苏云金芽孢杆菌的筛选和初步鉴定

采集四川温江昆虫孳生地的土壤样本94份,利用醋酸钠-抗生素法分离、筛选,共获得苏云金芽孢杆菌9株。镜检可观察到大菱形、小菱形、方形、圆形等四种主要形态的伴胞晶体;采用cryⅠ、cryⅡ、cryⅢ、cryⅤ基因的通用对9株Bt分离菌进行的PCR检测结果表明：9株菌全部含cryⅠ和cryⅤ基因,2株菌含cryⅡ基因,5株菌含cryⅢ基因,而且各菌株均包含2—3个基因型。利用这些菌株对菜青虫进行了室内和室外生物毒力测定,达到了较好的杀虫效果。     

产高活性抗凝溶栓双活性蛋白的短小芽孢杆菌的筛选及鉴定

【目的】筛选可产生抗血栓活性物质的细菌。【方法】利用VY/4平板、酪蛋白平板从水样、土样、兔粪、羊粪、朽木等20多个样品中筛选目的菌株;利用纤维蛋白平板和纤维蛋白试管检测抗血栓活性;利用形态学特征、理化性质、16S rRNA序列同源性鉴定目的菌株。【结果】得到5株可产生抗血栓活性物质的细菌,重点研究了菌株LDS33,发现其分泌的胞外蛋白在纤维蛋白平板上和纤维蛋白试管中均显示出强烈的溶栓活性,通过试管法发现此蛋白质同时具有较强的抗凝活性。结合形态学、理化性质、16S rDNA序列及进化树分析,发现该菌株属于硬壁菌门芽孢杆菌目芽孢杆菌科芽孢杆菌属的短小芽孢杆菌,将其命名为Bacillus pumilus LDS.33。【结论】短小芽孢杆菌LDS33可产生高活性的抗凝溶栓双活性蛋白。     

产高活性抗凝溶栓双活性蛋白的短小芽孢杆菌的筛选及鉴定

摘要:【目的】筛选可产生抗血栓活性物质的细菌。【方法】利用VY/4平板、酪蛋白平板从水样、土样、兔粪、羊粪、朽木等20多个样品中筛选目的菌株;利用纤维蛋白平板和纤维蛋白试管检测抗血栓活性;利用形态学特征、理化性质、16S rRNA序列同源性鉴定目的菌株。【结果】得到5株可产生抗血栓活性物质的细菌,重点研究了菌株LDS33,发现其分泌的胞外蛋白在纤维蛋白平板上和纤维蛋白试管中均显示出强烈的溶栓活性,通过试管法发现此蛋白质同时具有较强的抗凝活性。结合形态学、理化性质、16S rDNA序列及进化树分析,发现该菌株属于硬壁菌门芽孢杆菌目芽孢杆菌科芽孢杆菌属的短小芽孢杆菌,将其命名为Bacillus pumilus LDS.33。【结论】短小芽孢杆菌LDS33可产生高活性的抗凝溶栓双活性蛋白。     

抗动物病原菌芽孢杆菌的筛选、初步鉴定和抗菌活性

从鸡肠道分离、挑选的18株芽孢杆菌经培养特征、形态观察、生理生化实验,初步被鉴定为枯草芽孢杆菌(Bacillus subtilus)、地衣芽孢杆菌(Bacillus licheniformis)、蜡状芽孢杆菌(Bacillus cereus)、巨大芽孢杆菌(Bacillus megaterium)和凝结芽孢杆菌(Bacillus coagulans)。同时测定了它们对5种常见动物病原菌大肠杆菌(E.coli)、金黄色葡萄球菌(Staphylococcus aureus)、肠炎沙门氏菌(Salmonella typhimurium)、猪链球菌(Streptococcus suis)、奇异变形杆菌(Proteus mirabilis)的抑菌活性,其中有4株芽孢杆菌对5种病原菌都有抑制作用。还分别测定了它们产木聚糖酶(从0.895 U1到3.239 U1)和纤维素酶活性(分别为0.391 U2和0.465 U2)。结果表明,芽孢杆菌分离株BC17、BC32、BC106、BC228、BC247和BC261具有作为益生菌的开发潜力。     

产丁醇芽孢杆菌的分离、筛选与鉴定

通过富集培养和分离纯化等过程,从种植怀地黄的土壤中分离得到一株产丁醇兼性厌氧细菌菌株C2。以7%的玉米醪液为原料总溶剂(丙酮、丁醇、乙醇,ABE)产量可达17.17g/L,其中丁醇11.2g/L,占65.2%;发酵玉米秸秆糖化液(总糖浓度为25g/L)产总溶剂量为3.64g/L,其中丁醇2.63g/L,占72.3%。形态学、生理生化及系统发育研究表明该菌株为革兰氏阳性芽孢杆菌(Bacillus),与B.vallismortis、B.atrophaeus和B.mojavensis亲缘关系最近。     

转化异丁香酚生成香草醛纺锤芽孢杆菌的筛选

以底物异丁香酚为唯一碳源从七壤中筛选获得了一株能高效转化异丁香酚生成香草醛的芽孢杆菌。根据生理生化特性及16SrRNA序列分析鉴定其属于纺锤芽孢杆菌（Bacillus fusiformis）,初步试验表明该菌能转化2％异丁香酚生成4．20g/L香草醚。     

两株对虾育苗用益生芽孢杆菌的筛选和鉴定

从土壤和虾池中分离到22株具有不同特征的芽孢杆菌,分别将各菌株对数期约5×10^8个细胞添加到500mL预先加有凡纳滨对虾（Litopenaeus vannamei）无节Ⅲ期幼体的水体中,各菌株在初始水体中含量均约为106个细胞/mL,根据各菌株对无节Ⅲ期到蚤状Ⅱ期幼体变态成活率的影响,从中初步筛选出10株对幼体变态成活有极显著（p〈0.01）效果的芽孢杆菌。进一步又从初筛到的10个菌株中复筛到2株对幼体变态成活最为明显的芽孢杆菌,菌株zou4和zou8。两菌株与复筛的其他8株芽孢杆菌相比,均显著（p〈0.05）促进幼体变态存活率。根据形态和生理生化特征,菌株zou4和zou8分别初步鉴定为坚强芽孢杆菌（Bacillus firmus）和蜡状芽孢杆菌（B.cereus）。为进一步确定zou4和zou8的分类地位,测定了两菌株的16S rRNA基因序列,分析了相关细菌16S rDNA序列的同源性,构建了系统发育树。结果表明菌株zou4和zou8分别与坚强芽孢杆菌和蜡状芽孢杆菌的同源性最高,相似值均为99%以上。系统发育树上zou4和zou8也分别与坚强芽孢杆菌和蜡状芽孢杆菌聚为一类。综合上述结果,芽孢杆菌zou4和zou8菌株分别被鉴定为坚强芽孢杆菌和蜡状芽孢杆菌。     

炭疽芽孢杆菌检测鉴定技术研究进展

炭疽芽孢杆菌的感染,无论是自然感染,还是作为生物恐怖和生物战的手段。快速检验和鉴定疽芽孢杆菌是最为关键的,只有正确识别生物战剂的种类,才能为正确实施防治措施指明方向。本文讨论了炭疽芽孢杆菌的检验鉴定技术,包括常规的分析培养,免疫学技术,核酸分析技术,生物传感器,基因芯片技术的应用和新诊断分子,如肽核酸与适配子的应用。     

具有群体感应抑制效果芽孢杆菌的筛选与鉴定

细菌性疾病是危害水产业健康发展的主要原因之一,从抑制调控致病菌毒力表达的群体感应（Quorum sensing,QS）入手是水产动物疾病防治研究新的热点方向。研究以紫色杆菌 （Chromobacterium violaceum）为报告菌株,采用T型划线和滤纸片法,对分离自异育银鲫肠道及实验室保存的芽孢杆菌F3-1、F3-2、F6-4、F6-5、X77、X93、X3914进行分析测测试,筛选具有群体感应抑制效果的菌株。T型划线结果显示菌株F3-1能够抑制紫色杆菌紫色菌素的产生;滤纸片法结果显示抑制紫色杆菌紫色菌素产生的主要物质存在于菌株F3-1的胞外产物中。试验提取了菌株F3-1的培养物的粗提物,测定粗提物浓度分别在0、50、100、200、300 mg/L时紫色杆菌菌素在585 nm处的光密度值,结果显示随着粗提物浓度的增加其色素的产量显著降低（P0.05）,当粗提物浓度超过200 mg/L时,试管中观察不到紫色菌素存在;利用硫酸铵盐析的方法,提取了菌株F3-1的粗提物中蛋白产物,滤纸片扩散法结果显示表明其胞外蛋白是紫色杆菌QS系统的抑制物质。通过分子生物学16S rDNA鉴定,确定菌株F3-1为短小芽孢杆菌（Bacillus pumilus,GenBank accession No. JX984444）。电镜照片显示该菌具有短杆状特征,因此,分离自异育银鲫肠道的短小芽孢杆菌F3-1具有抑制细菌群体感应的作用。       

一株与中草药五倍子协同抑菌的芽孢杆菌筛选及体外益生效果评价

目的利用分子生物学方法对从猪肠道食糜分离的芽孢杆菌进行鉴定,并通过体外法评价其益生效果。方法实验从健康猪肠道食糜中筛选到1株能抑制大肠埃希菌、副伤寒沙门菌、金黄色葡萄球菌和巴氏杆菌生长,并与中草药五倍子具有协同抑菌作用的芽孢杆菌AP139。结果经16SrDNA同源性序列分析,结合形态和生理生化特点,鉴定为枯草芽孢杆菌(B.subtilis)。芽孢杆菌AP139联合中草药五倍子发酵结果表明:5%组对4种致病菌的抑菌圈直径分别扩大了1.20、14.45、23.54和6.09 mm,其抑菌效果优于未发酵,对巴氏杆菌PM2010和猪大肠埃希菌抑菌圈极显著提高(P0.01),对金黄色葡萄球菌抑菌圈有显著提高(P0.05)。经体外益生效果评价得知,AP139菌株在pH=5.0~8.0的条件下活菌数均较高,NaCl浓度为1%时生长较好,0.3%胆盐和人工肠液分别处理3h后存活率分别达84.26%和89.35%。结论芽孢杆菌AP139具有良好的协同抑菌作用,并具有益生菌的耐酸、耐胆盐、耐胃肠液等特性,可适应动物胃肠道内环境。     

新的产弹性蛋白酶菌株的筛选与鉴定

本研究从秸秆中分离得到一株弹性蛋白酶高产菌, 并对该菌株进行鉴定, 以期为实现其工业化生产提供理论依据。采用酪蛋白(脱脂奶粉)进行初筛后, 以弹性蛋白(牛筋)进行复筛, 并对筛选结果进行检验, 然后对所得菌株结合形态、生理生化特性和16S rRNA基因序列进行分类鉴定, 得到一株弹性蛋白酶高产菌株LSF-97。结果显示, 菌株LSF-97与短小芽孢杆菌同源率达到100%, 形态及生理生化特征与模式菌也显示高度一致性, 故将其确定为短小芽孢杆菌。     

Expression and secretion of the protective antigen of Bacillus anthracis in Bacillus brevis

We used the Bacillus brevis-pNU212 system to develop a mass production system for the protective antigen (PA) of Bacillus anthracis. A moderately efficient expression-secretion system for PA was constructed by fusing the PA gene from B. anthracis with the B. brevis cell-wall protein signal-peptide encoding region of pNU212, and by introducing the recombinant plasmid, pNU212-mPA, into B. brevis 47-5Q. The clone producing PA secreted about 300 microg of recombinant PA (rPA) per ml of 5PY-erythromycin medium after 4 days incubation at 30 degrees C. The rPA was fractionated from the culture supernatant of B. brevis 47-5Q carrying pNU212-mPA using ammonium sulfate at 70% saturation followed by anion exchange chromatography on a Hitrap Q, a Hiload 16/60 Superdex 200 gel filtration column and a phenyl sepharose hydrophobic interaction column, yielding 70 mg rPA per liter of culture. The N-terminal sequence of the purified rPA was identical to that of native PA from B. anthracis. The purified rPA exhibited cytotoxicity towards J774A.1 cells when combined with lethal factor. The rPA formulated in either Rehydragel HPA or MPL-TDM-CWS adjuvant (Ribi-Trimix) elicited the expression of a large amount of anti-PA and neutralizing antibodies in guinea pigs and completely protected them against a 100 LD50 challenge with fully virulent B. anthracis spores.     

Comparative study on the cell wall structure of protein-producing Bacillus brevis

Abstract Among eight strains of protein-producing Bacillus brevis , three morphological groups have been identified according to the structure of the cell walls.

• (I)

  Cell wall consisting of a peptidoglycanlayer
• (II)

  Two-layered cell wall consisting of a peptidoglycan-layer and an S-layer
• (III)

  Three-layered cell wall consisting of a peptidoglycan-layer and two S-layers

Group I and group II cell walls have not been described yet for protein-producing bacteria. The S-layers observed in this study all had hexagonal symmetry and lattice constants of approximately 18 nm. The immunological relation between the S-layer proteins of the newly isolated B. brevis strains and those of B. brevis 47 has been examined using antisera against both S-layer-proteins of B. brevis 47. S-layers from protein-producing B. brevis strains, which were adjacent to the peptidoglycan-layer, were similar to each other, whether they were the outermost cell wall layer (group II) or not (group III). However, no similarity was found between these layers and the outermost S-layer of B. brevis 47 (group III).     

一株来自蚯蚓的产纤溶酶蜡状芽孢杆菌Bacillus cereus的筛选及鉴定

对来自4个不同省份的5条蚯蚓的肠道及体表细菌进行分离,共获得122株细菌。通过脱脂奶粉平板法初筛,纤维蛋白平板法复筛,以透明圈为筛选标记,共筛选出产纤溶酶菌株12株,其中菌株SC-3-W-3的纤溶酶活力较高,达到了538.64 U/mL(相当于尿激酶的活力单位)。通过对其形态、培养、生理生化特征进行研究,发现其与蜡状芽孢杆菌Bacillus cereus Frankland的特征很相符。进一步对SC-3-W-3的16S rDNA序列及系统发育分析表明,该菌株与蜡状芽孢杆菌的同源性高达100%。综合生理生化及16S rDNA序列比对结果,将SC-3-W-3菌株鉴定为蜡状芽孢杆菌。     

Production and purification of Clostridium perfringens alpha-toxin using a protein-hyperproducing strain, Bacillus brevis 47

Abstract Clostridium perfringens alpha-toxin was produced in a protein-hyperproducing strain, Bacillus brevis 47, by cloning the gene into the constructed expression-secretion vector which has the multiple promoters and the signal peptide coding region of an outer cell wall protein gene. The amount of alpha-toxin produced by the B. brevis 47 transformant carrying the gene was approximately 10 times greater than that produced by a B. subtilis transformant carrying the toxin gene. Biological activities and the N-terminal amino acid sequence of the toxin secreted by the B. brevis 47 transformant were identical to those of wild-type alpha-toxin.     

Effective extracellular production of Bacillus stearothermophilus esterase by pH-stat modal fed-batch culture of recombinant Bacillus brevis

An automated two-component substrate feeding strategy with a pH-stat modal fed-batch culture using a high pH limit was developed to effectively porduce esterase from a hyperprotein exreting Bacillus brevis HPD31 harboring a plasmid pHSC131 which carries a Bacillus stearothermo philus esterase gene. First, the effect of single- and multi-substrate feedings on the growth and activity of the excreted esterase was investigated. Then a two-component (polypepton + glucose) feeding using different feed rates was studied. Highest activity of the excreted esterase (34 U/mL) was obtained when the concentrations of poly-pepton and glucose in the nutrient feed solution were 250 and 41.60 g/L respectively. The absence and excessive amount of glucose in the nutrient feed solution was ineffective for the exracellular esterase formation because without glucose the increase in cell concentration was minimum while excessive amount of glucose favored cell growth at the expense of the esterase production. It is believed that the mechanism of enzyme excretion is growth dependent and that a higher cell growth of the host is in effect unfavorable for the enzyme production. The feed rate, automatically controlled by the direct signal of the pH change, at 0.30 mL/pulse was found optimum for the esterase production while lower (0.15 mL/pulse) and higher (0.67 mL/pulse) feed rates did not produce good results. The activity of the excreted esterase was increased more than eight times from 4 U/mL obtained in the conventional batch culture to 34 U/mL obtained in this study. The esterase productivity was likewise increased more than threefold. (c) 1992 John Wiley & Sons, Inc.     

一株以葡萄糖为底物产2,3-丁二醇微生物菌株的筛选及鉴定

从自然界中筛选出一批以葡萄糖为底物发酵产2,3-丁二醇的菌株,经初步发酵测定发酵液中2,3-丁二醇含量,其中菌株6-7的2,3-丁二醇产量最高达49.6g/L。对其进行常规生理生化鉴定实验,并结合16SrDNA序列分析,比对结果表明,菌株6-7与Bacillus subtilis strain BIHB332相似性达99%。在细菌分类学上属于枯草芽孢杆菌属,将其命名为Bacillussubtilis6-7。其特点是属于环境友好和食品安全型菌株,因此,利用Bacillus subtilis6-7生产2,3-丁二醇具有良好的工业应用价值。