有机溶剂/去污剂病毒灭活处理对破伤风抗毒素质量的影响

目的 探索用有机溶剂/去污剂(solvent/detergent, S/D)病毒灭活处理对破伤风抗毒素质量的影响。方法 3批破伤风抗毒素样品,每批样品取3等份,向其中2份中分别加入磷酸三丁酯(tri- n -butylphosphate, TNBP)和吐温-80(Tween 80)至终质量分数为0.3%和1%;一份放置在(25±1)℃水浴中振摇6 h后取样,另一份放置在(30±1)℃水浴中振摇4 h后取样;向第3份破伤风抗毒素原液中加入等量的生理盐水混匀后室温放置作为对照。各样品经超滤后检测其效价和蛋白质含量,同时用SDS-PAGE电泳和凝胶过滤色谱柱测定。结果 破伤风抗毒素原液样品经过S/D病毒灭活处理后,其动物效价与对照相比差异无统计学意义( P >0.05),蛋白质含量、分子大小分布无明显变化,多聚体、二聚体及F(ab′) 2含量也无明显变化。结论 S/D病毒灭活处理对破伤风抗毒素的效价,蛋白质含量,分子大小分布,多聚体、二聚体及F(ab′) 2含量均无明显影响,可以作为破伤风抗毒素病毒灭活的候选方法。     

肝素钠树脂精制工艺的研究

利用 D2 5 4 树脂可以将原始粗品肝素钠的效价提高到 179.8U SP U/m g,进一步纯化可得 2 0 1.5U SP U/m g的精品 ,其 H2 O2 用量 ( 1.5% )和氧化时间 ( 2 4 h)均少于传统工艺 ,产品光吸收和收率 ( 85.8% )最优     

马免疫血浆的稳定性研究

目的了解不同温度条件对马免疫血浆外观和效价的稳定性影响,为马免疫血浆的采集、分离、贮存、运输及抗毒素生产提供数据支持。方法将破伤风类毒素及肉毒A、B、E、F型类毒素制备的马免疫血浆,分别放置于2~8℃、20℃、37℃3种温度下,并分别于0、1、3、6、12个月取样,依据《中国药典》三部(2010版)的检测方法和标准,对样品进行外观检查及效价检测。结果破伤风及肉毒A、B、E、F型马免疫血浆在2~8℃条件下,放置12个月稳定性良好,外观及效价均符合《中国药典》三部(2010版)规定要求。20℃与37℃下放置的马免疫血浆随着时间的延长,外观会发生不同程度的浑浊,效价也均有不同程度的降低,低效价组比中、高效价组的效价下降明显,且温度越高效价降低幅度越大。结论马免疫血浆在2~8℃条件下质量稳定。     

基于样品及点源光声信号逆卷积的光声成像方法

光声成像是一种新的生物组织成像方法,在目前的光声成像中,都是通过样品光声信号和超声探测器的脉冲响应来计算样品光吸收的投影,但是由于无法获得超声探测器较准确的脉冲响应,影响重建图像质量。提出一种新的计算样品光吸收投影的方法,从理论上给出了样品光吸收投影和样品及点源光声信号的关系,由样品及点源光声信号的逆卷积可直接计算样品光吸收的投影,点源光声信号通过聚焦入射激光直接测得。试验结果显示,重建图像和样品的相对位置、形状及尺寸完全吻合,成像系统空间分辨率达到0．3mm,证明这是一种有效的光声成像方法。     

小角X射线散射实验中蛋白质溶液的辐照损伤及防护方法

随着同步辐射光源（尤其是目前快速发展的第四代同步辐射光源）技术的进步,可用于实验的辐射通量越来越高,实验样品（特别是蛋白质等生物大分子样品）受到的辐照损伤也越来越严重。在全球现有的同步辐射装置上,蛋白质等生物大分子溶液专用小角X射线散射（SAXS）实验站的光子通量基本上都在1013cps量级。在如此高的通量下,蛋白质等生物大分子溶液样品在实验测量中受到的辐照损伤极其严重。如果没有有效的辐照防护措施,蛋白质溶液样品在毫秒级辐照时间内便会辐照损伤,导致不能获取有效的实验数据。辐照损伤严重制约了SAXS实验技术在蛋白质溶液样品方面的应用。因而,认识蛋白质溶液样品辐照损伤的产生机理、影响因素、判断标准,以及有效降低辐照损伤程度、延缓辐照损伤产生时间的方法,对于蛋白质等生物大分子溶液的散射实验具有重要的指导意义。本文在简要概述生物大分子溶液样品辐照损伤产生机理、影响因素、辐照剂量等基本概念的基础上,重点综述了同步辐射SAXS实验中辐照损伤的判断标准和防护措施。此外,本文还对比了各种防护措施的优缺点,讨论了在建HEPS新光源中SAXS束线可用的散射数据采集时间,指出辐照损伤防护剂是有价值的研究方向...     

制备用于结晶的蛋白质样品

制备高质量蛋白质晶体是通过X射线衍射解析蛋白质分子三维结构的关键环节,是结构生物学领域中的瓶颈问题之一。蛋白质的结晶受多因素控制,其中蛋白质样品自身的质量是影响蛋白质能否结晶及晶体质量好坏的关键因素。我们从蛋白质纯度、可溶性、均一性及表面修饰等方面介绍了如何获得适于结晶的蛋白质样品,以及如何借助相关仪器检测蛋白质样品的质量,预测蛋白质的可结晶性。     

紫外吸收法测定苏芸金杆菌伴孢晶体蛋白质的研究

本文介绍了用紫外光吸收法定量测定苏芸金杆菌制剂中伴孢晶体蛋白质的方法。本方法从样品处理到测定完成只需4个小时,方法简便准确。用此法俭出伴孢晶体蛋白质所指示的毒力效价,与昆虫生测之间有较好的相关性。     

TYXN—91智能血液凝集仪——(上海通用电机电技术研究和上海通用医用仪器公司研制生产)

TYXN—91智能血液凝集仪是根据光电比浊法原理而设计的,具有血小板聚集测定和凝血时间测定的两大功能,并自动描绘聚集曲线及打印实验报告的智能化临床检验仪器。 TYXN—91智能血液凝集仪的组成有: 1.光电检测系统:采用红外光谱(避免可见光干扰)的发射和接受元件组成光电比浊系统。当被测样品置于发射和接受元件之中,样品在实验中浊度的变化引起光电信号的变化。从该变化信号中取出在血浆状态下血小板聚集程度的变化量和血浆在凝血试剂参与下,生成纤维蛋白质和凝固时的光     

BrdU抗血清制备和应用的研究——Ⅰ.高度特异的BrdU抗血清的制备与特性

半抗原BrU通过与BSA偶联制备了完全抗原,经过光吸收、SDS聚丙烯酰胺凝胶电泳和琼脂糖凝胶电泳的测定表明,核苷-蛋白质复合物符合制备的要求,每个BSA上估计大约平均有10.3个BrU。用常规免疫的方法获得兔抗BrdU的抗血清,与BrU-EA的双向扩散效价高达32。抗血清稀释128万倍时仍可见明显的ELISA阳性反应。与以前所报道的BrdU抗血清不同,该抗血清具有高水平的识别能力,已达到BrdU单克隆抗体的识别水平,无须纯化即可用于染色体及核酸的研究。     

硫酸西梭霉素微生物检定法研究

以短小芽孢杆菌为检定菌,比较了培养基原料对抑菌圈的影响,进行了检定菌的分离和敏感活性测定,标准曲线的可倍限率检测;一剂量和二剂量法的可靠性试验;样品效价测定和统计分析,确认我们建立的硫酸西梭霉素的生测方法是准确、可行的。＊ｐ＜０．００１）,试品间和偏离平行不显著（＊ｐ＞０．０５）,可倍限率小于５％,本实验成立。按ｎ次实验结果合并计算,处理２＃、３＃两批样品的实验结果,见表４。经统计分析,２＃样品标定效价为６２９ｕｇ／ｍｇ,可倍限率４．１９％。３＃样品标定效价５７９ｕｇ／ｍｇ,可倍限率３．７１％。３结论３．１硫酸西梭霉素生物效价测定,美国药典［１］以表皮葡萄球菌作检定菌。我们用短小芽孢杆菌作检定菌,抑菌圈清晰,菌液易于保藏,测定结果准确,复现性强。因此,短小芽孢杆菌可作为硫酸西梭霉素的检定菌,培养时间１４～１６小时,培养温度３５～３７℃。３．２采用标准曲线法和二剂量法进行生物效价测定。前者标准品溶液校正点为５ｕｇ／ｍｌ,后者标准品溶液浓度分别为１０ｕｇ／ｍｌ,５ｕｇ／ｍｌ,剂距比２：１。３．３试验结果表明,培养基的原料直接影响检定菌的生长和样品扩散,从而影响抑菌圈的大小和清晰程度。蛋白陈和琼脂的质量尤为重量。?     

Occurrence of a novel nucleotide, Zpp5'A2'p, in rat liver extracts

The nucleotide Zpp5'A2'p has been isolated from rat liver. Z stands for an unknown compound, probably a nucleoside. The preliminary structure of Zpp5'A2'p has been elucidated by treatment with phosphodiesterase and/or alkaline phosphatase and analysis of the products of the reaction by high pressure liquid chromatography. The following ultraviolet absorption spectral characteristics were determined at pH 7.0: Zpp5'A2'p (lambda max = 265 nm; A250/A260 = 0.76; A280/A260 = 0.83); Zp (lambda max = 280 nm; A250/A260 = 0.88; A280/A260 = 1.46). The molar extinction coefficient found for Zp, at 280 nm, was (7.5 + 0.9) X 10(3) M-1 cm-1. The base of Zp could correspond to an indole derivative.     

短梗霉黑色素的分离纯化及结构的初步分析

采用热碱提取、水煮酸沉法从短梗霉发酵液中提取得到黑色素粗品,再经DMSO萃取、酸性甲醇(pH=2)沉淀得到不含多糖和蛋白的短梗霉黑色素。此黑色素不溶于水及常规有机溶剂,可溶于碱性溶液和DMSO;离子交换色谱分析表明黑色素组分均一,出峰时间26±0.5 m in;紫外光谱谱图最大吸收峰为215 nm左右,未见蛋白(280 nm)与核酸(260 nm)的特征吸收峰;红外光谱谱图具有黑色素3μm和6μm的特征吸收峰,并含大量的羟基、氨基,与核磁共振和液质联机谱图结合分析推出短梗霉黑色素可能含有酚羟基、羧基和吲哚等官能团,主要结构骨架为5,6-二羟基吲哚-羧酸和多巴醌,推断该黑色素为酪氨酸酶控制合成的真黑素。     

Spectrophotometric determination of the protein concentration in solutions

The spectrophotometric methods for determination of protein concentration in solution are analysed. According to the given data it has been concluded that the method based on the measurement of difference in light absorption by protein solution at 235 and 280 nm is more accurate as compared with the method of absorption determination at 260 and 280 nm. The linear regression coefficient is used in calculation.     

Studies on the toxin of Aspergillus fumigatus. VII. Purification and some properities of hemolytic toxin (asp-hemolysin) from culture filtrates and mycelia.

A hemolytic toxin has been obtained from mycelia and culture filtrates of Aspergillus fumigatus by the procedures that included precipitation with ammonium sulfate, chromatography of DEAE-Sephadex, affinity chromatography on Concanavalin A-Sepharose and gell filtration on Sephadex G-50, G-100 AND G-150. The purified homolytic toxin was homogeneous on immunological and disk electrophoretic analysis, and the toxin from culture filtrates was identical with that from mycelia by the immunodiffusion technique. The hemolytic toxin was obtained for the first time from fungi and designated as Asp-hemolysin. The molecular weight of Asp-hemolysin was estimated to be appoximately 30,000 by the gel-filtration technique and its isoelectric point was found to be around pH 4.0. This Asp-hemolysin contained large amounts of protein and very small amounts of carbohydrate. The UV absorption spectrum of Asp-hemolysin showed a maximum absorption at 280 nm and minimum absorption at 251 nm. The extinction coefficient at 280 nm and minimum absorption at 251 nm. The extinction coefficient at 280 nm, E 1% 1CM, was 12.4 and the ratio of absorbance at 280 nm to that at 260 nm was 2.3. The optimum pH for the hemolytic activity of the toxin toward chicken erythrocytes was 5.0 at room temperature and it was active in the pH range of 3.5 to 10.5. The optimum temperature was 21 C and about 50% of the activity was lost by incubation at 50 C for 5 min or 45 C for 23 min. The hemolytic activity was remarkably inhibited by Hg2+, Cu2+, Fe2+, Ag1+, iodine and p-CMB, but enhanced slightly by Zn2+ and Co2+.     

Isolation and Characterization of Subacute Sclerosing Panencephalitis Virus Nucleocapsids

Nucleocapsids from subacute sclerosing panencephalitis (SSPE) virus-infected CV-1 cells were concentrated by differential centrifugation employing sucrose cushion techniques and further purified by centrifugation through a linear CsCl density gradient. The bouyant density of (3)H-uridine-labeled nucleocapsids in CsCl was found to be 1.31 g/cm(3). Ultraviolet absorption spectra of the purified SSPE nucleocapsid showed an absorption maximum at 260 to 265 nm and a 280/260 ratio that corresponded to a nucleic acid content of approximately 4.3%. Negatively stained preparations of SSPE nucleocapsids were found to have a width of 18 +/- 1 nm, a periodicity of 5 to 6 nm, and a length between 1-1.4 mum, with the greatest number at 1.3 mum.     

Purification of C-phycocyanin from Spirulina (Arthrospira) fusiformis

C-phycocyanin was purified from Spirulina (Arthrospira) fusiformis by a multi-step treatment of the crude extract with rivanol in a ratio 10:1 (v/v), followed by 40% saturation with ammonium sulfate. After removal of rivanol by gel-filtration on Sephadex G-25, the pigment solution was saturated to 70% with ammonium sulfate. After the last step of purification, C-phycocyanin had an emission and absorption maxima at 620 and 650 nm, respectively and absorbance ratio A(620)/A(280) of 4.3, which are specific for the pure biliprotein. Its homogeneity was demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, yielding two bands of molecular masses 19500 and 21500 kDa, corresponding to alpha and beta subunits of the pigment, respectively. The yield of C-phycocyanin was approximately 46% from its content in the crude extract.     

蛋白质二硫键异构酶的纯化和活力测定

 从500g新鲜牛肝制得蛋白质二硫键异构酶(PDI,EC 5.3.4.1)98mg。该酶制剂在SDS-聚丙烯酰胺凝胶电泳中表现为亚基分子量62,000的均一条带。在260nm追踪,因二硫键错接而失活的牛胰核糖核酸酶A,经PDI作用使其二硫键重排恢复活力,从而催化酵母RNA的水解来测定PDI活力。这种单波长法比文献中介绍的追踪A_(260)—A_(280)的双波长法更为灵敏方便。酶的克分子消光系数ε_M=1.03×10~5(pH7.5),其比活性为1400单位/克蛋白质。     

Use of the diaminobenzoic acid fluorescence assay in conjunction with uv absorbance as a means of quantifying and ascertaining the purity of a DNA preparation

In the course of a study conducted to determine the correlation between covalently bound DNA-ethyl adducts and specific locus mutation induction in maize (W. E. Schy and M. J. Plewa Mutat. Res., 211, 231-241), it was necessary to accurately quantify and ascertain the purity of small amounts of DNA isolated from germinating maize kernels. DNA was purified from leaf primordial tissue that was dissected from germinating maize kernels and quantified by measuring its absorbance at 260 nm. Its absorbance at 260 nm relative to its absorbance at 280 nm (A260/A280 ratio) fell within the range of values that indicated a pure preparation. An attempt to verify the quantity of DNA using a second independent method specific for DNA, the diaminobenzoic acid dihydrochloride fluorescence assay, revealed a significant discrepancy between the two methods. The difference appeared to result from impurities present within the DNA preparation, despite a A260/A280 ratio that indicated otherwise. We found the A260/A280 ratio to be a poor indicator of the purity of DNA preparations, and determined that significant error may result from quantifying DNA using spectrophotometric methods alone. We propose as an alternative, quantifying DNA using the diaminobenzoic acid dihydrochloride assay in conjunction with uv absorbance at 260 nm and using a FLUOR/A260 ratio as an indicator of DNA purity.     

Participation of π-Electrons of Phospholipid Molecules in Absorption of Ultraviolet Light in the Range of 260–280 nm

The UV-spectra (230–260 nm) of the rat brain lipid extract and of individual extract lipid fractions obtained on a column with silica gel (10 µm) were studied. It was found out that hydration of the lipid extract led to a decrease of the absorption intensity of the UV-spectrum by 70% as compared with the initial intensity. Addition of silica gel to the lipid extract decreases twice the UV-spectrum intensity, whereas the repeated addition of silica gel does not decrease intensity of the UV absorption, with no changes of the amount of phospholipids in the lipid extract. Some lipid fractions isolated from the column shift the UV-spectra towards the shorter wavelength region, while the fractions containing phosphatidylcholine shift the spectrum towards the longer wavelength region. It has been established that the phosphatidylcholine fractions containing different amounts of polyenic acids differ by the UV-spectrum intensity. It was concluded that chromophore groups of polyunsaturated acids of phospholipids participated in absorption of energy in the range of 260–280 nm, which lead to excitation of valence electrons of multiple (double) bonds. Energy of such electrons can be used in interactions with other molecules, in particular, for energy transfer inside the membrane monolayer.__________Translated from Zhurnal Evolyutsionnoi Biokhimii i Fiziologii, Vol. 41, No. 3, 2005, pp. 236–239.Original Russian Text Copyright © 2005 by Zabelinskii, Chebotareva, Shukolyukova, Furaev, Krivchenko.     

鼠肝中金属硫蛋白的纯化

旨在探索鼠肝中金属硫蛋白(MT)提取工艺并加以改进。按照经典方法工艺提取MT,用中空纤维柱超滤方法加以改进,依据MT自身特点进行分离和鉴定。结果显示,粗品符合MT特点:分子量在6.5kD左右;无280nm特征吸收峰,加酸后紫外吸收(200-300nm)肩峰消失;通过离子交换可以实现MT1、MT2的分离;通过原子吸收测定,含有锌、铜、镉3种金属,锌含量最高,具有重要的病理生理意义。因此,与其它方法比较,改进后方法更适宜实验室制备。