CpG-DNA对大肠杆菌诱导的大鼠实验性乳腺炎的影响

72只雌性大鼠随机分成对照组和实验组(n =36)。对照组产后0 h于左后腿胫骨前肌肌肉注射0.01 mol/L，pH 7.2 磷酸盐缓冲液(PBS)100 μL,实验组肌注CpG-DNA 200 μg/只，72 h后经乳头管灌注2×1012 cfu (colony forming unit/mL)/mL,大肠杆菌(Escherichia coli )100 μL/侧到第4对(两侧)乳腺内，分别于灌注前0 h，灌注后8，16，24，48和72 h(n = 6)颈静脉放血处死。组织学观察显示大鼠感染后16 h为乳腺腺泡嗜中性粒细胞(polymorphonuclear leukocytes，PMN)浸润高峰，实验组PMN浸润迅速， 72 h已无浸润。乳腺组织细菌数在16 h上升至最高，实验组大鼠感染后16、24和48 h乳腺组织细菌数较对照组有显著下降。CpG-DNA能极显著地提高感染前乳腺组织中IL-2水平。乳腺组织IL-6在不同时间点均有显著上升，在16 h达最高。CpG-DNA能显著地提高16 h乳腺组织IL-6水平。实验组乳腺组织N-acetyl-β-D-glucosaminidase (NAGase)无显著变化。表明CpG-DNA可加速炎症初期PMN的快速浸润，促进IL-2和IL-6的产生，减少细菌数量，减轻炎症介质对细胞的损伤并缩短炎症过程，提示CpG-DNA对大肠杆菌诱发的大鼠实验性乳腺炎有一定的预防作用。     

Protozoan predation of Escherichia coli in hydroponic media of leafy vegetables

Multiple outbreaks of food poisoning associated with fresh vegetable consumptions have occurred in many countries. Numerous reports have described human pathogenic bacteria, such as Escherichia coli O157:H7 and Salmonella spp., that can internalize into fresh vegetables via root or leaf surfaces. While attempting to obtain the threshold concentration of internalization of E. coli inoculated into hydroponic medium during vegetable cultivation, we observed a rapid decrease in E. coli numbers. In the present study, we determined that the rapid decline in E. coli was not due to a physiological change into a viable but non-culturable (VNC) state. The population crash was instead caused by true bacterial death, as the rapid descent was also confirmed by micro-colony fluorescence in situ hybridization, a culture-independent method that can detect VNC cells. We next monitored the number of E. coli inoculated into intact or filter-sterilized hydroponic medium after cultivation of various types of plants. We found that the number of E. coli in intact hydroponic medium decreased markedly, whereas the level in filter-sterilized hydroponic medium was completely unchanged. This result suggests that biotic factors were present that could be eliminated by filtering. Robust predation of E. coli by protozoa (ciliates and flagellates) was observed using fluorescently labeled bacteria incorporated into the hydroponic medium. Finally, morphological identification of flagellates by scanning electron microscopy revealed the presence of a species of Stramenopiles. These findings suggest the importance of protozoa as bacterial feeders in hydroponic systems and hence the use of these organisms as potential control agents of human pathogenic bacteria.     

在大肠杆菌内高效表达大分子量拟蜘蛛牵丝蛋白

依据已报道的蜘蛛(Nephilaclavipes)牵丝蛋白部分cDNA序列,设计合成两种不同结构的拟蜘蛛牵丝蛋白基因单体,大小分别为360和390bp。多聚化得到8倍体和16倍体后,克隆到表达载体pET-30a,得到4种表达载体,转化大肠杆菌(Escherichiacoli)BL21(DE3)诱导表达。用自制的抗蜘蛛牵丝蛋白血清Westernblot检测,表达产物呈梯度排列,主带与预计大小一致。蜘蛛牵丝蛋白表达量最高为800mg/L。     

大肠杆菌海藻糖磷酸合酶基因的克隆

根据报道的大肠杆菌（Escherichia coli)海藻糖－6－磷酸合酶基因（otsA)，设计引物，通过PCR技术从大肠杆菌XLI菌株的总DNA中扩增到一个1．4kb片段。经克隆、测序分析，该片段长1425bp并含一完整的开放读框（ORF）。在核苷酸水平上，该ORF与已报道的otsA基因具有99．86％的同源性。在氨基酸水平上，其推断性的编码产物蛋白与OtsA具有100％一致性。     

白颈长尾雉圈养种群大肠杆菌耐药性及其耐药基因

对白颈长尾雉圈养条件下的38个样品进行大肠杆菌分离及PCR检定,并采用肠杆菌基因间重复共有序列PCR指纹法（ERIC）剔除各个样品的重叠分离株,检测获得的170个大肠杆菌分离株对9种抗生素的耐药性、Ⅰ型整合子携带率及其可变区抗性基因,结果显示：（1）来自白颈长尾雉的分离株对实验用的9种抗生素的抗性比率和多重耐药性远高于环境源和人源者（来自白颈长尾雉的分离株100%耐受3种及以下的抗生素,而环境源者为50.7%,人源者66.7%）;（2）来自白颈长尾雉的分离株的Ⅰ型整合子携带率（92%）高于环境源（87%）和人源（78%）;（3）来自白颈长尾雉和来自人的大肠杆菌分离株的Ⅰ型整合子可变区抗生素抗性基因检出率相同（36%）,但高于环境源（24%）;（4）携带Ⅰ型整合子的分离株对实验用的抗生素的抗性百分率一般高于不携带者,只有个别种类抗生素这种差异为非显著性差异;（5）Ⅰ型整合子可变区基因盒的基因为3类,即aadA、dfrA和未知功能的orfF;aadA、dfrA的频率相同;3类基因均以基因盒形式存在,分别是dfrA17-aadA5、dfrA12-ofrF-aadA2、dfrA12-aadA2。     

Occurrence and survival of coliform bacteria, Escherichia coli and Salmonella in various manure and compost

pp. 865–874
Occurrence and survival of fecal-contamination indicator bacteria (coliform bacteria, Escherichia coli and Salmonella ) in various manure and compost samples collected from 23 composting facilities mostly in Kyushu were investigated by using selective media. Coliform bacteria were detected on desoxycholate agar from 11 (38%) of 29 product samples (15 cow dung manure, 4 poultry manure, 2 biosolid compost and 8 food waste compost) at a range of 10² to 10⁶ cfu g¹ dry matter. From positive samples, 21 isolates of possible coliform bacteria were purified. Among them, species of coliform bacteria ( E. coli , E. vulneria , Pantoea sp. and Buttiauxella agrestis ) were identified whereas isolates of Serratia marcescens , not coliform bacteria, were also obtained, suggesting that careful observation was necessary to avoid false positive counting due to the presence of a red colony of S. marcescens that resembled coliform bacteria. Isolates of E. coli were tested for slide aggregation with a set of antiserum against pathogenic E. coli serotypes and negative reaction was obtained for all the isolates tested. Direct detection of E. coli on Chromocult coliform agar and Salmonella on MLCB agar resulted in none and 2 (17%) of 12 samples tested, respectively. The fate of fecal-contamination indicator bacteria as above was followed during compost production on 7 cases at 6 compost facilities and 4 patterns were observed: fecal-contamination indicator bacteria 1) decreased and finally disappeared, 2) decreased once but re-growth was occurred on products, 3) decreased to some extent but remained in products, 4) was not detected throughout production. These results suggest that some fecal-contamination indicator bacteria may survive compost production and appropriate temperature control would be significant for hygiene control of manure and compost.     

大肠杆菌分子伴侣表达的辅助载体构建及应

异源蛋白在大肠杆菌不能正确折叠表达成包涵体,分子伴侣能在细胞内帮助异源蛋白折叠,改善蛋白的聚集。本研究以大肠杆菌(Escherichia coli)DH5α菌株DNA为模板,利用PCR技术扩增出6个分子伴侣编码基因groEL、groES、dnaK、dnaJ、grpE和clpB,分别将groEL、groES和grpE(Ⅰ组)插入pCDFDuet-1载体,将dnaK、dnaJ和clpB(Ⅱ组)基因插入pRSFDuet-1,构建辅助载体pR-GESP和pC-DJKL,每个重组载体中含有一个人工操纵子,每个基因上游含有T7启动子。SDS-PAGE结果显示,诱导后的重组大肠杆菌上清除了DnaJ外其它5个蛋白明显表达。SDS-PAGE分析了共表达分子伴侣对玉米(Zea mays)四吡咯分子合成的谷氨酸-1-半醛氨基转移酶、尿卟啉原Ⅲ脱羧酶和西罗叶绿三酸:铁螯合酶在大肠杆菌的折叠和聚集影响,结果显示,GroEL、GroES和GrpE能防止玉米西罗叶绿三酸:铁螯合酶在大肠杆菌的聚集,但DnaK、DnaJ和ClpB分子伴侣对该酶则没有作用;GroEL、GroES和GrpE能部分抑制玉米谷氨酸-1-半醛氨基转移酶在大肠杆菌的聚集...     

大肠杆菌yqhD基因的克隆与表达

以大肠杆菌E scherich ia coli K-12基因组DNA为模板,利用PCR技术扩增得到假定的氧化还原酶(pu tative ox idoreductase)基因yqhD,将它连接到克隆质粒pGEM-3zf(+)上,得到重组质粒pGEM-yqhD,对此重组质粒进行序列测定,对其DNA序列分析表明,yqhD基因全长为1 164 bp。再将yqhD基因插入表达载体pSE 380,构建成重组子pSE 380-yqhD,并在E.coli BL 21中获得表达。研究表明,以1,3-丙二醇为底物时,基因工程菌在30°C下,以1.0 mm o l/L IPTG诱导12 h的酶活力达到3.13 U/mL,比对照菌株提高4.4倍。     

Displacement of native rhizobia nodulating chickpea (Cicer arietinum L.) by an inoculant strain through soil solarization

Summary Soil solarization greatly reduced the native chickpea Rhizobium population. With inoculation, it was possible to increase the population of the Rhizobium in solarized plots. In the 1st year, 47% nodulation was obtained with chickpea inoculant strain IC 59 when introduced with a cereal crop 2 weeks after the soil solarization and having a native Rhizobium count of <10 g^-1 soil, and only 13% when introduced 16 weeks after solarization at the time the chickpeas were sown, with 2.0×10² native rhizobia g^-1 soil. In the non-solarized plots inoculated with 5.6×10³ native rhizobia g^-1 soil, only 6% nodulation was obtained with the inoculant. In the succeeding year, non-inoculated chickpea was grown on the same plots without any solarization or Rhizobium inoculation. The treatment that showed good establishment of the inoculant strain in year 1 formed 68% inoculant nodules. Other treatments indicated a further reduction in inoculant success, from 1%–13% to 1%–9%. Soil solarization thus allowed an inoculant strain to successfully displace the high native population in the field and can serve as a research tool to compare strains in the field, irrespective of competitive ability. In year 1, Rhizobium inoculation of chickpea gave increased nodulation and increased plant growth 20 and 51 days after sowing, and increased dry matter, grain yield, and grain protein yield at maturity. These beneficial effects of inoculation on plant growth and yield were not measured in the 2nd year.Submitted as Journal Article No. JA 945 by the International Crops Research Institute for the Semi-Arid Tropics (ICRISAT), Patancheru, Andhra Pradesh 502 324, India     

猫白介素-18基因的克隆、序列分析及其表达

用猫白介素18(interleukin,IL-18)基因特异性引物对刀豆蛋白(ConA)刺激后猫外周血单核细胞(PBMCf)总RNA进行了RT-PCR扩增,并将扩增产物纯化后克隆入pMD18-T中进行核苷酸序列测定。结果该基因全长579bp,编码192个氨基酸(GenBank登录号:DQ100372)。在推导的猫IL-18氨基酸序列中,无信号肽序列和潜在的N-联糖基化位点,但存在4个Cys残基。与不同物种IL-18相比,猫IL-18与犬、羊、牛和猪IL-18核苷酸序列有较高的同源性,分别为89.8%、88.6%、88.4%和88.1%,但与小鼠和鸡IL-18有明显的种属差异。将目的基因片段进一步亚克隆到大肠杆菌(Escherichia coli)表达载体pET28a中构建了重组质粒pETIL-18,转化大肠杆菌BL21(DE3),并用IPTG诱导。结果重组菌菌体裂解物经SDS-PAGE电泳可检测到分子量为27.5kD的重组目的蛋白。经凝胶薄层扫描,目的蛋白表达量可占菌体蛋白的13.6%。     

克雷伯氏菌1,3-丙二醇氧化还原酶基因在大肠杆菌中的克隆与表达

以基因组DNA为模板,利用PCR技术从克雷伯氏菌(Klebsiella pneumoniae)中扩增得到含有1,3-丙二醇氧化还原酶基因(dhaT)的DNA片段,将其定向连接到克隆质粒pGEM-3xf上,得到重组质粒pGEM-dhaT。将此重组质粒转化到受体菌Escherichia coli DH5α中,通过蓝白斑鉴定挑选出阳性菌株。DNA序列分析表明其基因全长为1185bp。将该片段插入表达载体pSE380,构建成重组子pSE-dhaT,并在E．coli JM109中获得表达,经SDS-PAGE检测,表达产物分子质量与天然纯1,3-丙二醇氧化还原酶(DHAT)相同,约为43ku。     

近年来大肠杆菌K88ac菌毛的变异和生物学特性

2000~2005年，从猪大肠杆菌病 (colibacillosis)中分离到一些表达K88菌毛的大肠杆菌(Escherichia coli )，这些分离株只与K88 a因子单抗反应，而不与K88 b、c和d因子单抗反应。分离的菌株 (13/16)以O149为常见血清型，且全部拥有STb毒素基因，通过K88常规血清交叉吸收试验、SDS-PAGE和Western印迹，表明这些菌株不仅与K88ac参考菌株C83907制备的c因子血清反应，而且与以分离株SEC586制备且经K88ab、K88ac和K88ad参考菌株吸收后的血清也反应，表明这些分离株仍为K88ac大肠杆菌。对新近分离的SEC464、SEC525、SEC586、SEC799和EC910株及80年代我国分离的TM128株的K88主要亚单位结构基因faeG进行克隆、测序，发现新近分离株的faeG基因由846对核苷酸组成，编码菌毛主要亚单位的261个氨基酸及21个氨基酸的信号肽，比国内外报道的K88ac FaeG亚单位 (262个氨基酸)少了一个氨基酸，比K88ab、K88ad (264个氨基酸)少了3个氨基酸。TM128株的FaeG氨基酸序列与K88ac(M29375)的同源性为100.0%，SEC464、SEC525、SEC586、SEC799和SEC910株的FaeG亚单位氨基酸序列的同源性为97.7%~99.6%，它们与K88ac的同源性为94.6%~96.6%；与K88ab的同源性为90.0%~91.6%；与K88ad的同源性为87.0%~88.9%。 结果表明新分离的K88ac大肠杆菌黏附素主要亚单位已发生了部分变异。     

地衣芽孢杆菌GXN151的纤维素酶基因cel12A的序列分析及其在大肠杆菌中的表达

地衣芽孢杆菌菌株GXNl51的cell2A基因为783bp,可编码含261个氨基酸的羧甲基纤维素酶Cel12A,Cel12A的N-末端第1～28氨基酸具典型的信号肽特征、第9～31氨基酸具跨膜功能域特征、第104～261氨基酸形成家族12糖基水解酶(glycosyl hydrolase)功能域。将编码其第73-261氨基酸的DNA序列克隆到大肠杆菌表达载体pET一30a( )得表达质粒pGXNl2A,用1mmol／IPTG诱导处理JMl09(DE3)／pGXNl2A的培养液6h pGXNl2A的表达量达到最高,在JMl09(DE3)和BL21(DE3)pLysS中该表达蛋白质可分别占菌体胞内总蛋白的54．3％和20．9％。在含羧甲基纤维素的LA平板上JMl09(DE3)／pGXNl2A和BL21(DE3)pLysS／pGXNl2A表现有较弱的羧甲基纤维素酶活性,说明重组的Cell2A的催化功能域具有独立的催化活性。包含体检测表明pGXNl2A在JMl09(DE3)中的表达产物大部分形成不溶性包含体。     

Expression of the Truncated vip3A Gene at the 3'-terminus from Bacillus thuringiensis in Escherichia coli

将苏云金芽孢杆菌(Bacillus thuringiensis)缺失C端154个氨基酸编码区的vip3A基因(vip3T)插入原核表达载体pQE30,构建了重组表达载体pQEvip3T,并转化大肠杆菌(Escherichia coli ) M15进行IPTG诱导表达,比较了完整的Vip3A蛋白和C端缺失的蛋白Vip3T的可溶性和杀虫活性。与Vip3A不同,融合蛋白Vip3T以不可溶的包含体形式存在,诱导表达的菌液中没有检测到可溶性Vip3T蛋白。生物测定结果表明,M15(pOTP)诱导表达的Vip3A蛋白对初孵斜纹夜蛾(Spodoptera litua)和甜菜夜蛾(S. exigua)幼虫具有较高的杀虫活性,其提纯的包含体无毒,但包含体的碱性裂解液却又恢复了对夜蛾科害虫的活性;M15(pQEvip3T)菌液、包含体及其碱性裂解液对这两种昆虫幼虫则完全无毒,说明在大肠杆菌中,Vip3A蛋白C端氨基酸可能对Vip3A蛋白的可溶性和杀虫活性具有重要的影响。     

Use of soil solarization to control root rots in gerberas (Gerbera jamesonii)

Summary An investigation was conducted during the summer months of 1986–1987 and 1987–1988 in Western Australia to evaluate the effect of soil solarization on the control of root rot of gerbera an also on the microbial and nutrient status of the soil. Infested soil cores were sampled from a site where root-rot was a severe problem and were removed to a non-infested site where they were subjected to soil solarization or fumigation. Soil solarization resulted in reduced root rot (root disease index 28.6%) in comparison to the untreated control (52.0%) 8 months after planting. Plants in the fumigated plots had 15.8% less disease than those in solarized plots. Solarization increased the total numbers of bacteria and actinomycetes, and the proportion of bacteria and fungi antogonistic to Fusarium oxysporum, F. solani and Rhizoctonia solani. The proportion of actinomycetes antagonistic to these fungi, however, did not differ between solarized and control soil treatments. There was a significant reduction in disease in plants grown in infested fumigated soil to which a 10% concentration of solarized soil had been added, suggesting the development of microbial suppression in solarized soil. Phytophthora cryptogea was eradicated to 30 cm by solarization as well as by fumigation. Solarization eliminated R. solani but not F. oxysporum to a soil depth of 10 cm. Solarization increased the levels of NO _n3 ^– -N and NH₄ ⁺-N in soil, but did not affect the concentrations of PO₄ ^3–, K⁺, Fe²⁺, organic C and pH. Yield (as number of flowers per plant) was increased by soil solarization and by fumigation.Increased yields and decreased disease severity in the solarized plots could have been caused by (1) a reduction in the infectivity of the infested soils, (2) an increase in the suppressiveness of the soil, and (3) an increased available of plant nutrients.     

Microbial community response to soil solarization in Nepal's rice-wheat cropping system

The Indo-Gangetic Plains of South Asia support 13.5 million hectares of rice-wheat cropping systems, which currently feed over one billion people. Intensified agriculture has resulted in a more than two-fold increase in rice and wheat yields since the 1970s; however, this continuous cropping has also exacerbated weed, pest and disease problems. Soil solarization is an accessible, low-risk management practice for small-holder farmers that has ameliorated these problems in some settings and has the potential to dramatically improve yields. Field trials were conducted at two sites in Nepal to test whether soil solarization: (i) had a lasting effect on soil bacterial, fungal and nematode communities; (ii) altered the rhizosphere communities of rice nursery seedlings and (iii) improved crop growth and yield in the rice-wheat cropping system. Rice seedlings were grown in nursery plots that were solarized for 28 days or left untreated and were transplanted to field plots that were also either solarized for 28 days or not in a randomized complete block design with four replications. Rice was grown to maturity and harvested, followed by a complete wheat cropping cycle. Solarization of main field plots increased counts of fungal propagules and decreased root galling and nematode counts and decreased weed biomass. Terminal restriction fragment length polymorphism (T-RFLP) analyses of extracted soil DNA revealed significant shifts in fungal community composition following soil solarization, which was sustained throughout the entire rice cropping cycle at both field sites. The bacterial community composition was similarly affected, but at only one of the two sites. Despite the observed changes in soil microbial community composition over more than one cropping period, solarization had no impact on crop productivity at either site. Nevertheless, such changes in soil microbial communities in response to solarization may be responsible for increased yields observed at other sites with greater pathogen pressure. This practice has shown promising results in many farmers’ fields in South Asia, but further elucidation of the mechanisms by which solarization increases productivity is needed.     

酸碱度对沼液土壤理化性质及粪大肠杆菌消长的影响

以猪粪沼液和棕壤为试材,采用土柱培养法,共设土壤pH分别为5、7、9的3个处理,探究土壤酸碱度对粪大肠杆菌随沼液施入土壤后消长变化的影响机理。结果表明,所有处理粪大肠杆菌均呈先增后减的趋势,在培养后的第8 d达到峰值并在培养第60 d数量降至105数量级。pH为7(中性土壤)最有利于粪大肠杆菌生存,pH 为9(碱性土壤)抑制作用最强,在培养后的第60 d,pH为7处理的粪大肠杆菌数量分别比pH为5和9处理升高了10.06%和48.85%。随土壤深度的增加,土壤pH为5、7、9处理的粪大肠杆菌分别呈现先增后减、先减后增、先减后增的趋势,均在15 cm处出现拐点。神经网络模型分析结果表明,影响土壤中粪大肠杆菌生存的关键因素是土壤硝态氮,其次为土壤有机质,土壤铵态氮的重要性最低,而土壤酸碱度对粪大肠杆菌生存的影响不仅体现在酸碱度的直接作用上,还通过酸碱度的变化导致土壤理化性质改变所产生的间接作用。综合分析,pH为7(中性土壤)最有利于粪大肠杆菌生存,pH为9(碱性土壤)对粪大肠杆菌灭活效果最好。     

Fluctuations in the abundance of ammonia oxidizers and the contents of inorganic nitrogen in solarized soil

Solarization makes a great impact on the abundance of ammonia oxidizers and nitrifying activity in soil. To elucidate fluctuations in the abundance of ammonia oxidizers and nitrification in solarized soil, copy numbers of amoA gene of ammonia-oxidizing bacteria (AOB) and archaea (AOA), viable number of ammonia oxidizers and inorganic nitrogen contents were investigated in greenhouse experiments. The copy number of amoA gene and the viable number of ammonia oxidizers were determined by the quantitative polymerase chain reaction and most probable number methods, respectively. Abundance of AOB based on the estimation of amoA gene copy numbers and viable counts of ammonia oxidizers was decreased by the solarization treatment and increased during the tomato (Solanum lycopersicum L.) cultivation period following the solarization. Effect of solarization on the copy number of amoA gene of AOA was less evident than that on AOB. The proportion of nitrate in inorganic nitrogen contents was declined by the solarization and increased during the tomato cultivation period following the solarization. Positive correlations were found between the proportion of nitrate in inorganic nitrogen content and the copy number of bacterial or archaeal amoA gene or the viable number of ammonia oxidizers; the copy number of bacterial amoA gene showed a strong correlation with the viable number of ammonia oxidizers. The present study revealed influences of solarization on the fluctuation in the abundance of ammonia oxidizers and dynamics of inorganic nitrogen contents in soil and the results indicate that the determination of amoA gene of AOB is possibly a quick and useful diagnostic technique for evaluating suppression and restoration of nitrification following solarization.