染色体结构差异,探索染色体重排在鼠疫菌中发生的原因及用于菌株遗传特征识别和亲缘关系分析的可能性.方法 以完成全基因组测序的首株鼠疫菌株CO92(美国)为参考株,以从中国分离出的已完成全基因组测序的4株鼠疫菌株(内蒙古91001、云南剑川D182038、云南玉龙D106004、西藏那曲Z176003)为对比株,根据美国国立生物技术信息中心网站(http://www.ncbi.nlm.nih.gov/genome)上公布的这5株鼠疫菌的染色体序列及染色体上所有编码基因序列间的相似性,将鼠疫菌染色体人为的划分成多个大的DNA节段(染色体板块),确定这些板块在不同鼠疫菌株间排列方式的差异以及板块之间的断裂区片段的基因组成;根据菌株两两比较所得的重排差异结果,分析菌株间的亲缘关系.结果 除Z176003菌株外,CO92、D182038、D106004、91001菌株可被分成44个相对独立的染色体板块,板块内部基因组成和排列高度稳定,板块之间可以相对移动.与参考株CO92菌株比较,D182038、D106004菌株染色体均存在13处重排,Z176003菌株染色体存在14处重排,91001菌株染色体存在37处板块排列顺序的改变.菌株间两两比较,D106004与Z176003菌株之间仅有8处染色体板块排列方式不同,表明二者的亲缘关系最为接近.CO92菌株染色体上的相邻板块之间存在43个断裂区,有39个断裂区的DNA片段含有插入序列,其中25个插入序列为IS100.结论 鼠疫菌基因组具有一定的流动性,不同菌株间的染色体结构存在较大差异,染色体重排事件发生的原因与其拥有的插入序列密切相关.菌株间染色体的重排差异,能用于菌株遗传特征的识别和亲缘远近关系的分析.     

鼠疫菌6MD质粒在假结核菌中的表达及其对假结核菌毒力的影响

使用电穿孔转化技术,将鼠疫耶尔森氏菌所携带的6MD 质粒转移到假结核耶尔森氏菌中,获得了杂交菌株。该杂交菌株能够表达鼠疫菌素Ⅰ及凝固酶,但其毒力却仍与作为受体的假结核菌株类似,而没有向鼠疫菌的毒力水平接近。本文对这种现象的可能原因进行了分析。     

鼠疫菌pYC质粒对菌体蛋白质组影响的初步研究

目的 了解鼠疫菌pYC质粒对菌体蛋白质组的作用。方法 将含有pYC质粒的鼠疫菌与不含该质粒的鼠疫菌通过双向电泳的方法进行蛋白质组比对,并将差异蛋白进行质谱鉴定。结果 含有pYC质粒的鼠疫菌与不含该质粒的鼠疫菌的蛋白图谱可识别的蛋白点均在500个以上,两者总体上相似性较高;前者较后者在大小约70×103、等电点5.0左右处,明显多一个蛋白点簇,该蛋白簇经质谱鉴定均为伴侣蛋白GroEL,但该蛋白并非是pYC质粒编码蛋白。结论 pYC质粒对菌体蛋白质组有影响,其编码蛋白pYC_p10与pYC_p11,可能调控GroEL高表达。     

胶体金免疫层析法诊断鼠疫的特异性试验

目的研究胶体金免疫层析法用于鼠疫快速诊断的特异性。方法采用胶体金免疫层析测试条和鼠疫反向间接血凝试验(RIHA)平行检测。结果检测鼠疫菌F1抗原具有很强的特异性,不与包括小肠结肠炎和假结核耶尔森等其他细菌发生交叉免疫反应。结论该方法简便、快速、敏感,适合现场鼠疫监测。     

Stone Age Yersinia pestis genomes shed light on the early evolution,diversity, and ecology of plague

The bacterial pathogen Yersinia pestis gave rise to devastating outbreaks throughout human history, and ancient DNA evidence has shown it afflicted human populations as far back as the Neolithic. Y. pestis genomes recovered from the Eurasian Late Neolithic/Early Bronze Age (LNBA) period have uncovered key evolutionary steps that led to its emergence from a Yersinia pseudotuberculosis-like progenitor; however, the number of reconstructed LNBA genomes are too few to explore its diversity during this critical period of development. Here, we present 17 Y. pestis genomes dating to 5,000 to 2,500 y BP from a wide geographic expanse across Eurasia. This increased dataset enabled us to explore correlations between temporal, geographical, and genetic distance. Our results suggest a nonflea-adapted and potentially extinct single lineage that persisted over millennia without significant parallel diversification, accompanied by rapid dispersal across continents throughout this period, a trend not observed in other pathogens for which ancient genomes are available. A stepwise pattern of gene loss provides further clues on its early evolution and potential adaptation. We also discover the presence of the flea-adapted form of Y. pestis in Bronze Age Iberia, previously only identified in in the Caucasus and the Volga regions, suggesting a much wider geographic spread of this form of Y. pestis. Together, these data reveal the dynamic nature of plague’s formative years in terms of its early evolution and ecology.

The earliest known cases of human infection with the plague pathogen, Yersinia pestis, date to around 5,000 y ago (1–4). Analyses of ancient Y. pestis genomes from this period suggest that the time window between 6,000 and 4,000 y ago was critical and formative for the evolution and ecology of Y. pestis as we know it today. Four ancient Y. pestis lineages have been identified so far, which can be genomically distinguished based on their adaptations to the flea, the main vector of modern plague. Today, fleas are known to play a central role in the transmission of plague within rodent populations, which can act as reservoirs from where spillovers to human populations typically occur (5, 6). The transmission of Y. pestis by the flea is either facilitated by a blockage of the foregut (proventriculus), where the bacterium produces a biofilm (7), or in a biofilm-independent manner, also known as early-phase transmission (8, 9). The oldest lineages of Y. pestis (2, 4) (hereafter referred to as preLNBA−), and the Late Neolithic and Early Bronze Age (LNBA−) lineage (1, 3) present a genetic background that has been interpreted as being incompatible with flea transmission via the blockage of the foregut (indicated in the naming by the minus sign). While a recently identified ancient lineage also dating to the Bronze Age presents all the genetic adaptations for this highly efficient form of flea transmission (10) (LNBA+; the plus sign indicates the adaptation to the flea vector). Intriguingly, both variants coexisted for millenia and they might have occupied different niches. However, it remains unclear how the different forms of Y. pestis infected humans during prehistory and how the resulting diseases manifested in the human population. Whether plague ecology and transmission as we know it today can serve as a model to understand its manifestation in the past remains also unknown.Elucidating the ecology and transmission will be crucial for understanding how the LNBA+/− lineages of plague, which were widespread across Eurasia for thousands of years (1, 3, 10, 11), have impacted human societies, and how changes in human subsistence and economy have shaped the early evolution of this pathogen. It is currently unknown whether and which types of animal populations served as potential reservoirs of the disease and their identification will be essential for characterizing past Y. pestis transmission dynamics. The absence of an adaptation to the flea vector in some plague lineages suggests that the transmission dynamics were complex. Today, the flea-mediated model is not the only documented form of plague transmission: pneumonic plague can be acquired via respiratory droplets from close human-to-human contact. However, only a few reported outbreaks have been attributed to this transmission mode and usually in contexts of poor ventilation and direct contact with infected individuals (12–17). Additionally, plague has been documented in humans who handled or ingested parts of infected animals (18–22).Changes in human behavior may also have contributed to a higher risk of plague infection. During the LNBA period, archeological evidence attests to technological advances, such as the spread of oxen-drawn carts and wagons (23) and horse domestication (24), which enabled increased human mobility and exploitation of new habitats, such as the Eurasian steppe belt. This ultimately led to the establishment of long-distance networks, in which raw materials such as copper were circulated (25, 26). However, periods of unrest and war could also have played a role in the extended human mobility during the LNBA period. While earlier studies hypothesized that increased mobility was the cause for an early spread of Y. pestis across Eurasia (1, 3), it could also have been its effect. It is also during this period that animal husbandry and mobile pastoralism intensified in the steppe (27), thus facilitating the overlap of ecological niches for zoonoses to occur. The aforementioned changes could have played a role in the likelihood of transmission to humans and the long-distance spread of plague during its early evolution.Here we expand the number of Y. pestis genomes from the LNBA period to offer a higher genomic resolution for important stages in the evolution of the bacterium, as well as its diversity and geographical distribution in the past. By linking the genomic evidence with the available archeological context, we discuss potential transmission mechanisms of plague during its early evolution.     

南方鼠疫菌对18种抗菌素的敏感性试验

目的 通过鼠疫菌对新型抗菌药物的敏感性实验,寻找比链霉素敏感性更高且毒性小的药物.方法 选用22株鼠疫菌对18种活性较高的抗菌素进行了纸片法药物敏感性试验.结果 头孢菌素类、青霉素类和喹诺酮类等多种药物对鼠疫菌的敏感性较链霉素好.结论 本试验结果可为鼠疫临床治疗选择新药提供参考资料.     

从基因水平看鼠疫菌的侵袭致病性

目的：阐述鼠疫菌的侵袭致病性。方法：从自然界生物进化的角度，分析鼠疫菌致病基因表达受环境因素调节，成功实现在宿主(包括侵入皮下组织和进一步扩散到深层组织)和蚤体内的寄生。结果：环境因素调节鼠疫菌基因的表达和致病性，结论：在其生命循环过程中，与环境相适应的基因表达促进了致病性。     

鼠疫菌对抗生素的敏感性综合评价

目的 建立鼠疫菌对抗生素敏感性综合评价方法,并用该方法就鼠疫菌对6种抗生素的敏感性进行综合评价。方法 通过查阅文献,收集鼠疫菌对抗生素敏感性实验数据,以抗生素对强毒141菌株、弱毒EV76paris菌株和云南分离菌株的抑菌情况,即抑菌环直径作为评价指标,用层次分析法综合评价鼠疫菌对所选6种抗生素（头孢他啶、氨苄青霉素、头孢唑啉、环丙沙星、诺佛沙星、链霉素）的敏感性。结果 鼠疫菌对不同抗生素的敏感性存在差异,鼠疫菌对所选6种抗生素的敏感性由高到低依次为头孢他啶、氨苄青霉素、头孢唑啉、环丙沙星、诺佛沙星、链霉素,综合评分指数分别为1.730 77、1.631 77、1.581 95、1.567 80、1.449 48、0.999 99。结论 运用层次分析法评价鼠疫菌对不同抗生素的敏感性可以取得合理、客观和较为准确的评价结果,可以利用该方法及其评价结果为进一步筛选适宜的鼠疫治疗药物提供参考依据。