N-V蛋白酶两种ELISA检测方法的建立及其初步应用

发布时间：2018-04-15 12:27

本文选题：N-V蛋白酶 + 多克隆抗体　；参考：《吉林大学》2006年硕士论文

【摘要】： N-V蛋白酶是从一种我国天然海洋生物体内发现的具有强烈纤维蛋白溶解作用的纤溶酶,对血栓分解具有潜在的作用[1]。为了进一步研究N-V蛋白酶的作用机理和它的代谢过程以及此药在临床上的应用,本实验初步建立了ELISA的两种检测方法并对N-V蛋白酶在动物体内的代谢过程作了初步研究。 N-V蛋白酶是一种氨基肽酶[2],通过对N-V蛋白酶多克隆抗体的制备,我们对氨基肽酶这一酶类多克隆抗体的免疫制备过程有了初步了解,采用N-V蛋白酶加入弗氏佐剂免疫家兔的方法得到免疫血清[3],利用蛋白质等电点、盐析和疏水特性,运用硫酸铵沉淀法和phenyl-sepharose疏水色谱柱结合法提纯免疫血清。比一般的DEAE-纤维素层析、分子筛和硫酸铵-辛酸沉淀法获得的抗体相比,具有纯度高、实验步骤少、活性好、回收率高等优点。分别用双向免疫扩散电泳和SDS-PAGE电泳测效价和纯度,成功的用过碘酸钠氧化法把HRP标记于N-V蛋白酶多克隆抗体以及二抗即羊抗兔抗体,且酶标记质量完全符合以后的实验要求。我们成功建立N-V蛋白酶的两种ELISA检测方法,分别为夹心ELISA和间接ELISA法。并将此方法与其他蛋白定量方法作了比较。该方法为N-V蛋白酶的药代和毒理研究提供技术手段,为临床中用药提供参考和临床检验提供技术,并初步确定正常家兔对N-V蛋白酶的体液免疫应答特点以及N-V蛋白酶在家兔体内的代谢过程。
[Abstract]:N-V protease is a fibrinolytic enzyme with strong fibrinolysis found in natural marine organisms in China. It has a potential role in thrombolysis [1].In order to further study the mechanism of N-V protease and its metabolic process and its clinical application, two methods for the detection of ELISA were established and the metabolic process of N-V protease in animals was studied.N-V protease is an aminopeptidase. Through the preparation of polyclonal antibody against N-V protease, we have a preliminary understanding of the preparation process of aminopeptidase, an enzyme polyclonal antibody.Immunized rabbits were immunized with N-V protease and Freund's adjuvant. Using protein isoelectric point, salting out and hydrophobic properties, the immunized serum was purified by ammonium sulfate precipitation method and phenyl-sepharose hydrophobic chromatographic column method.Compared with the conventional DEAE-cellulose chromatography, molecular sieve and the antibody obtained by ammonium sulphate-octanoic acid precipitation method have the advantages of high purity, less experimental steps, good activity and high recovery.The titer and purity of N-V protease polyclonal antibody and sheep anti-rabbit antibody were detected by two-dimensional immunodiffusion electrophoresis and SDS-PAGE electrophoresis, respectively. The HRP was successfully labeled with N-V protease polyclonal antibody and sheep anti-rabbit antibody by sodium periodate oxidation method.We successfully established two ELISA methods for the detection of N-V protease, sandwich ELISA and indirect ELISA, respectively.The method was compared with other protein quantification methods.This method provides a technical means for the pharmacokinetic and toxicological study of N-V protease, and provides a reference for the clinical use of drugs and techniques for clinical testing.The humoral immune response of normal rabbits to N-V protease and the metabolic process of N-V protease in rabbits were preliminarily determined.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R392.1

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