实验8 质粒DNA的提取

一、氯化铯超速离心提取法 1.在LB培养基(10克蛋白胨,5克酵母膏,10克氯化钠,15克琼脂粉,溶解于水后,用1N NaOH调节pH至中性,加水至1,000毫升,高压蒸汽灭菌)上划线接种含有待抽提的质粒的细菌。37℃培养过夜。如果质粒上带有抗性基因,则在培养基内加入相应的抗菌素,例如20微克氨苄青霉素/毫升,12.5—15微克四环素/毫升。 2.挑出单菌落接种在10毫升LB培养液(除不加琼脂粉外,成份与LB培养液相同)中,37℃培养过夜。     

中华眼镜蛇膜毒素的体外溶吉田肉瘤细胞作用

本文介绍了中华眼镜蛇膜毒素(CT)在试管中破坏吉田肉瘤腹水细胞(YSC)的行为特征:(1)在缺钙溶液中固定细胞浓度为4～5×10~6个/毫升时,癌细胞会迅速破坏;(2)在2微克/毫升毒素蛋白浓度以下YSC 破坏速度及破坏程度都与CT 浓度成正比,倘以3～6分钟的破坏程度计量,每毫克毒蛋白每分钟可破坏癌细胞0.7×10~9个,T_(50)≈1微克/毫升;(3)由YSC 浓度增大能对抗溶胞的实验可推算出在此过程中与每个癌细胞作用的毒蛋白量为2.8×10~6～25×10~6个分子:(4)虽然镁离子对此反应是部分必需的,但钙离子却有强烈的抑制作用,这些作用都可被金属螫台剂所去除。作者对CT 不能在体内抗癌作了解释,并对毒蛋白一级结构中第25位酪氨酸对细胞毒力的重要性作了讨论。     

中华眼镜蛇膜毒素的体外溶吉田肉瘤细胞作用

本文介绍了中华眼镜蛇膜毒素(CT)在试管中破坏吉田肉瘤腹水细胞(YSC)的行为特征:(1)在缺钙溶液中固定细胞浓度为4～5×10~6个/毫升时,癌细胞会迅速破坏;(2)在2微克/毫升毒素蛋白浓度以下YSC破坏速度及破坏程度都与CT浓度成正比,倘以3～6分钟的破坏程度计量,每毫克毒蛋白每分钟可破坏癌细胞O.7×10~9个,Y_(50)≈1微克/毫升;(3)由YSC浓度增大能对抗溶胞的实验可推算出在此过程中与每个癌细胞作用的毒蛋白量为2.8×10~6～25×10~6个分子;(4)虽然镁离子对此反应是部分必需的,但钙离子却有强烈的抑制作用,这些作用都可被金属螯合剂所去除。作者对CT不能在体内抗癌作了解释,并对毒蛋白一级结构中第25位酪氨酸对细胞毒力的重要性作了讨论。     

酪氨酸对未成年大鼠卵巢Δ~5-3β-羟类固醇脱氢酶活性的影响

利用PMSG(妊娠母马血清促性腺激素)及HCG(人羢膜促性腺激素)处理的未成年大鼠卵巢匀浆,通过测定Δ~5-3β-羟类固醇脱氢酶(HSD)活性,研究了酪氨酸影响孕酮的代谢机理。结果表明,同一浓度的酪氨酸(60微克/毫升)加到排卵后不同时间(12—72小时)的卵巢匀浆中,Δ~5-3β-HSD活性增加,并随着时间延长有逐渐增加的趋势。酪氨酸组同对照组间差异非常显著(P<0.001),不同浓度的酪氨酸(30—160微克/毫升)加到排卵后同一时间(24小时)的卵巢匀浆,也能提高Δ~5-3β-HSD活性,酪氨酸组与对照组间差异非常显著(P<0.001)。     

15甲基前列腺素 F_(2α)和13去氢ω—乙基前列腺素 F_(2α)对妊娠大鼠血中孕酮的影响

15-甲-PGF_(2α)和13-去氢-ω 乙-PGF_(2α)在相同剂量时均能使妊娠7天的大鼠血浆孕酮浓度下降。15-甲-PGF_(α2)组于用药后4、8和24小时血浆孕酮浓度下降,分别为用药前的56.6%、43.7%和13.3%。在给药后72小时所有动物子宫中胚胎已被吸收。13-去氢ω-乙-PGF_(2α)组于用药后4、8和24小时血浆孕酮浓度分别为用药前的63.5%、34.4%和51.9%,给药后72小时大多数动物子宫中仍有胚胎,但胚胎比对照组显著为小,且多游离于子宫中。在给15-甲-PGF_(2α)前30分钟和第二次给15-甲-PGF_(2α)的同时,肌注 HOG 20国际单位,能完全对抗15-甲-PGF_(2α)的降低妊娠大鼠血浆孕酮浓度和抗早孕作用,给药后24小时内血浆孕酮浓度与对照组相似,全部动物维持妊娠,胚胎大小和数目也与对照组相似。恒速静注15-甲-PGF_(2α)(20微克)于麻醉妊娠大鼠,30分钟后已使子宫卵巢静脉血中孕酮含量由用药前1.271±0.154微克/10分钟下降到0.279±0.083微克/10分钟,给药后60分钟仍维持于低水平。如预先静注 HOG 20国际单位,可使子宫卵巢静脉血中孕酮含量由用药前1.123±0.162微克/10分钟升高到1.496±0.018微克/10分钟,在 HCG作用的基础上再静脉恒速注入15-甲-PGF_(2α),虽可使子宫卵巢静脉血中孕酮含量下降到1.179±0.042微克/10分钟,但不能降低到     

DNA重组技术：Ⅲ.重组质粒在大肠杆菌中的转化

氯化钙法是用质粒DNA转化细菌细胞,尤其是大肠杆菌细胞最常使用的方法。实验操作如下: (1) 将受体菌接种在平板培养基上划线,37℃过夜,进行活化。 (2) 挑选单菌落,接于5ml LB培养液,37℃震荡过夜。 (3) 从上述培养液中取0.5ml,转入50mlLB培养液,37期震荡培养2～4小时,到细菌密度大约为5×10~7/ml,OD_(575)约为0.8。     

大肠杆菌及聚肌胞核苷酸对柞蚕、家蚕蛹诱导产生溶菌酶、抗菌肽及凝集素的动力学

大肠杆菌D₃₁诱导柞蚕蛹产生抗菌多肽。同时,溶菌酶和凝集素活性都比诱导前有明显增高.其活力高峰、抗菌多肽在第7天左右、溶菌酶在第5天,而凝集素在第3天即达最高水平。不同品种的柞蚕蛹,经诱导产生的三种活性物质其活力差异不明显,但741、河四和小混品种中的抗菌多肽P_9A及P_9B组成比例较高。上述三种活性物质的诱导变化与性别有关,雄性高于雌性。比较了柞蚕蛹和家蚕经细菌诱导后上述三种活性物质的变化,家蚕凝集素活力很低,诱导后活力增高不明显。抗菌活力及溶菌酶活力的提高程度柞蚕也高于家蚕。聚肌胞核苷酸(Poly I:C)也能诱导两种蚕产生抗菌多肽及溶菌酶,但活力提高的显著程度都不及大肠杆菌诱导,凝集素活力变化也不显著。     

柞蚕滞育蛹的体液防卫反应

本文对柞蚕Antheraea pernyi滞育蛹体液防卫的某些特性做了报道。结果表明：(1)经注射大肠杆菌诱导6h后,体液中出现了抗菌活性物质。诱导7d后,抗菌活力达到峰值,15d后活力消失。用生理盐水诱导的反应较快,4d后达到峰值,10d后活力消失。(2)经大肠杆菌诱导后的体液对苏芸金杆菌、大肠杆菌、枯草杆菌及金黄葡萄球菌均有杀菌效应。在最初5min内杀菌效率较高。注射生理盐水诱导的体液较大肠杆菌诱导的杀菌效果低。(3)用昆虫病原菌苏芸金杆菌及非病原菌大肠杆菌作不同的诱导源,诱导1d后的体液对大肠杆菌的抗菌活力差别不显著,但对柞蚕蛹的生活力及发育有明显影响;用苏芸金杆菌诱导的柞蚕滞育蛹3d后死亡,用大肠杆菌诱导的蛹能正常羽化。(4)在一定范围内用不同剂量的大肠杆菌进行诱导,其抗菌活力不受影响,但超过一定限度,既使是非病原菌也可以突破昆虫的防卫功能。     

酵母脯氨酸合成酶基因(Pro2)的分离及Leu~+ Pro~+表型共转导

酵母7209-1 A(a)DNA,用pHC79为载体,以BamHⅠ、PstⅠ和Hind Ⅲ酶切,经体外重组和包装后,转导至大肠杆菌HB101和SC294中。在我们的实验条件下(对酵母DNA适度酶解,F/V值为15,连接时DNA总浓度为200—250微克/微升以及BHB 2688/BHB2690比值为5),重组建库效率达到10~6CFU/微克载体DNA,重组子双抗值降低到10％或5％以下。 大肠杆菌HB101和SC 294的10~6个重组体克隆中,观察到酵母基因对leuB 6及proA2(在HB101中),leu6,metB1,argG,his1(在SC294中)等营养缺陷型的互补(校正)效应。由于互补频率(10~(-3)—10~(-4))比上述基因突变自发回复率高2—3个数量级,因此可以说,酵母的上述基因在大肠杆菌中获得了功能表达。对pYeleu5和pYeleu7单克隆DNA的再次包装转导和再次互补测试表明,我们已使酵母leu~+的互补效率提高10~4倍。本文还报道了在HB101中的酵母基因文库Leu~+ Pro~+的表型共转导现象,频率为30％以上。     

革兰氏阳性菌苏云金芽孢杆菌和革兰氏阴性菌大肠杆菌诱导的柞蚕蛹生理指标变化

【目的】明确柞蚕Antheraea pernyi对外源微生物防御性生理变化规律,为柞蚕的病害防治和合理饲养提供理论依据。【方法】本研究选用革兰氏阳性菌苏云金芽孢杆菌Bacillus thuringeinsis(Bt)和革兰氏阴性菌大肠杆菌Escherichia coli(Ec)为外源诱导微生物,调整至10~6～10~8cfu/m L菌液,灭活后处理柞蚕蛹,诱导24,48,72和96 h后不同时间测定血淋巴蛋白含量、PO活性、CAT活性、抗菌活性和溶菌酶活性等生理指标。【结果】Ec和Bt诱导柞蚕蛹导致各生理指标出现显著变化,但两种菌株诱导生理指标变化规律差异明显,Ec高浓度诱导72 h会增加血淋巴蛋白含量,而Bt各浓度诱导会在24,48和96 h增加血淋巴蛋白含量。免疫防御关键酶系PO和CAT活性变化规律在不同菌株诱导后差异更明显,Ec诱导后,PO活性随着时间增加表现为先升高后降低的趋势,CAT活性呈现"升高-降低-升高"的规律;而Bt诱导后PO活性表现为"升高-降低-升高"的规律,CAT活性随诱导时间增加变化规律不明显,但有随菌液浓度增加而降低的趋势。对抗菌活性测定表明,Ec和Bt诱导都会显著增加蛹粗酶液抗菌活性,溶菌酶活性也会极显著增加,但2个指标高峰值出现的时间会有明显差别。【结论】本研究结果表明Ec和Bt不同处理均可诱导柞蚕蛹产生明显防御反应,但柞蚕蛹生理指标变化规律与不同种类微生物及处理时间和浓度有关,推测革兰氏阳性菌和革兰氏阴性菌具有不同的诱导防御反应机制。研究结果可以为外源微生物侵染柞蚕后的免疫防御反应规律提供理论指导。     

家蚕抗菌肽CM4对大肠杆菌超微结构影响的研究*(简报)

The effect of antibacterial peptide CM4 of Bombyx mori against E. coll K12 was investigated using scanning electron microscopy(SEM) and transmission electron microscopy (TEM). The ultrastructural changes of E. coli K12 were observed by the challenge of the purified antibacterial peptide CM4. The results showed that the antibacterial peptide caused a series of pathological changes on E. coli. SEM and TEM revealed aggregates of bacteria and SEM revealed wrin-kled bacterial surfaces in the early stage. Thereafter, plasmolysis was observed with irregular holes appearing in the two ends of bacteria and the cytoplasmic contents of the cells leaking out. Finally, bacteria became empty vesicles and disintegrated into small fragments subsequently. Comparatively, the bacterial membrane was normal and the bacterial structure remained intact in the control group.     

Effect of lactobacilli onE. coli adhesion to caco-2 cellsin Vitro

Inhibitory effect of various lactobacilli against pathogenic strains of E. coli in model system Caco2 cells was determined by enumerating the number of adhering E. coli after pre-incubation (exclusion), post-incubation (displacement) or co-incubation (competition) with lactobacilli. Porcine E. coli strain F107 (F18ab, Stx2v) in the competition assay with porcine lactobacillus strain P10 gave bacterial counts 7.25 (log CFU per well); in the exclusion test it was only 7.05 while in displacement test it reached 7.29. The lowest E. coli counts adhering to Caco-2 cells were in exclusion assay (pre-incubation, Lactobacillus inoculated as the first). Pre-treatment of E. coli with our lactobacilli strains reduced the cultivable E. coli numbers.     

柞蚕抗菌肽D中精氨酸、赖氨酸和色氨酸等氨基酸残基的化学修饰

用不同的化学试剂修饰了柞蚕抗菌肽D分子中的色氨酸、精氨酸和赖氨酸等氨基酸残基。NBS修饰抗菌肽D,以及氨肽酶M对抗菌肽D作用的结果表明色氨酸残基对抗菌肽D抑制E.coli D31的作用影响不大。CHD和MLH对精氨酸和赖氨酸残基的修饰,导致抗菌肽D失去抑制E.coli的作用,但可逆地消除CHD和MLH的修饰作用后,抗菌肽D恢复了对E.coli D31的抑菌作用。这些结果初步认为,抗菌肽D抑菌作用与分子中的荷电性有关,改变了分子的电荷,也就同时失去了其抑菌功能。 此外,对精氨酸残基修饰的结果还表明,抗菌肽D的免疫原性与精氨酸残基有关。但是,抗菌肽D的免疫决定簇与其生物活性中心并不完全平行。     

Studies on the cellular defense reactions of the Madeira cockroach, Leucophaea maderae: nodule formation in response to injected bacteria

Nodules were formed in the Madeira cockroach, Leucophaea maderae, in response to injections of low doses (3 x 10(4) bacteria/insect) of three strains of Bacillus cereus and Escherichia coli K12 D31. The most pathogenic strain of bacteria used, B. cereus B1, produced the greatest cellular response, while the least pathogenic, E. coli K12 D31, injected at the same dose, caused little nodule formation. Similarly, nodules were generally found to be larger following injection of pathogenic bacteria such as B. cereus B1 than to the weak pathogen, E. coli K12 D31. There was, however, no difference in the extent of nodule formation with the four bacterial strains/species if they were heat killed prior to injection. Histologically, the nodules formed in response to all bacterial species employed were similar, with a central necrotic core enclosing cell debris and occasional bacteria, and an outer, thin sheath of plasmatocyte-like hemocytes. Possible reasons for the enhanced cellular reactivity observed in L. maderae to pathogenic bacteria are discussed.     

Immobilization of Escherichia coli cells by use of the antimicrobial peptide cecropin P1

An immobilization scheme for bacterial cells is described, in which the antimicrobial peptide cecropin P1 was used to trap Escherichia coli K-12 and O157:H7 cells on microtiter plate well surfaces. Cecropin P1 was covalently attached to the well surfaces, and E. coli cells were allowed to bind to the peptide-coated surface. The immobilized cells were detected colorimetrically with an anti-E. coli antibody-horseradish peroxidase conjugate. Binding curves were obtained in which the signal intensities were dependent upon the cell concentration and upon the amount of peptide attached to the well surface. After normalization for the amount of peptide coupled to the surface and the relative binding affinity of the antibody for each strain, the binding data were compared, which indicated that there was a strong preference for E. coli O157:H7 over E. coli K-12. The cells could be immobilized reproducibly at pH values ranging from 5 to 10 and at ionic strengths up to 0.50 M.     

重组大肠杆菌利用木糖发酵产高纯度L-乳酸

对重组大肠杆菌JH16利用木糖产高纯度的三一乳酸进行研究。通过无氧管驯化EscherwhiacdiJH12菌株得到E．coliJH16,驯化后的菌株茵体浓度提高了31％,乙酸积累减少了43％;在摇瓶中考察不同Mg2＋浓度对EcoliJHl6产三一乳酸的影响,确定最适Mg2＋质量浓度为0．25g/L;EcoEJH16以60g/L木糖为C源,在7L全自动发酵罐中添加0．25g／LMg2＋,乳酸积累量提高了18％,达38．18g/L,乳酸纯度高达95％;E．coliJH16在30g／L木糖和30g／L葡萄糖混合C源中,优先利用葡萄糖,当葡萄糖质量浓度低于1．56g/L后,菌体开始利用木糖进行乳酸发酵,最终得到39g／L乳酸。     

Displacement of Escherichia coli O157:H7 from rumen medium containing prebiotic sugars

A fed-batch, anaerobic culture system was developed to assess the behavior of Escherichia coli O157:H7 in a rumen-like environment. Fermentation medium consisted of either 50% (vol/vol) raw or sterile rumen fluid and 50% phosphate buffer. Additional rumen fluid was added twice per day, and samples were removed three times per day to simulate the exiting of digesta and microbes from the rumen environment under typical feeding regimens. With both types of medium, anaerobic and enteric bacteria reached 10(10) and 10(4) cells/ml, respectively, and were maintained at these levels for at least 5 days. When a rifampin-resistant strain of E. coli O157:H7 was inoculated into medium containing raw rumen fluid, growth did not occur. In contrast, when this strain was added to sterile rumen fluid medium, cell densities increased from 10(6) to 10(9) CFU/ml within 24 h. Most strains of E. coli O157:H7 are unable to ferment sorbitol; therefore, we assessed whether the addition of sorbitol as the only added carbohydrate could be used to competitively exclude E. coli O157:H7 from the culture system. When inoculated into raw rumen broth containing 3 g of sorbitol per liter, E. coli O157:H7 was displaced within 72 h. The addition of other competitive sugars, such as L-arabinose, trehalose, and rhamnose, to rumen medium gave similar results. However, whenever E. coli O157:H7 was grown in sterile rumen broth containing sorbitol, sorbitol-positive mutants appeared. These results suggest that a robust population of commensal ruminal microflora is required to invoke competitive exclusion of E. coli O157:H7 by the addition of "nonfermentable" sugars and that this approach may be effective as a preharvest strategy for reducing carriage of E. coli O157:H7 in the rumen.     

Antimicrobial activity of organic extracts isolated from Aplysina fistularis (Demospongiae: Aplysinidae)

Organic extracts of the sponge Aplysina fistularis (Pallas 1766) were tested for antimicrobial activity against Gram positive bacteria (Staphylococcus aureus) and Gram negative bacteria (Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa). The minimal inhibitory concentration (MIC) and toxic activity of extract were determined. Susceptibility trials of organic fractions obtained by VLC: Hexane, EtOAc and CHCl3 showed that EtOAc fraction has antibacterial activity against E. coli, while CHCl3 fraction inhibited E. coli and S. aureus growth. The later refractioning of EtOAc fraction and the biodirected assays showed that fractions F12 and F13 of EtOAc/Hex and EtOAc F14 were bioactive against Gram positive and Gram negative bacteria. Only EtOAc/MeOH Sf2 from subfractionig of EtOAc F14 produced inhibition for E. coli and S. aureus. In Sf2 EtOAc/MeOH, MIC was moderate for S. aureus (MIC > 256 g/ml). F4 CHCl3/MeOH produced a high inhibition in S. aureus (MIC = 0.125 g/ml) and for E. coli (MIC > 16 g/ml). F10 CHCl3/MeOH showed a moderate activity against S. aureus (MIC > 128 g/ml) and low activity against E. coli (MIC = 512 g/ml). F10 CHCL3/MeOH did no present toxic activity against Artemia salina. The fractiorts F4 CHCL3/MeOH and Sf2 EtOAc/MeOH were toxic for this organism when the concentration was higher than 100 microg/ml. LC50 in both cases was 548.4 and 243.4 microg/ml respectively. Secondary metabolites of medium polarity obtained from A. fistularis have a wide spectrum of anti bacterial activity. Toxicity analysis suggests that only F10 CHCL3/MeOH has potential as an antimicrobial agent for clinical use.     

The in-vitro effect of the leukocytic cationic protein preparation Intercide on Escherichia coli]

Intercide is a cationic protein with the molecular weight of 11.0-11.5 kD from human leukocytes. The in vitro effect of its different concentrations (0.6 to 1.8 mg/ml) on populations of Escherichia coli M17 and K12 and 120 E.coli isolates from various sources such as water, feces of healthy humans and patients with extraintestinal escherichiosis was studied. The experiments with the bacterial suspensions and broth cultures demonstrated that Intercide had an antibacterial action on both the stationary and growing cells. However, some strains of E.coli were resistant to the lethal effect of Intercide. It was observed for the first time that in a concentration of 1.8 mg/ml Intercide was able to stimulate the biomass growth of some E.coli strains in broth culture. The factor analysis showed that the Intercide stimulating effect was more often evident with respect to extraintestinal escherichiosis pathogens with high anti-Intercide and antilysozyme activities.     

Chemotaxis by Bdellovibrio bacteriovorus toward prey.

A chemotaxis assay system that uses a modified Boyden chamber was characterized and used for measurements of chemotaxis by Bdellovibrio bacteriovorus strain UKi2 toward several bacterial species. Bacteria tested included both susceptible and nonsusceptible cells (Escherichia coli, Pseudomonas fluorescens, Bacillus megaterium, and B. bacteriovorus strains UKi2 and D). None was attractive to bdellovibrios when present at densities below 10(7) cells per ml. Chemotaxis toward E. coli was studied most extensively; under conditions that minimized effects of osmotic shock to the cells, E. coli and exudates from E. coli at densities as high as 10(8) cells per ml failed to elicit a chemotactic response. Cell-free filtrates from mixed cultures of bdellovibrios and E. coli neither attracted nor repelled bdellovibrios. The data indicate that bdellovibrios do not use chemotaxis to locate prey cells.