角蛋白酶基因kerB的提取、 克隆及表达

从角蛋白酶产生菌株地衣芽孢杆菌L-25中提取基因组DNA， 借助特定引物通过聚合酶链反应(PCR)扩增得到kerB基因片段. 扩增后的kerB片段通过分子生物学方法克隆 到产酶缺陷型枯草芽孢杆菌DB104菌株敏感细胞中进行表达. 结果表明， 表达成功的FD-8菌 株（DB104/pLK18）可以在羽毛培养基上生长并完全水解羽毛.     

产CTX-M型超广谱β-内酰胺酶肺炎克雷伯菌耐药性分析

目的了解吉林市区肺炎克雷伯菌CTX-M型超广谱β-内酰胺酶（ESBLs）的基因型分类及耐药情况,并对其分子流行病学特征进行分析.方法采用PCR扩增法检测118株肺炎克雷伯菌产CTX-M型ESBLs基因型情况,调查其阳性株对14种常见抗菌药物的耐药情况,对产CTX-M型肺炎克雷伯菌进行AP-PCR同源性分析,并绘制基因分析图谱.结果 118株肺炎克雷伯菌中共扩增出CTX-M型菌株25株,占21.19%;其中CTX-M-1组13株,占11.02%;CTX-M-9组16株,占13.56%;同时含CTX-M-1组和CTX-M-9组菌株4株,占3.39%;扩增未发现CTX-M-2组、CTX-M-8组和CTX-M-25组阳性株.25株产CTX-M型超广谱β-内酰胺酶均为多重耐药菌（耐3种以上抗菌药物）,对红霉素耐药率高达100%;其次耐药率依次为头孢噻肟92%,氨苄西林92%,四环素88%;对亚胺培南耐药率较低,对美罗培南均敏感.25株CTX-M型阳性菌株做随机扩增多态性分析,共发现13种RAPD.结论吉林市区产CTX-M型超广谱β-内酰胺酶菌株均为多重耐药,且对红霉素、头孢噻肟和氨苄西林类耐药率均很高.CTX-M酶在一定程度上存在克隆传播.     

成都地区60株铜绿假单胞菌中第I类整合子的鉴定及特性分析

采用两种PCR法(整合酶PCR法和长片段整合子PCR法)检测60株成都地区4家医院分离的铜绿假单胞菌的第Ⅰ类整合子,并对扩增产物进行序列分析.对60株铜绿假单胞菌第Ⅰ类整合酶基因的检测,发现有28株扩增结果为阳性(46.7%),所扩增片段的大小为110 bp.测序结果与GenBank中已报道的ⅠntI1基因核酸序列同源性为100%.长片段PCR法扩增Ⅰ类整合子的可变区,60株铜绿假单胞菌中有18株检测到不同大小的Ⅰ类整合子基因盒,占标本总数的30.0%.插入的基因盒大小从300 bp到超过2000 bp不等,18株整合子阳性菌中,有两株菌同时含有2种大小不同的整合子.序列分析结果表明,菌株R03的1900 bp 整合子携带aacA4基因盒,菌株W13的1900 bp整合子中含dfr17和aadA5基因盒.     

植病生防木霉菌的RAPD分析

对分离自不同植物根际的38株木霉菌菌株(包括哈茨木霉菌，绿色木霉菌，康宁木霉菌和绿木霉菌)进行了棉花立枯病盆栽防治试验以及用290条随机引物进行了RAPD分析。结果表明，38株木霉菌都能够降低病害严重度，按照防治效果的高低，38个菌株可以分为6个组。共有59条引物产生多态性扩增条带，根据UFGMA聚类分析，其中9条引物(S1，S42，S60，S122，S146，S147，S171，S180，S181)在防效最高的菌株中产生相近的与其他菌株不同的扩增条带，表明这RAPD技术可以用于本霉菌类生防菌株的筛选。     

宁夏牛源克雷伯菌和大肠埃希菌分离株产超广谱β-内酰胺酶基因型检测

为了解宁夏牛源产超广谱β-内酰胺酶（ESBLs）肺炎克雷伯菌（Klebsiella pneumonia）和大肠埃希菌（Esche-richia coli）的耐药性和基因型分布,采用美国临床实验室标准化研究所（CLSI）2008年推荐的双纸片确证试验,对宁夏地区100株克雷伯菌和大肠埃希菌进行产ESBLs确定,并采用PCR扩增法和克隆测序法对产ESBLs菌株进行基因分型.结果显示,宁夏地区有42株产ESBLs菌株,阳性率为42%;其中31株菌扩增呈阳性,产TEM型菌17株,产SHV型菌14株,产CTX型菌15株;同时产3种基因型菌1株,同时产2种基因型菌13株,产单一基因型菌17株,分别占产ESBLs菌株的2.38%,30.95%,40.48%;多表现为多重耐药.     

不同花色金盏菊的RAPD分析

利用RAPD分析技术,选取13个具有10个碱基长度的随机引物,对开淡黄色花和橙色花的金盏菊进行了扩增反应,在对扩增产物进行琼脂糖电泳后显示：有9个随机引物扩增出条带,一共扩增出条带89条,平均每个随机引物扩增约10条,在9个随机引物中,有3个引物扩增的条带在两种不同花色金盏菊中有差异,此项研究结果将为分析控制淡黄色和橙色花色有关的基因提供一点新的思路。     

用PCR法检测苏云金杆菌的溶原性噬菌体

根据苏云金杆菌(Bacillus thuringiensis,B.t)溶原性噬菌体G IL01(GenBank Accession Number:AJ536703)的同源序列设计一对引物,用35株B.t标准菌株和生产菌株MZ1(血清型H3 a3b)的过夜培养物作模板,结果有16株菌株包括MZ1能扩增出大小约为750 bp的预期产物,推测这16株菌为溶原菌。MZ1菌株的过夜培养液与指示菌ZK1(血清型H25)混匀并在双层琼脂上培养12 h后出现了滴度为2×107pfu的噬菌斑,从而证明生产菌株MZ1确为溶原菌;又以G IL01同源性较高的其它四个基因设计引物,分别以MZ1的培养物及其溶原性噬菌体基因组为模板进行PCR扩增,结果两者得到几乎一致的带型,进一步证明了生产菌株MZ1为溶原菌,说明利用PCR法检测鉴定苏云金杆菌的溶原性是可行的。比较指示菌方法、直接电镜法和DNA鉴定法,PCR法快速简便,对B.t生产上快速检测其溶原性噬菌体有实用意义。     

苯酚降解菌的富集、分离与鉴定

 用苯酚作为唯一碳源富集培养化工厂、废水处理厂、造纸废水样品,从中分离得到4株具有苯酚降解能力的菌株.用编码苯酚羟化酶大亚基基因的保守序列设计特异引物进行检测,共有3株菌扩增出相应的产物.对其中降解能力最强的1株菌的16SrDNA序列进行系统发育分析,确定其为假单胞菌.与以前方法相比,本方法可检测到目前几乎所有的苯酚羟化酶基因,从而实现快速、准确、方便地从苯酚污染的环境中检测苯酚降解菌.     

啤酒酵母XB05及其突变株JY2-2的RAPD分析

菌株JY2-2是啤酒酵母(Saccharomyces cerevisiae)XB05经半导体激光诱变筛选得到的优良菌株.为了从分子水平考察菌株JY2-2与XB05的差异,为酵母菌株JY2-2的鉴定提供分子依据,我们运用随机扩增多态性DNA(RAPD)技术对啤酒酵母XB05和JY2-2进行全基因组分析.用27条随机引物对这两株酵母菌进行扩增,结果得到5条可产生较多扩增条带,并具有较丰富多态性的随机引物,每条引物可扩增8～20条DNA条带,分子量大小在100～3000bp之间.菌株XB05和JY2-2的DNA遗传相似系数为0.794,可以认为菌株JY2-2是啤酒酵母XB05的突变株.     

喀斯特地貌高海拔自然保护区土壤放线菌多样性研究

为了揭示贵州喀斯特地貌高海拔读取土壤放线菌多样性,通过在贵州喀斯特地貌高海拔自然保护区采集土壤,对放线菌进行分离、提纯和培养;并对已获得的放线菌菌株的16Sr DNA、18Sr DNA、ITS以及26S通过引物进型PCR扩增,扩增后行进DNA序列测定以及菌株鉴定。结果表明:该区域土壤中包含了1株为疑似新型放线菌、1株为链孢囊菌属,为较稀有菌属。4株为拟诺卡氏菌属,11株为链霉菌属、为常见菌属。此次研究初步揭示了贵州喀斯特地貌高海拔地区土壤中放线菌多样性。     

A draft genome of Yersinia pestis from victims of the Black Death

Technological advances in DNA recovery and sequencing have drastically expanded the scope of genetic analyses of ancient specimens to the extent that full genomic investigations are now feasible and are quickly becoming standard. This trend has important implications for infectious disease research because genomic data from ancient microbes may help to elucidate mechanisms of pathogen evolution and adaptation for emerging and re-emerging infections. Here we report a reconstructed ancient genome of Yersinia pestis at 30-fold average coverage from Black Death victims securely dated to episodes of pestilence-associated mortality in London, England, 1348-1350. Genetic architecture and phylogenetic analysis indicate that the ancient organism is ancestral to most extant strains and sits very close to the ancestral node of all Y. pestis commonly associated with human infection. Temporal estimates suggest that the Black Death of 1347-1351 was the main historical event responsible for the introduction and widespread dissemination of the ancestor to all currently circulating Y. pestis strains pathogenic to humans, and further indicates that contemporary Y. pestis epidemics have their origins in the medieval era. Comparisons against modern genomes reveal no unique derived positions in the medieval organism, indicating that the perceived increased virulence of the disease during the Black Death may not have been due to bacterial phenotype. These findings support the notion that factors other than microbial genetics, such as environment, vector dynamics and host susceptibility, should be at the forefront of epidemiological discussions regarding emerging Y. pestis infections.     

Genome sequence of Yersinia pestis, the causative agent of plague

The Gram-negative bacterium Yersinia pestis is the causative agent of the systemic invasive infectious disease classically referred to as plague, and has been responsible for three human pandemics: the Justinian plague (sixth to eighth centuries), the Black Death (fourteenth to nineteenth centuries) and modern plague (nineteenth century to the present day). The recent identification of strains resistant to multiple drugs and the potential use of Y. pestis as an agent of biological warfare mean that plague still poses a threat to human health. Here we report the complete genome sequence of Y. pestis strain CO92, consisting of a 4.65-megabase (Mb) chromosome and three plasmids of 96.2 kilobases (kb), 70.3 kb and 9.6 kb. The genome is unusually rich in insertion sequences and displays anomalies in GC base-composition bias, indicating frequent intragenomic recombination. Many genes seem to have been acquired from other bacteria and viruses (including adhesins, secretion systems and insecticidal toxins). The genome contains around 150 pseudogenes, many of which are remnants of a redundant enteropathogenic lifestyle. The evidence of ongoing genome fluidity, expansion and decay suggests Y. pestis is a pathogen that has undergone large-scale genetic flux and provides a unique insight into the ways in which new and highly virulent pathogens evolve.     

我国鼠疫研究概况

主要从鼠疫的发生与鼠疫耶尔森菌的发现、区域分布、易感宿主与控制等方面对我国鼠疫研究进行了综述。     

Xenorhabdus nematophila BP品系xptB1基因的克隆和序列分析

采用PCR方法,从该室构建的Xenorhabdus nematophila BP品系粘粒文库的一个对棉铃虫有强口服杀虫活性的粘粒cos83中扩增出全长的xptB1基因,将扩增片段回收纯化后,连接到pMD-18T载体,转化大肠杆菌TG1后进行序列测定和分析。结果显示,该基因全长为3051bp,编码1016个氨基酸,其编码的毒素BP XptB1与X.nematophila PMF1296品系的XptB1的氨基酸序列同源性平均为95.8%,两侧高度一致,中间第607—738位差异明显。结构分析显示,两者在跨膜区,α螺旋等方面也有差异。BP XptB1与Photorhabdus luminescens的TccC毒素,Serratia entomophila的SepC,以及Yersinia pseudotubercusis和Y.pestis等的杀虫毒素均有58%以上的同源性。分析结果预示了XptB1可能是一个杀虫毒素。     

Evolutionary genetics: Ambiguous role of CCR5 in Y. pestis infection

Mecsas and colleagues suggest that a deficiency in the chemokine receptor CCR5 in humans is unlikely to confer protection against plague, based on their study of Yersinia pestis infection in Ccr5-deficient mice. They were testing the hypothesis that a mutation in the CCR5 gene, frequently found in Caucasians, may have been selected for in the past because it provided protection against (bubonic) plague; the mutation, called CCR5Delta32, is characterized by a 32-base-pair deletion. We have also tested this hypothesis by using Y. pestis infection in mice and, in addition, we have done phagocytosis experiments with macrophages from wild-type and Ccr5-deficient mice. Although, like Mecsas et al., we did not see any difference in the survival of the two groups of mice, we did find that there was a significantly reduced uptake of Y. pestis by Ccr5-deficient macrophages in vitro. Our results indicate that the role of Ccr5 in Y. pestis infection may therefore be more complex than previously thought.     

Increased virulence of Yersinia pseudotuberculosis by two independent mutations

A chromosomally encoded protein, which mediates invasion into HeLa cells was recently identified in Yersinia pseudotuberculosis. The role of this protein (invasin) in the virulence process was not, however, investigated. We show that mutation of the invasin gene in Y. pseudotuberculosis abolishes the ability of the bacteria to invade HeLa cells. When mice were challenged by intraperitoneal injection both the mutant and the wild-type strain produced infections of similar virulence but mutant showed a slower rate of infection after oral challenge. A double mutant, carrying an additional mutation in the gene coding for the Yop1 protein, was also constructed. The double mutant was significantly more virulent than either the wild-type or the corresponding single mutants. Y. pestis, in contrast to Y. pseudotuberculosis lacks the ability to express either invasin or Yop1, sequence analysis of the yopA gene from both Y. pestis and Y. pseudotuberculosis shows that the yopA gene of Y. pestis contains a point-mutation leading to a reading-frame shift. When the yopA+ gene was introduced into Y. pestis the virulence of this strain was reduced. These results may provide insight into the rise and fall of plague epidemics caused by Y. pestis.     

Caenorhabditis elegans: plague bacteria biofilm blocks food intake

Bubonic plague is transmitted to mammals, including humans, by the bites of fleas whose digestive tracts are blocked by a mass of the bacterium Yersinia pestis. In these fleas, the plague-causing bacteria are surrounded by an extracellular matrix of unknown composition, and the blockage depends on a group of bacterial genes known as the hmsHFRS operon. Here we show that Y. pestis creates an hmsHFRS-dependent extracellular biofilm to inhibit feeding by the nematode Caenorhabditis elegans. Our results suggest that feeding obstruction in fleas is a biofilm-mediated process and that biofilms may be a bacterial defence against predation by invertebrates.     

Evolutionary genetics: CCR5 mutation and plague protection

A recent and prevalent mutation in the chemokine receptor CCR5 in humans of northern European ancestry has been proposed to provide protection against bubonic plague. Here we infect both normal and CCR5-deficient mice with the bacterium Yersinia pestis, the cause of the plague epidemics that wiped out one-third of Europeans in the Middle Ages, and find no difference in either bacterial growth or survival time between the two groups. Unless the pathogenesis of Yersinia infection differs markedly between mice and humans, our results indicate that CCR5 deficiency in people is unlikely to protect against plague.     

低温脂肪酶产生菌筛选与鉴定、产酶条件及酶学性质研究

 昆明东站一家屠宰厂冷库中筛选到14株产脂肪酶低温菌株.选取1株胞外低温脂肪酶高产菌,进行显微形态及生理生化特征、16S rRNA基因序列分析,将其初步鉴定为Yersinia enterocolitica的一株菌,命名为KM1.对该菌发酵产酶条件研究,发现其最佳产酶发酵条件为:培养温度13℃,pH7.2,摇瓶培养时间54h.在最佳产酶发酵条件下酶活由1.8U/mL提高到3.1U/mL,提高了72%.对该菌脂肪酶的酶学性质研究表明:在0℃时仍具有最高酶活的20%,最适反应温度37℃,最适反应pH为9.0.在25℃以下,pH7.2～10范围内均能保持良好的稳定性.该酶在有机溶剂中较稳定,即使在50%的甲醇中仍能保持60%以上的活性,该酶的最适底物为C₈的酯化物,且对C₄～C₁₂的酯化物均有较好的催化能力.