大肠杆菌AS1.357 L-天门冬酰胺酶的选择性化学修饰

用九种化学修饰剂研究了大肠杆菌AS1.357 L-天门冬酰胺酶分子中的五种不同氨基酸侧链基团与催化活性的关系。结果说明,渡酶活力与硫氧墓完全无关;与色氨酸、精氨酸和组氨酸亦无直接联系;而酪氨酸残基和羧基的修饰引起酶活力急剧下降。其中酪氢酸残基巳被证实是该酶活力的必需基团,处于该酶分子的活性部位。     

琥珀酸弧菌L-天门冬酰胺酶基因的初级克隆和表达

本文以表达型噬菌体λgtll为载体,以及¹²⁵I标记的放射免疫抗体为探针,从EcDR I酶切的琥珀酸弧菌(Vyibrto succinogenes)染色体DNA片段中克隆得到携带天门冬酰胺酶基因的目的片段,在宿主菌E．Coil Y 1090 中得到表达。经酶解和凝胶电泳分析表明该插入DNA片段的分子量为5．8kb．重组DNA感染另一宿主菌E．ColiYl089后所产生的酶蛋白具有L-天门冬酰胺酶活力。用重组DNA(λgt11-AS8)为探针进行southern DNA杂交,琥珀酸弧菌染色体DNA的Ec0R I酶切片段中,出现一条位置在5．8kb处的杂交带,证明我们克隆到的携带L-天门冬酰胺酶基因的目的片段来自琥珀酸弧菌。     

R质粒对大肠杆菌L-天门冬酰胺酶活力的影响

对含有不同R质粒的6株大肠杆菌J53和不含质粒的大肠杆菌J53所产生的L-天门冬酰教酶的活力进行了比较,前者的L-天门冬酰胺酶活力比后者的降低了约1/2—3/4。消除大肠杆菌J53细胞中的R质粒后,L-天门冬酰胺酶活力明显增加并与不含质粒的大肠杆菌J53的相近。结果表明,在大肠杆菌内存在的R质粒对寄主的L-天门冬酰胺酶活力具有明显的抑制作用。     

左旋门冬酰胺酶治疗小儿急性淋巴细胞白血病的护理

左旋门冬酰胺酶（L-ASP）是治疗急性淋巴细胞白血病的主要药物之一。临床用于治疗儿童急性淋巴细胞白血病（ALL）的完全缓解率达95％以上,长期无事件生存率达75％以上[1]。然而L-ASP不良反应较多,包括急性胰腺炎、药物性糖尿病、变态反应、凝血功能障碍、肝功能损害等[2]。因此在用药过程中对不良反应的观察及精心细致的护理极为重要。现将我科2008年1月～2010年1月收治的30例患儿应用L广ASP治疗护理报告如下。     

培门冬酶治疗儿童急性淋巴细胞性白血病的不良反应观察

目的:观察左旋门冬酰胺酶两种常用剂型在急性淋巴细胞性白血病患儿联合化疗中的治疗反应.方法:本院2010-3-2010.12行长春新碱+吡柔比星+门冬酰胺酶+强的松方案化疗的急性淋巴细胞白血病患儿,使用含有聚乙二醇化的左旋门冬酰胺酶剂型培门冬酶即VDPAP者作为A组(40例)与既往使用含大肠杆菌剂型即VDLP者B组(60例)对比,观察不良反应.化疗前后检测血象,肝功能,凝血功能,观察过敏情况等,记录化疗后第28天的各项指标.结果:A组过敏发生2例(5%)而B组过敏发生13例(21.67%),P值0.045,有统计学意义.既往对大肠杆菌剂型发生过敏的13例患儿首剂使用PEG-Asp均无过敏,2例于第二剂时出现皮试阳性.A组(＞2级)白细胞减少16例,中性粒细胞减少26例,血红蛋白降低0例,血小板减少5例,纤维蛋白原降低1例,部分凝血活酶时间延长0例;B组(＞2级)白细胞减少28例,中性粒细胞减少46例,血红蛋白降低2例,血小板减少16例,纤维蛋白原降低2例,部分凝血活酶时间延长0例.A组(未分级)抗凝血酶Ⅱ降低23例,D二聚体升高4例,谷丙转氨酶升高5例:B组(未分级)抗凝血酶Ⅱ降低33例,D二聚体升高8例,谷丙转氨酶升高5例..血液学不良反应差异无统计学意义.A组平均住院日11.14天.B组平均住院日18.47天.结论:左旋门冬酰胺酶两种剂型不良反应相当,但培门冬酶具有使用次数少,使用方便,过敏率低,缩短住院目的优点.     

培门冬酶治疗儿童急性淋巴细胞性白血病的不良反应观察

目的：观察左旋门冬酰胺酶两种常用剂型在急性淋巴细胞性白血病患儿联合化疗中的治疗反应。方法：本院2010.3-2010.12行长春新碱＋吡柔比星＋门冬酰胺酶＋强的松方案化疗的急性淋巴细胞白血病患儿,使用含有聚乙二醇化的左旋门冬酰胺酶剂型培门冬酶即VDPAP者作为A组（40例）与既往使用含大肠杆菌剂型即VDLP者B组（60例）对比,观察不良反应。化疗前后检测血象,肝功能,凝血功能,观察过敏情况等,记录化疗后第28天的各项指标。结果：A组过敏发生2例（5%）而B组过敏发生13例（21.67%）,P值0.045,有统计学意义。既往对大肠杆菌剂型发生过敏的13例患儿首剂使用PEG-Asp均无过敏,2例于第二剂时出现皮试阳性。A组（〉2级）白细胞减少16例,中性粒细胞减少26例,血红蛋白降低0例,血小板减少5例,纤维蛋白原降低1例,部分凝血活酶时间延长0例;B组（〉2级）白细胞减少28例,中性粒细胞减少46例,血红蛋白降低2例,血小板减少16例,纤维蛋白原降低2例,部分凝血活酶时间延长0例。A组（未分级）抗凝血酶Ⅲ降低23例,D二聚体升高4例,谷丙转氨酶升高5例;B组（未分级）抗凝血酶Ⅲ降低33例,D二聚体升高8例,谷丙转氨酶升高5例。.血液学不良反应差异无统计学意义。A组平均住院日11.14天。B组平均住院日18.47天。结论：左旋门冬酰胺酶两种剂型不良反应相当,但培门冬酶具有使用次数少,使用方便,过敏率低,缩短住院日的优点。     

用盐酸胍和Triton X-100从大肠杆菌细胞内释放L-天门冬酰胺酶

研究了用盐酸胍和TritonX 10 0处理ATCCEscherichiacoli 1130 3细胞 ,使细胞内的L-天门冬酰胺酶 (EC 3.5 .1.1)释放到细胞外的方法 ,发现盐酸胍和TritonX 10 0结合使用的效果好。考察了盐酸胍浓度、TritonX 10 0浓度、大肠杆菌细胞浓度、处理时间、pH值等因素对L -天门冬酰胺酶释放的影响。结果表明 ,0 .75～ 1mol/L盐酸胍、2 %TritonX 10 0、细胞浓度 3.2× 10 8/ml、pH 7.0、15℃处理 16～ 2 0h后 ,酶的释放率达到 6 0 %左右。这种方法操作简便 ,酶的释放率较高 ,但对酶无明显的选择性。     

利用Red同源重组技术构建产L-苏氨酸的基因工程菌

利用Red重组技术构建不同基因突变的L-苏氨酸工程菌大肠杆菌ITHR,研究单敲除metA、ilvA和双敲除metA、ilvA基因后对L-苏氨酸积累的影响。应用质粒pKD46介导的Red同源重组系统,通过第一次同源重组将拟敲除基因替换为氯霉素抗性基因,再通过重组酶在FRT位点发生第二次同源重组,消除抗性基因,成功敲除了菌株ITHR体内苏氨酸合成的代谢旁路途径中的metA和ilvA基因,构建了三株不同的基因突变株。将携带苏氨酸操纵子的工程质粒pWYE065电转化入敲除不同基因的突变株中,构建基因工程菌。经5 L发酵罐发酵产酸实验,未敲除任何基因的菌株ITHR/pWYE065 L-苏氨酸的产量为5.55±0.51 g/L,metA基因单敲除菌株ITHR△metA/pWYE065 L-苏氨酸产量为9.77±1.83 g/L,ilvA基因单敲除菌株ITHR△ilvA/pWYE065 L-苏氨酸产量为8.65±1.42 g/L,同时敲除ilvA和metA基因的菌株ITHR△metA△ilvA/pWYE065 L-苏氨酸的产量增加到13.6±1.14 g/L。通过敲除L-苏氨酸的旁路代谢途径中的关键酶的基因,可以增强L 苏氨酸积累的效果,为L-苏氨酸工程菌的进一步改造奠定了基础。     

氮源对重组大肠杆菌发酵产L-精氨酸的影响

考察有机氮源种类、蛋白胨用量以及(NH4)2SO4用量对重组E.coli发酵产L-精氨酸的影响.结果表明:以蛋白胨作为有机氮源且用量在10 g/L,( NH4 )2SO4用量在15 g/L时,摇瓶发酵产L-精氨酸产量最高,达到9.4g/L.在5L发酵罐进行补料分批培养,通过补加(NH4)2 SO4,L-精氨酸产量可以达到18.8 g/L,比未补加提高了108.9％.     

New PEG2 type polyethylene glycol derivatives for protein modification

Although proteins with 2,4-bis (o-methoxypolyethylene glycol)-6-chloro-s-triazine (PEG2-Cl) as a divalent PEG modification have some advantages compared to proteins with the linear PEG modification, PEG2Cl cannot react with amino groups at neutral pH. Therefore, we have prepared new PEG2 derivatives that have an activated ester as the functional group. We confirmed that these derivatives are useful for the divalent modification of proteins, such as bSOD and rhG-CSF. © Rapid Science Ltd. 1998     

Modification of recombinant asparaginase from Erwinia carotovora with polyethylene glycol 5000

A method for polyethylene conjugation with recombinant asparaginase has been developed to improve therapeutically important properties of enzyme. Methoxy-p-nitrophenyl carbamate of polyethylene glycol with molecular weight 5000 was employed as the modification reagent. Optimization of the pegylation procedure resulted in high level of enzyme modification. Under 4.5 molar excess of the modification reagent more than 10 molecules of methoxy-polyethylene bound per one asparaginase molecular. The modified asparaginase retained 57% of initial activity. A simple and efficient pegylation procedure described in this work can be used for production of asparaginase with improved therapeutic properties.     

Enhanced activity of Candida rugosa lipase modified by polyethylene glycol derivatives

The derivatives of polyethylene glycol (PEG) were prepared by reacting PEG with propylene oxide to enhance its hydrophobicity and introduce a branched structure. The PEG derivatives were activated with cyanuric chloride and used to modify the lipase fromCandida rugosa. The maximum specific activity of lipase modified with the PEG derivatives was about 2-fold of that modified with PEG for the esterification of oleic acid and lauryl alcohol in hexane.     

新一代PEG在修饰抗原和药物缓释中的应用

聚乙二醇及其衍生物是具有许多优良性质的高分子化合物,由于良好的生物相容性、无毒、无免疫原性,广泛用于生物医学领域。本文总结聚乙二醇的发展历史和新一代聚乙二醇的特点,阐述聚乙二醇化修饰的目的,特别是在抗原修饰、血型改造和细胞移植等方面的应用,重点对聚乙二醇在药物缓释方面的应用进行了系统的综述。     

Chemical modification of Vibrio vulnificus metalloprotease with activated polyethylene glycol

Abstract Vibrio vulnificus , an opportunistic human pathogen causing septicemia, produces a metalloprotease which is suspected to be a virulence determinant, but which is labile in vivo due to inactivation by α -macroglobulin. To obtain a derivative which is stable in vivo, the metalloprotease was modified with activated monomethoxy polyethylene glycol. The modified protease retained full activity to a peptide substrate and 10–20% activity to protein substrates, and was resistant to entrapment by α -macroglobulin because of the increased molecular size (approx. 90 kDa). These findings suggest that the modified protease is stable in vivo and may be used to investigate the pathological actions of the protease in the bloodstream.     

Refolding and proton pumping activity of a polyethylene glycol-bacteriorhodopsin water-soluble conjugate.

Bacteriorhodopsin (BR), from the purple membrane (PM) of Halobacterium halobium, was chemically modified with methoxypolyethylene glycol (m-PEG; molecular weight = 5,000 Da) succinimidyl carbonate. The polyethylene glycol-bacteriorhodopsin (m-PEG-SC-BR33) conjugate, containing one polyethylene glycol chain, was water soluble. The secondary structure of the conjugate in water appeared partially denatured, but was shown to contain alpha-helical segments by circular dichroism spectroscopy. The isolated bacteriorhodopsin conjugate, with added retinal, was refolded in a mixed detergent-lipid micelle and had an absorption maximum at 555 nm. The refolded conjugate was transferred into vesicles that pumped protons, upon illumination, as efficiently as did native BR. Modification of the PM with m-PEG did not alter the native structure or inhibit proton pumping, and therefore it is suggested that the glycol polymer is present as a moiety covalently linked to residues unnecessary for proton pumping and proper folding. The site of attachment of m-PEG was determined to be at either Lys 129 or Lys 159, with position Lys 129 the most probable site of attachment. The m-PEG-SC-BR33 could be stepwise refolded to the native conformation by the addition of trifluoroethanol to lower the dielectric constant, simulating the insertion of the BR into the phospholipid bilayer.     

聚乙二醇修饰牛血清白蛋白的反应与分析

采用N,N′-羰基二咪唑活化法活化单甲氧基聚乙二醇5000分子一端的羟基,对活化后的单甲氧基聚乙二醇分子进行了元素分析。用该活化产物对牛血清白蛋白的赖氨酸侧链氨基进行化学修饰。应用毛细管电泳对聚乙二醇修饰后的产物进行了分析,并与高效液相色谱分析结果作了对照研究,表明毛细管电泳对修饰后的牛血清白蛋白有更好的分析效果。     

Time sequence and morphological evaluations of cells fused by polyethylene glycol 6000

Summary Mouse L cells (clone 1D) were fused with polyethylene glycol (PEG). The fusion sequence was determined by using sequential light microscopy of the same group of cells, scanning electron microscopy (SEM), transmission electron microscopy, and freeze-etching. The cells were found to fuse only 1 min after PEG had been washed off at small localized areas. Larger fusion images were found after 3 min. Intramembrane particles were observed to have a tendency to aggregate after PEG treatment, but a direct correlation of this activity with the fusion process could not be made. No pathological changes were noted at longer times after PEG removal, except for the extensive widening of the rough-surface endoplasmic reticulum (RER) in some cells. It is proposed that fusion does not occur if apposing cells have many microvilli at the area of apparent contact. Presented in the formal symposium on Somatic Cell Genetics at the 27th Annual Meeting of the Tissue Culture Association, Philadelphia, Pennsylvania, June 7–10, 1976. This work was supported by U.S. Public Health Service research grants CA 10815 from the National Cancer Institute and GM 21615 from the Institute of General Medical Sciences.     

Enhancement effect of polyethylene glycol on enzymatic synthesis of cephalexin

In an enzymatic synthesis of cephalexin (CEX) using an acylase from Xanthomonas citri, the effect of polyethylene glycol (PEG) on the synthetic reaction of 7-amino-3-deacetoxycephalosporanic acid (7-ADCA) and D-alpha-phenyl-glycine methyl ester (PGM) to CEX was investigated. The addition of PEG (MW 300-20,000) increased the yield significantly. This yield enhancement effect tended to increase with the increasing molecular weight of PEG. Addition of PEG to the reaction system did not affect both the CEX and PGM hydrolytic reactions. The PEG added to the reaction medium used in these experiments did not depress the water activity significantly, and the product yield improvement could not be explained by the activity alone. The PEG stabilized the enzyme activity to some extent, but this stabilizing effect was only partially attributable to the yield enhancement of CEX. The enhancing effect of PEG on the synthetic yield increased with the increasing PEG molecular weight or the length of the poly(oxy-1,2-ethanediyl) chain, which increases the hydrophobicity of PEG. This finding consequently has led to the conclusion that the PEG structure renders the affinity between enzyme and 7-ADCA, which is a hydrophobic substrate. The microenvironmental hydrophobicity of PEG and its interaction with the hydrophobic substrate was found to be the main reason for the improvement of the CEX yield. In fact, the Michaelis-Menten kinetic constant for 7-ADCA, K(7-ADCA) in the presence of PEG was smaller than that in the control system (without PEG addition). (c) 1993 John Wiley & Sons, Inc.     

Glucose-sensing carbon paste electrode containing polyethylene glycol-modified glucose oxidase

Polyethylene glycol-modified glucose oxidase (PEG-GOD) was prepared. Carbon paste (CP) containing PEG-GOD retained enzyme activity of 0·02 U cm⁻². Anodic and cathodic peak currents of modified GOD in CP matrix were observed on the differential pulse voltammograms at the potential of −0·36 and −0·36 V vs. Ag/AgCl, respectively. The addition of glucose to a test solution brought about an increase in the anodic current on the PEG-GOD-based electrode at the potential as low as 0·0 V vs. Ag/AgCl. The current increase was proportional to the concentration of glucose up to 50 mM.