坛紫菜藻红蛋白分离纯化研究

[目的]建立一种新型、高效、快速的藻红蛋白分离纯化方法,并对其分离机理进行探讨。[方法]以坛紫菜的藻红蛋白搅切法提取物为原料,首先通过100和500 kDa的超滤离心管对其进行超滤分离,再在Superose 12色谱柱上以不同种类和不同浓度的流动相对藻红蛋白粗提物进行等度洗脱,分析其分离机理,并优化分离条件。[结果]在0.05 mol/L NaCl等度洗脱的条件下,藻红蛋白在Superose12色谱柱上表现为典型的氢键吸附模式;在优化的条件下,通过超滤-Superose 12氢键吸附分离的方法,可以使坛紫菜藻红蛋白提取物纯度(A565/A280)达到7.98,总回收率52.9%。[结论]该研究结果表明藻红蛋白在Superose 12色谱柱上存在氢键吸附作用,同时,该研究所建立的超滤-Superose 12氢键吸附分离方法是一种新型、高效、快速的藻红蛋白分离纯化方法。     

DNA recovery from agarose gels with a simple centrifuge-driven sephadex filtration

Conventional methods of DNA recovery from agarose gel generally require expensive equipment, extended elution times, or considerable handling of the sample after elution. We developed a simple protocol for a quick and effective recovery of DNA from agarose gels with good yield and quality. Using a Sephadex resin filled spin column, DNA fragments of 500 bp to 6 kb in an agarose gel slice were easily recovered by a 2 min centrifugation. The recovery efficiencies were over 40%-50% and the eluted DNA can be used directly for downstream application, such as polymerase chain reactions (PCR) and restriction enzyme digestion. This method could also be used to recover large DNA fragment (48 kb) without degradation. The use of Sephadex helps to remove small molecular impurities from agarose and it also reduces the chance of clogging the column filter caused by direct contact with agarose.     

A quick method for the assessment of activity and inhibition of fish amylases

A sensitive and quick method was developed to determine the presence of α-amylase in the gut of aquatic organisms, as well as its sensitivity to inhibitors. The assay is based on the utilization of Petri dishes filled with starch–agarose gel as a substrate for the enzyme solution, which is placed in small wells punched in the surface. Circular zones produced by the action of amylase remain colourless after staining with lugol. Pure commercial porcine amylase was used to fit the better conditions for developing the assay (1 g L^–1 starch in the gels, 4 h of incubation). The diameter of the cleared zones were related to the activity of enzyme and the method detected linearly amylase activity in a range of 2–20 U well^–1, so it was used to reveal the presence of amylase in digestive extracts obtained from different sparid fish. The method was also used to evaluate the effect produced by a specific inhibitor on fish amylases, showing a linear response when the ratio inhibitor:enzyme (in units) changed from 20:1 to 2:1. Comparison of the cleared zones produced by amylases of sparid fish in the presence or absence of inhibitor, revealed differences in their sensitivity to inhibition, which ranged from 15 to 50% of total activity. The assay is proposed for a preliminary evaluation of possible inhibitors contained in feedstuffs used in fish feeding.     

不溶性草酸氧化酶的包埋及草酸快速检测方法研究

通过研究不溶性草酸氧化酶包埋方法和草酸快速检测方法,建立氧电极法测定草酸的便捷体系。以大麦麸皮为原料制备不溶性草酸氧化酶,采用5%的琼脂糖包埋,检测包埋酶的稳定性和抗离子干扰能力;以包埋酶为工具酶,通过氧电极法检测草酸含量。试验结果表明:包埋酶连续反应2 h酶活性下降低于5%,贮存30 d活性下降低于8%;草酸检测线性范围0.1～0.8mmol/L,外标法草酸回收率为83%～97%。用氧电极法与显色法测定小麦草酸含量,两种方法的测定值无显著性差异。但氧电极法测定耗时短,反应体系简单,易于推广应用。     

琼脂及琼脂糖中3,6-内醚半乳糖的测定

本文研究了间苯二酚法对琼脂及琼脂糖样品中的3,6-内醚半乳糖的测定方法。实验中,根据间苯二酚试剂与3,6-内醚半乳糖和果糖的浓度在0.25μmol/mL以下时,具有近乎相同的标准比色曲线的原理,分别对测定波长、显色温度、显色时间和比色时间等检测条件进行优化。结果显示,用间苯二酚法测定3,6-内醚半乳糖的最佳检测条件为:测定波长554 nm、显色温度80°C、显色时间15 min;在该条件下,对琼脂及琼脂糖样品中的3,6-内醚半乳糖进行测定,方法的相对偏差为0.94%～1.27%,加标回收率为105.4%～111.04%。表明该方法操作简便,具有较高的准确性和重复性。     

中国矮马血红蛋白多态性的初步研究

采用等电聚焦电泳对中国矮马进行了血红蛋白多态性的检测。结果表明 :在该位点共发现有 2种基因型 ,由 2个等位基因HbBⅡ 、HbBⅠ 控制。基因型BⅡ /BⅡ、BⅠ /BⅡ的频率分别为 0 62 5 0、 0 375 0 ,其中纯合型BⅡ /BⅡ为优势基因型。基因频率HbBⅡ 、HbBⅠ 分别为 0 81 2 5、0 1 875。其基因杂合度 (H)、个体鉴别概率 (Dp)和亲仔关系排除概率 (PE)分别为 0 30 4 7、 0 4690和 0 1 2 90。     

Agarose gel electrophoretic fractionation of serum proteins in adult cattle. II. A study of cows with different diseases.

In 283 dairy cows suffering from internal disorders the serum proteins were studied by agarose gel electrophoresis supplemented by total protein and albumin determinations. The clinical diagnoses could be grouped according to protein pattern. Group 1 (abomasal displacement and traumatic muscle injury) did not appreciably affect the serum protein concentrations and represented primarily non-inflammatory diseases or diseases of non-infectious origin. Group 2 (leukosis) occupied an exceptional position, with heavy lowering of albumin unaccompanied by a corresponding rise of total globulin. The γ-globulin concentration was significantly lowered. Group 3 (acute traumatic peritonitis) represented an acute inflammatory process with increase chiefly of the α-globulins, while the γ-globulin concentration was normal. Group 4 (chronic traumatic peritonitis, summer mastitis, chronic subclinical mastitis, chronic laminitis and polyarthritis, urinary tract infection, abscess, and sub-acute-chronic pneumonia) comprised diseases chiefly of chronic inflammatory character and with infectious origin. Especially characteristic was the heavy rise of globulins in general and of γ-globulin in particular. In most cases there was a large increase also of the α- and p-globulin fractions.     

小鼠附植前胚胎超薄切片的琼脂糖预包埋制备方法

将小鼠附植前胚胎分别经戊二醛一锇酸固定后,采取琼脂糖预包埋法使胚胎凝聚成团,然后对含胚胎的琼脂糖块进行脱水、渗透和包埋。结果显示,Epon812包埋块中的胚胎排列紧密,半薄切片胚胎的整体形态完整,胚胎超微结构保存良好。说明琼脂糖预包埋法是制备小鼠附植前胚胎超薄切片的有效方法。     

Isolation of new pregnancy-associated glycoproteins from water buffalo (Bubalus bubalis) placenta by Vicia villosa affinity chromatography

The present study describes the isolation and characterization of new pregnancy-associated glycoprotein molecules (PAG) from midpregnancy and late-pregnancy placentas in the water buffalo (Bubalus bubalis). After extraction, the homogenates are subjected to acid and ammonium sulfate precipitations followed by DEAE chromatography. Subsequently, the water buffalo PAG (wbPAG) from these solutions are enriched by Vicia villosa agarose (VVA) affinity chromatography. As determined by western blotting with anti-PAG sera, the apparent molecular masses of the immunoreactive bands from the VVA peaks range from 59.5 to 75.8 kDa and from 57.8 to 73.3 kDa in the midpregnancy and late-pregnancy placentas, respectively. Amino-terminal microsequencing of the immunoreactive proteins has allowed the identification of three distinct wbPAG sequences, which have been deposited in the SwissProt database: RGSXLTIHPLRNIRDFFYVG (acc. no. P85048), RGSXLTILPLRNIID (acc. no. P85049), and RGSXLTHLPLRNI (acc. no. P85050). Their comparison to previously identified proteins has shown that two of them are new because they have not been described before. Our results confirm the suitability of VVA chromatography for the enrichment of the multiple PAG molecules expressed in buffalo placenta.     

Protein characterization using electrophoresis and immunofixation; a case‐based review of dogs and cats

Protein electrophoresis and immunotyping can be a useful adjunct to the standard biochemical techniques for characterizing serum and urine proteins. This paper reviews currently available and commonly used methods for diagnostic protein electrophoresis, including both agarose gel and capillary zone electrophoretic techniques and total protein assessments. Immunofixation and immunosubtraction methods for identification of immunoglobulin location and class are also presented. Practical application of quality assurance and quality control strategies in compliance with American Society of Veterinary Clinical Pathology (ASVCP) best practices are discussed. Commonly encountered serum and urine electrophoretic diagnostic patterns, including electrophoretically normal, acute‐phase protein responses, polyclonal gammopathies, restricted polyclonal/oligoclonal gammopathies, paraproteinemias (monoclonal or biclonal gammopathies), and Bence‐Jones proteinurias are also reviewed using relevant case material. Cases in which immunofixation electrophoresis are particularly useful are highlighted, and methodologies to more accurately quantify serum monoclonal proteins (M‐proteins), monitoring tests commonly used in human medicine, are discussed.