大孔吸附树脂吸附链霉菌702抗真菌活性物质工艺

研究大孔吸附树脂吸附链霉菌702抗真菌活性物质的工艺条件。采用5种不同大孔吸附树脂吸附链霉菌702发酵液提取液中抗真菌活性物质,选择吸附效果较佳树脂进行吸附条件优化,以桔青霉为指示菌,纳他霉素为对照抗生素．采用“管碟法”测定抗真菌活性物质含量。结果发现,XAD18树脂吸附效果较好,获得优化吸附条件：上样液pH6,NaCl质量浓度10g/L,上样量22．63mg／g湿树脂,吸附流速2．5mL／min,水洗体积180mL,洗脱流速1．5mL／min,洗脱剂为体积分数10％、50％和90％的甲醇,洗脱方式为梯度洗脱。在确定的工艺条件下XAD18对链霉菌702抗真菌活性物质的吸附率可达90％,洗脱率可达75％,回收率可达80％。     

木鳖子提取物对朱砂叶螨的触杀活性

测定了木鳖子提取物对朱砂叶螨的触杀活性。用甲醇、氯仿、石油醚3种不同极性的溶剂提取,石油醚提取率最高为30.79%,且对朱砂叶螨成螨和卵的触杀活性均高于其他两种溶剂的提取物,24 h校正死亡率分别为77.52%和72.04%。用甲醇对石油醚提取物萃取,发现甲醇萃取物活性明显高于石油醚部分,对成螨和卵处理24 h后的校正死亡率分别为89.60%和74.65%,产卵抑制率为62.74%,驱避率为58.23%。柱层析对甲醇萃取物进行分离得到10种组分,组分5活性最高,浓度为2 mg/mL时,24 h校正死亡率为86.15%。用薄层层析和气相色谱质谱联用仪分别检测组分5纯度和主成分,初步确定活性成分为α-菠菜甾醇。     

亚洲玉米螟滞育关联蛋白的分离和纯化

用6%聚丙烯酰胺凝胶电泳, 在亚洲玉米螟Ostrinia furnacalis河南种群滞育幼虫血淋巴中发现了2种滞育关联蛋白, 它们的等电点分别为5.3和4.7; 而在亚洲玉米螟上海种群滞育幼虫血淋巴中发现了1种滞育关联蛋白,等电点为4.7。亚洲玉米螟河南种群滞育关联蛋白仅在滞育幼虫血淋巴中存在。亚洲玉米螟上海种群滞育关联蛋白在滞育和非滞育幼虫血淋巴中均存在,只是它们在滞育幼虫血淋巴中的含量明显高于非滞育幼虫血淋巴中的含量。用30%～50%丙酮沉淀、SephaDex G-100凝胶过滤和MONOQ RH 5/5阴离子交换快速液相法,纯化到了高纯度(≥98%)亚洲玉米螟上海种群滞育幼虫血淋巴中的滞育关联蛋白。     

斑点野生稻拒食活性组分的分离及其对斜纹夜蛾消化酶活性的影响

为了进一步评估斑点野生稻Oryza punctata的抗虫性,采用液 液分配萃取和硅胶柱层析的方法,从斑点野生稻甲醇提取物中分离获得石油醚、氯仿、乙酸乙酯、正丁醇和水的萃取物,测定了其对斜纹夜蛾Spodoptera litura 3龄幼虫拒食活性。结果显示,氯仿萃取物比其他4种萃取物具有更高的拒食活性,在25 mg/mL的浓度下,24 h和48 h的拒食率分别为55.62%和52.66%。氯仿萃取物经硅胶柱层析后,得到14个组分。比较14个组分的拒食活性,发现组分4和10为主要的活性组分。这两个组分对斜纹夜蛾幼虫中肠脂肪酶和α-淀粉酶的活性都具有一定的抑制活性,其中组分10对脂肪酶具有显著的抑制效果,以1 mg/mL的浓度活体处理48 h和72 h抑制率分别达19.82％和34.60％; 对α-淀粉酶的影响在48 h和72 h内都具有显著的抑制效果,随着处理时间的延长,其抑制率逐步提高,72 h的抑制率分别为25.06％和27.40％。结果提示斜纹夜蛾幼虫中肠脂肪酶和α-淀粉酶可能是斑点野生稻拒食活性成分的作用靶标。     

大孔吸附树脂对家蝇抗菌肽的吸附与分离纯化

通过比较6种不同型号的大孔吸附树脂对家蝇蛋白的吸附解吸特性,发现D101大孔吸附树脂的性能优于其他5种,吸附流速、浓度影响大孔吸附树脂的动态吸附性能。D101大孔吸附树脂对未经诱导的家蝇蛋白的吸附量可达217．18mg／g（以干树脂总量为基准）,洗脱率为76．48％。吸附后的大孔吸附树脂用15％、35％、55％的乙醇溶液阶段洗脱,各洗脱组分的疏水性逐渐增大,蛋白质含量也明显增加。用E.coli、S．aureus和B．subtilis对各洗脱组分进行抑菌活性测定,抑菌活性随洗脱组分的疏水性增加而增大。测得55％乙醇洗脱组分的抑菌活性最大,其中对E.coli的抑菌圈直径达5．8mm。     

人GDNF在甲醇酵母及家蚕幼虫中的表达

将人胶质细胞源性神经营养因子(GDNF)基因克隆入酵母分泌型表达载体pPIC9K中,酶切线性化后电穿孔导入酵母细胞进行整合,经G418筛选得到多拷贝转化子,甲醇诱导表达。将人GDNF基因克隆入昆虫病毒转移载体pBacPAK8中,与线性化Bm-BacPAK6修饰病毒基因组DNA共转染家蚕细胞,经体内重组,筛选到重组病毒。用重组病毒感染家蚕幼虫,5d后收集血淋巴。SDS-PAGE和蛋白质印迹杂交结果证实了酵母培养上清液及家蚕幼虫血淋巴中含有GDNF蛋白。活性研究表明,甲醇酵母及家蚕幼虫表达的GDNF蛋白能促进多巴胺能神经元的存活和突起生长。     

HPLC-PAD法优化乌骨鸡活性肽分离纯化

乌骨鸡是一种具有药用价值的鸡种,乌骨鸡的鸡肉蛋白通过酶促水解后可获得多肽产物乌骨鸡活性肽,营养价值高,分离纯化活性肽是研究乌骨鸡应用价值的基础。利用装置C18反相色谱柱或TSKgel凝胶色谱柱的高效液相色谱仪进行乌骨鸡活性肽的分离优化,针对流动相、流速进行优化的同时,参考波长、进样量,以出峰数为主,结合分离度等考察分离效果。结果表明:C18柱的出峰数随甲醇含量及流速的降低而增加;三氟乙酸或磷酸盐作缓冲液时出峰数基本一致,但分离度不同;综合因素得pH 6.0磷酸盐/6%甲醇/水体系的流动相、0.2 mL/min的流速为该实验下的最佳分离条件。TSKgel柱的乙腈分离效果优于甲醇,梯度洗脱效果较好;综合因素得出0.1%三氟乙酸/(10%～25%)乙腈/水体系的流动相、0.5 mL/min的流速为该实验下的最佳分离条件。     

Ni2+胁迫对斜纹夜蛾幼虫血淋巴中能量物质水平的适应性调节

为明确食物中重金属胁迫对昆虫血淋巴能量物质和生长的影响,本研究测定了饲料中不同浓度的Ni²⁺ （1~40 mg/kg）对连续3个世代斜纹夜蛾Spodoptera litura Fabricius 5龄始龄（0 h）至6龄末幼虫（分别记为0,24,48,72,96和120 h幼虫）血淋巴中能量物质总糖、蛋白质和脂肪含量,血淋巴中热量值及5龄和6龄幼虫体重的影响。结果表明：第1和2代48 h和96 h幼虫血淋巴的总糖含量均低于对照;而连续3代其他处理幼虫血淋巴中的总糖含量均在低浓度Ni²⁺（1~5 mg/kg）胁迫下增高,在高浓度Ni²⁺ （20~40 mg/kg）胁迫下降低。连续3个世代的Ni²⁺胁迫均显著降低了0~48 h幼虫血淋巴中的蛋白含量;低浓度Ni²⁺（1~5 mg/kg）胁迫增加了连续3代72~120 h幼虫血淋巴中的蛋白含量,而高浓度Ni²⁺（20~40 mg/kg）胁迫只降低了第1代72~120 h幼虫以及第2代72 h幼虫血淋巴中的蛋白含量。第1代0 h,48~72 h和120 h幼虫血淋巴中的脂肪含量在1~20 mg/kg Ni²⁺胁迫下均高于对照,但24 h和96 h幼虫血淋巴中的脂肪含量均低于对照中的含量。连续3代0~48 h幼虫血淋巴中的热量值均随饲料中Ni²⁺浓度的增加而降低;低浓度Ni²⁺胁迫（1~5 mg/kg）增加,而高浓度Ni²⁺（20~40 mg/kg）的胁迫降低第2和3代72 ~120 h幼虫血淋巴中热量值。同时,低浓度Ni2+胁迫增加而高浓度Ni²⁺胁迫降低了第3代5龄和6龄幼虫的体重。因此推测,Ni²⁺胁迫对S. litura幼虫血淋巴的能源物质含量以及热量值的影响与能量物质的种类、虫体的发育阶段和Ni²⁺的胁迫世代等都有一定的联系。     

真菌提取物JN219抗人巨细胞病毒机制的初步研究

目的探讨真菌提取物JN219的抗病毒机制。方法(1)样品对病毒入侵宿主细胞的抑制作用:将样品(JN219 50μg/ml、柱洗脱组分5 g/L)分别与人巨细胞病毒(HCMV半数组织感染量TCID50为105/ml)等容量混合,不同时间(混合后立刻、混合后37℃作用1 h)以不同稀释度、100μl/孔接种至多孔板人胚肺细胞(HEL)上;(2)样品对病毒复制的抑制作用:将HCMV(100个TCID50)100μl/孔接种于HEL上1 h后,加不同稀释度的样品;(3)灭活病毒对样品抗病毒作用的抑制作用:将紫外线灭活的HCMV与JN219、柱洗脱组分等容量混合,37℃作用1 h,加等容量HCMV(TCID50为105/ml)混合37℃作用1 h,以不同稀释度、100μl/孔接种于96孔板内的HEL上。以上各实验组同时设细胞对照组、病毒对照组、空白对照组、相应稀释度的样品对照组,37℃、5%CO2培养,每日观察细胞CPE,当病毒对照病变90%以上,终止培养,中性红染色,在540 nm读取吸光度A值,用Reed-Muench法计算细胞半数有效浓度(IC50)和病毒TCID50。通过比较组间差异,探讨在哪一复制周期样品对病毒产生抑制作用。结果(1)样品对病毒入侵宿主细胞的抑制作用:JN219、柱洗脱组分与HCMV等容量混合后立刻接种细胞,测得病毒TICD50为104.8/ml,与病毒对照组比较(TCID50为105/ml)差异无显著性;与病毒作用1 h后接种细胞,JN219、柱洗脱组分IC50分别为0.168μg/ml、0.185μg/ml,第一孔JN219 TCID50为101.6/ml,柱洗脱组分标本未测得病毒,可使HCMV得到抑制;(2)样品对病毒复制的抑制作用:先将病毒接种细胞1 h,然后加JN219、柱洗脱组分,IC50分别为3.29μg/ml、13.1 g/L,样品不能完全抑制人巨细胞病毒病变的发生;(3)灭活病毒对样品抗病毒作用的抑制作用:紫外线灭活的病毒与样品作用1 h,再加等容量病毒后接种细胞,样品JN219、柱洗脱组分与病毒混合物TCID50分别为103.5、103.75,样品抗病毒位点已被紫外线灭活病毒外膜蛋白占据,抗病毒效果降低。结论JN219抗人巨细胞病毒作用机制主要是阻断病毒入侵宿主细胞,作用位点主要为人巨细胞病毒外膜蛋白。     

Zn~(2+)胁迫对黑水虻幼虫血淋巴能量物质影响的研究

为了研究Zn~(2+)胁迫对黑水虻Hermetia illucens L.幼虫能量物质的影响,本文在人工饲料中添加不同浓度(75、150、300、600、1200 mg/kg)的Zn~(2+)对黑水虻幼虫进行胁迫。利用紫外分光光度法测定了连续5代Zn~(2+)胁迫对黑水虻幼虫血淋巴中三大能量物质(总糖、脂肪及蛋白质)含量的影响,并计算了血淋巴中的热量值。结果表明,黑水虻幼虫血淋巴中的总糖、脂肪、蛋白质含量及热量值有相似的变化趋势:对各处理浓度而言,低浓度Zn~(2+)(75-300 mg/kg)处理使血淋巴中三大能量物质含量及热量值随处理浓度增加而升高;高浓度Zn~(2+)(600-1200 mg/kg)胁迫则随处理浓度增加而下降。在第1、3代黑水虻幼虫中,与对照相比,75-300mg/kg的Zn~(2+)处理浓度增加了三大能量物质含量及热量值,300 mg/kg处理浓度时达到最高,且与对照相比显著增加;而600-1200 mg/kg的高剂量Zn~(2+)处理浓度降低了三大能量物质含量及热量值。第5代幼虫血淋巴中三大能量物质含量及热量值均低于对照组,并低于同一处理浓度下的第1、3代幼虫血淋巴中的数值。因此,Zn~(2+)胁迫影响了黑水虻幼虫血淋巴中三大能量物质含量及热量值,并存在发育阶段特异性和世代累积效应。     

An evaluation of the use of Sep-Pak C18 cartridges for the extraction of vitamin D3 and some of its metabolites from plasma and urine

The use of Sep-Pak C18 cartridges for the extraction of vitamin D and some of its metabolites from plasma and urine has been evaluated by studying the recovery of added tritiated secosteroids. The preparation of the cartridges, recoveries, extraction and elution with a number of solvents, effect of varying flow rates for application and elution, and the effect of increasing volumes of plasma and urine have been investigated. Two methods for the application of secosteroids present in plasma to Sep-Pak C18 cartridges have been examined, using methyl cyanide extracts removing precipitated protein by centrifugation, and using acidified methanolic plasma. Methyl cyanide extracts applied to Sep-Pak C18 cartridges and eluted with methanol or methyl cyanide gave the cleanest extracts suitable for direct HPLC. Acidified methanolic plasma, applied to Sep-Pak C18 cartridges and eluted with methanol or methyl cyanide gave extracts which could not be applied directly to an HPLC--further fractionation using Sep-Pak SIL cartridges was necessary. Recoveries of added tritiated secosteroids using both methods were greater than 80% with the exception of vitamin D itself which was poorly recovered--methyl cyanide extraction giving only 30% recovery and use of acidified methanolic plasma giving 66% recovery.     

Superoxide Dismutases: II. Purification and Quantitative Relationship with Water-soluble Protein in Seedlings

Superoxide dismutase was purified from pea (Pisum sativum L., cv. Wando) seeds and corn (Zea mays L., cv. Michigan 500) seedlings. The purified pea enzyme eluting as a single peak from gel exclusion chromatography columns contained the three electrophoretically distinct bands of superoxide dismutase characterizing the crude extract. The purified corn enzyme eluted as the same peak as the pea enzyme, and contained five of the seven active bands found in the crude extract. The similar molecular weights and the cyanide sensitivities of these bands indicated that they are probably isozymes of a cupro-zinc superoxide dismutase. One of the remaining corn bands was shown to be a peroxidase.     

Soluble starch synthases and starch branching enzymes from developing seeds of sorghum

Soluble starch synthases and branching enzymes have been partially purified from developing sorghum seeds. Two major fractions and one minor fraction of starch synthase were eluted on DEAE-cellulose chromatography. The minor enzyme eluted first and was similar to the early eluting major synthase in citrate-stimulated activity, faster reaction rates with glycogen primers than amylopectin primers, and in K_m for ADP-glucose (0.05 and 0.08 mM, respectively). The starch synthase peak eluted last had no citrate-stimulated activity, was equally active with glycogen and amylopectin primers, and had the highest K_m for ADP-glucose (0.10 mM). Four fractions of branching enzymes were recovered from DEAE-cellulose chromatography. One fraction eluted in the buffer wash; the other three co-eluted with the three starch synthases. All four fractions could branch amylose or amylopectin, and stimulated α-glucan synthesis catalysed by phosphorylase. Electrophoretic separation and activity staining for starch synthase of crude extracts and DEAE-cellulose fractions demonstrated complex banding patterns. The colour of the bands after iodine staining indicated that branching enzyme and starch synthase co-migrated during electrophoresis.     

Bioactive fractions containing methyl eugenol-derived sex pheromonal components in haemolymph of the male fruit fly Bactrocera dorsalis (Diptera: Tephritidae)

Sex pheromonal components of the tephritid fruit fly Bactrocera dorsalis (Hendel), 2-allyl-4,5-dimethoxyphenol and (E)-coniferyl alcohol, are biosynthesized from a highly potent male attractant, methyl eugenol, then sequestered and stored in the rectal gland prior to their release during courtship at dusk. These sex pheromonal components have been detected in the haemolymph and crop organ. Hence, attempts were made to separate and identify the haemolymph fractions which contained the sex pheromonal components. Identification of these bioactive fractions in methyl eugenol-fed male flies using gel filtration column chromatography and biodetection using live male flies showed two fractions as highly attractive to conspecific males. These fractions show a significant increase in protein absorbance in the elution profile of haemolymph from methyl eugenol-fed males compared with that from methyl eugenol-deprived males. The molecular mass of these bioactive fractions as determined by using gel filtration was in the peptide range of 3.3 to 5.5 kDa. Subsequent gas chromatography-mass spectrometry analyses further confirmed the presence of the pheromonal components in the bioactive fractions. The presence of these methyl eugenol-derived sex pheromonal components in specific haemolymph fractions suggests the involvement of a sex pheromone binding complex.     

Purification of Antibiotics from Physarum Gyrosum by High Pressure Liquid Chromatography

A family of five antibiotic substances was isolated from the slime mold Physarum gyrosurn by high pressure liquid chromatography (HPLC). For this purpose, mold was cultured for two weeks in a liquid medium. Soluble products were harvested by rotary evaporation of medium and extraction with 1-butanol. Paper chromatography in ethyl acetate :pyridine:water (2:2:1 v/v) was used for preliminary fractionation. Active components were separated by HPLC with a reverse-phase column packed with Bondapack C₁₈/Porasil B (Waters Associates) and were eluted with a linear gradient of methanol:water increasing from 70 to 100% methanol over 90 minutes. Puri-fication was completed by rechromatographing individual fractions. Purity of the active components was verified by HPLC and thin layer chromatography. Activity assays against Bacillus cereus showed these materials to be bacteriostatic rather than bacteriocidal.     

Extraction of Mullerian inhibiting substance from newborn calf testis.

Using a dissociative solvent and a protease inhibitor, Mullerian inhibiting substance, a testicular substance responsible for regression of the Mullerian ducts in the mammalian male embryo, has been extracted from newborn calf testis. Data are presented which demonstrate that fractions with biological activity for Mullerian inhibiting substance can be extracted from whole tissue and that activity can be blocked by antisera raised to extracted testes components. Following extraction in guanidine hydrochloride the extract was fractionated by density gradient sedimentation, gel filtration chromatography, and ion-exchange chromatography. Fractions were subjected to amino acid and carbohydrate analyses and peptide constituents were determined by SDS gel electrophoresis. Fractions were dialyzed, concentrated, filtered, and added to an organ culture assay to detect Mullerian inhibiting substance activity, which was found (1) in the guanidine extract, (2) in a protein fraction of the cesium chloride gradient, (3) in constituents eluted with K_av values between 0.19 and 0.38 on gel filtration chromatography using a Bio-Gel A-0.5 M column, and (4) in constituents eluted between 0.15 and 0.20 M NaCl on ion-exchange chromatography using a DEAE Bio-Gel A-50 ion exchanger. Sequentially this scheme effected a 30-fold purification of a fraction with Mullerian inhibiting substance activity. Biological activity was lost when positive extracts were absorbed with antiserum raised to guanidine extract. The strong dissociative conditions employed in the gradient and extraction procedures make it likely that the distribution of activity obtained in the density gradient procedure was due to a macromolecule, and not to an interaction between an active low molecular weight component and an inactive macromolecule acting as a carrier. Further fractionation on the Bio-Gel column using a dissociative solvent also indicated that the active component was a macromolecule. Amino acid and carbohydrate analyses indicate that the active fractions are composed of proteins and glycoproteins.     

Purification of a delayed hypersensitivity-inducing protein from Listeria monocytogenes

Abstract Cell-envelope fragments were prepared from Listeria monocytogenes L242, serotype 4b. Delayed hypersensitivity (DH)-inducing proteins were extracted with deoxycholate and separated into two fractions by filtration through a Sephacryl S-200 column equilibrated with deoxycholate buffer. The second peak eluting from the Sephacryl column was fractionated using ion exchange chromatography on a DEAE Sepharose CL-6B column in the presence of 6 M urea. A purified 20 400-Da protein which induced DH against L. monocytogenes was obtained by isocratic elution. Three other DH-inducing fractions containing several protein bands were eluted by a gradient of potassium thiocyanate (KSCN) in urea buffer. Our results indicate that denaturing conditions can be employed for the fractionation and purification of DH inducing proteins from L. monocytogenes . In addition, it is suggested that the procedure described might also be useful for the purification of other antigens involved in cellular immune reactions.     

粘虫咽侧体静止激素的初步分离纯化

用放射化学的方法,检测粘虫Mythimna separata幼虫脑提取物中咽侧体静止激素(Allatostatin,AS)样的活性物质。发现粘虫幼虫脑中含有AS样的活性物质,可抑制离体咽侧体(Corporaallata,CA)的保幼激素(Juvenile hormone,JH)的生物合成。用1个脑当量的幼虫脑提取物,对CA的JH合成的抑制率达51%。幼虫脑提取物经胰蛋白酶水解后,AS活性显著降低,表明幼虫脑中的活性物质是肽类或蛋白质类物质。幼虫脑提取物用Sep-Pak柱初步纯化,有活性的组分经高压液相色谱(HPLC)分离,洗脱组分的AS活性测定表明,有AS活性的组分主要集中在组分1～20和组分30～60,其中对离体CA的JH合成的抑制大于50%的组分有3、5、11、40、54和60。     

Influence of the Growth Phase and Fimbriae on the Gradient Elution of Salmonella typhimurium from DEAE-cellulose

Initial investigations showed that certain populations of Sytyphimurium cells could be resolved into three fractions by eluting the cells from DEAE-cellulose with a NaCl gradient in 0·05 Mphosphate buffer (pH 6·0). The three fractions were eluted with 0, 60 and 190 mmol/l NaCl. The results obtained with two strains indicate that non-fimbriate, exponential phase cells; fimbriate, stationary phase cells; and non-fimbriate, stationary phase cells are eluted with 0, 60 and 190 mmol/l NaCl in 0·05 M-phosphate buffer (pH 6·0), respectively. These conclusions appear to be consistent with current views on the effects of fimbriae and the exponential growth phase in reducing the electrical charge carried by bacterial cells.     

Chemical characterization of rat cholecystokinin-58

Cholecystokinin-58 (CCK-58) was purified from rat intestines using an extraction method that yields large amounts of this peptide. Greater than 30% of total CCK immunoreactivity eluted before CCK-39 upon gel permeation chromatography (Sephadex G-50) if extracts were loaded onto Sep Pak cartridges before freezing. If the extracts were frozen and stored at −70°C for six weeks, only 20% of the material eluted in this region and total immunoreactivity was reduced by 50%, suggesting that proteases were active under these storage conditions. This early eluting peak was purified by reverse phase and ion-exchange HPLC to a single absorbance peak. Microsequence analysis of this peak detected AVLRPDSEP which is the amino terminus of rat CCK-58 predicted from the rat preprocholecystokinin cDNA. Because degradation of CCK-58 occurred in these extracts, it is possible that CCK-58 is the predominant molecule form in the rat small intestine.