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101.
102.
本实验室已得到的亮氨酰-tRNA合成酶(LeuRS)基因,与文献相比,67位氨基酸残基由His变为Arg,此酶被定名为LeuRS67R。我们从该基因与pUC19重组质粒的大肠杆菌TG1转化子TG1-91中得到LeuRS的高表达,粗抽液中LeuRS的表达量在转化子中比在宿主菌TG1中高20倍以上。用三步拉层析得到电泳一条带的酶,其比活为1789单位/毫克。测定其动力学常数,氨酰化活力对Leu、ATP的Km值分别为0.027mmol/L、0.47mmol/L,Kcat值分别为3.5~5.1s-1。ATP-PPi交换活力对Leu、ATP的Km值分别为0.03mmol/L、1.0mmol/L,Lcat值分别为140~155s-1。此结果与从野生型大肠杆菌K-12中提纯的LeuRS的动力学常数差别很小,67位氨基酸残基在与活性中心无直接关系的域可能是大肠杆菌的种间差异。 相似文献
103.
本文从含ArgRS306KR基因args306KR的pUC18重组质粒的大肠杆菌TG1转化子中经DEAE-Sephacel和Blue-Sepharose两步柱层析,得到电泳一条带的ArgRS306KR。纯酶的比活为2790单位/毫克。该酶氨酰化和ATP~PPi交换活力的最适pH分别为pH8.3和pH7.5。氨酰化活力对ATP、Arg和tRNA的Km分别为2.6mmol/L、14.0μmol/L和5.0μmol/L:Vmax为7630单位/毫克;koat为9S-1。ATP~PPi交换活力对ATP和Arg的Km分别为8.3mmol/L和99μmol/L;Vmax为16320单位/毫克;kcat为18S-1。 相似文献
104.
实验中观察到,用MUG培养基对植物药中的大肠杆菌定量时多发生荧光猝灭现象,影响检测结果。本文对此现象产生的原因与克服方法进行了系统的考察,发现以一种简便的转接方法可排除植物药介质对菌检的干扰。该方法由两组检验系列构成,当怀疑正常稀释系列(第一系列)40h培养液的荧光结果可能因猝灭现象呈假阴性时,立即分别将该系列的1—3号管培养液以0.5ml的接种量转接入新鲜的MUG培养基(第二系列),重新培养24h,荧光猝灭现象即可克服。综合两系列的荧光、产气和吲哚三项生化特征得出检品中大肠杆菌含量。实际应用表明,此法能显著提高使用该培养基时菌检结果的可靠性。 相似文献
105.
通过DNA体外重组技术,以pET-3b为表达载体,构建了重组表达质粒pET-6R(B)和PET-6R(B)4,分别编码28kD的hIL-6R配基结合区片段及其53kD的二联体蛋白,并为酶切分析和DNA序列分析所证实。SDS-PAGE分析表明,含有重组表达质粒的菌株可分别表达出28kD的蛋白rIL6R-28和53kD的rIL6R-53。重组蛋白分别占菌体总蛋白的45%和29%左右。重组蛋白主要以包涵体形式存在,Western印迹表明重组蛋白具有IL-6R的抗原性。 相似文献
106.
将油桐尺蠖(Buzurasuppressaria)核多角体病毒晚期基因──多角体蛋白基因启动子及5′端编码区,以两种不同方式置于缺乏启动子的氯霉素乙酰基转移酶(CAT)基因上游,使其分别终止在不同翻译终止位点,其宿主菌具有明显不同的氯霉素抗性,最高达200mg/L LB培养基以上,表明昆虫病毒启动子能启动原核基因表达。对多角体蛋白基因启动子能在大肠杆菌中有效工作的原因进行了讨论。 相似文献
107.
Roberto E. Izquierdo Kimberly Breese Shalini Jain Daniel Carestio Lawrence Jung James Figge 《In vitro cellular & developmental biology. Animal》1995,31(1):71-76
Summary Gene transfer techniques can be used to encode the production of a polypeptide product, such as human growth hormone (hGH),
that is missing in an acquired or inherited disease state such as growth hormone deficiency. In one model system, engineered
C2C12 myoblasts are injected intramuscularly into a mouse and subsequently secrete hGH into the circulation. In this regard,
a gene-expression regulatory system that functions in myoblasts would be of interest. We demonstrate that theEscherichia coli lac operon system can be used to stringently regulate the expression of hGH in engineered C2C12 myoblasts in tissue culture.
A DNA segment encoding hGH was linked to a DNA segment containing an SV40 enhancer and promoter. The latter components were
positioned between two syntheticlac operators.Lac repressor expression was driven by a simian cytomegalovirus promoter. In transient co-transfection assays, hGH expression
from cultured C2C12 myoblasts could be modulated up to 60-fold (P = 0.002) with the inducing agent, isopropyl-β-d-thiogalactoside (IPTG). In the absence of IPTG, hGH expression was almost fully repressed. These results show that the components
of theE. coli lac operon provide a stringent regulatory system for use in myoblasts. The system might prove to be useful for the regulation
of transferred genes in animals. 相似文献
108.
Axenic Cultivation of Entamoeba dispar Brumpt 1925, Entamoeba insolita Geiman and Wichterman 1937 and Entamoeba ranarum Grassi 1879 总被引:2,自引:0,他引:2
C. GRAHAM CLARK 《The Journal of eukaryotic microbiology》1995,42(5):590-593
ABSTRACT. Three species of Entamoeba have been grown in axenic culture for the first time. In two cases, novel methods for adapting the organisms to growth without bacteria were employed. While E. ranarum was axenized by the classic technique of Diamond, from a monoxenic culture with Trypanosoma cruzi as the associate, both E. dispar and E. insolita were first grown in axenic culture medium supplemented with lethally irradiated bacteria. From there, E. insolita was axenized directly, but E. dispar initially required the presence of fixed bacteria. After prolonged culture under this technically axenic but unwieldy culture system, E. dispar was eventually adapted to growth in the absence of added bacteria. 相似文献
109.
Orazio Cantoni Giorgio Brandi Amedeo Albano Flaminio Cattabeni 《Free radical research》1995,22(3):275-283
Cystine markedly enhanced the cytotoxic response of Escherichia coli cells to concentrations of hydrogen peroxide resulting in mode one killing, but displayed little effect in mode two killed cells. The effect of cystine was concentration-dependent over a range of 5-50 μM and did not further increase at higher levels. Cystine had similar effects in other bacterial systems.
In order to sensitize the cells to the oxidative injury, the amino acid must be present during exposure to the oxidant since no enhancement of the cytotoxic response can be observed in cystine pre-loaded cells. In addition, no further enhancement of cytotoxicity could be detected when cystine was added before and left during challenge with the oxidant. The enhancing effect of cystine on oxidative injury of E. coli cells appears to be directly mediated by the amino acid and in fact cysteic acid, the most likely oxidation product, had no effect on the killing of bacterial cells elicited by hydrogen peroxide. Other disulfide compounds such as oxidized glutathione, cystamine and dithionitrobenzoic acid only slightly increased the susceptibility of bacteria to the oxidant. The effect of the disulfides was not concentration-dependent over a range of 200-800 μM and was statistically significant only for cystamine.
Taken together, these results indicate that cystine markedly increases the cytotoxic response of bacteria to hydrogen peroxide and suggest that the amino acid might impair the cellular defence machinery against hydrogen peroxide. This effect may involve a thiol-disulfide exchange reaction at the cell membrane level. 相似文献
In order to sensitize the cells to the oxidative injury, the amino acid must be present during exposure to the oxidant since no enhancement of the cytotoxic response can be observed in cystine pre-loaded cells. In addition, no further enhancement of cytotoxicity could be detected when cystine was added before and left during challenge with the oxidant. The enhancing effect of cystine on oxidative injury of E. coli cells appears to be directly mediated by the amino acid and in fact cysteic acid, the most likely oxidation product, had no effect on the killing of bacterial cells elicited by hydrogen peroxide. Other disulfide compounds such as oxidized glutathione, cystamine and dithionitrobenzoic acid only slightly increased the susceptibility of bacteria to the oxidant. The effect of the disulfides was not concentration-dependent over a range of 200-800 μM and was statistically significant only for cystamine.
Taken together, these results indicate that cystine markedly increases the cytotoxic response of bacteria to hydrogen peroxide and suggest that the amino acid might impair the cellular defence machinery against hydrogen peroxide. This effect may involve a thiol-disulfide exchange reaction at the cell membrane level. 相似文献
110.
Susann Teneberg Timothy R. Hirst Jonas Ångström Karl-Anders Karlsson 《Glycoconjugate journal》1994,11(6):533-540
The binding specificities of cholera toxin andEscherichia coli heat-labile enterotoxin were investigated by binding of125I-labelled toxins to reference glycosphingolipids separated on thin-layer chromatograms and coated in microtitre wells. The binding of cholera toxin was restricted to the GM1 ganglioside. The heat-labile toxin showed the highest affinity for GM1 but also bound, though less strongly, to the GM2, GD2 and GD1b gangliosides and to the non-acid glycosphingolipids gangliotetraosylceramide and lactoneotetraosylceramide. The infant rabbit small intestine, a model system for diarrhoea induced by the toxins, was shown to contain two receptor-active glycosphingolipids for the heat-labile toxin, GM1 ganglioside and lactoneotetraosylceramide, whereas only the GM1 ganglioside was receptor-active for cholera toxin. Preliminary evidence was obtained, indicating that epithelial cells of human small intestine also contain lactoneotetraosylceramide and similar sequences. By computer-based molecular modelling, lactoneotetraosylceramide was docked into the active site of the heat-labile toxin, using the known crystal structure of the toxin in complex with lactose. Interactions which may explain the relatively high toxin affinity for this receptor were found.Abbreviations CT
cholera toxin
- CT-B
B-subunits of cholera toxin
- LT
Escherichia coli heat-labile enterotoxin
- hLT
humanEscherichia coli heat-labile enterotoxin
- pLT
porcineEscherichia coli heat-labile enterotoxin
- EI
electron ionization 相似文献