【作者】 伊光辉；

【导师】 林天龙； 熊邦喜；

【作者基本信息】 华中农业大学 ， 水产养殖， 2001， 硕士

【摘要】 本文通过饱和硫酸铵分步盐析结合Sepharose-4B凝胶柱以及DEAE—52阴离子交换柱层析分别提取纯化非免疫和温和气单胞菌免疫的斑点叉尾鮰血清免疫球蛋白(Ig)，采用单克隆抗体(MAF-13株)结合酶联免疫吸附(ELISA)检测提取物中血清免疫球蛋白的活性。结果表明非免疫斑点叉尾鮰的血消Ig主要存在于35％～45％的硫酸铵沉淀区间，而免疫斑点叉尾鮰的血清Ig则分布在35％～55％的硫酸铵沉淀区间内。在Sepharose-4B凝胶柱和DEAE—52阴离子交换柱层析纯化中Ig均出现在第一个蛋白峰。十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)进行纯度鉴定，结果发现经过系列提纯的Ig样品杂蛋白条带逐渐减少，DEAE—52纯化后的Ig仅在重、轻链位置出现明显条带，表明纯化后的血清免疫球蛋白纯度较高。纯化的血清Ig用以制备兔抗斑点叉尾鮰血清Ig的多克隆抗体．采用ELISA方法分别测定单克隆抗体和多克隆抗体的特异性，结果表明抗斑点叉尾鮰血清Ig的多克隆抗体和单克隆抗体均与欧洲鳗鲡和日本鳗鲡有免疫交叉反应。应用变性还原(denatured and reduced)、变性非还原(denatured but non-reduced)和非变性非还原条件下(non-denatured and non-reduced)的免疫印迹(western blot)试验对纯化的Ig进行结构分析，以抗斑点叉尾鮰血清Ig的单克隆抗体和多克隆抗体碱性磷酸酶标记物为探针进行检测。结果表明免疫斑点叉尾鮰血清Ig的重链分子量为72KD，轻链有三条，分子量分别为21KD、23.5KD利26KD；而非免疫斑点叉尾鮰血清Ig中发现有2条重链，分子量分别为72KD和55KD。梯度PAGE和Western blot测得非还原非变性条件下免疫球蛋白的分子量约为900KD。在SDS变性非还原梯度PAGE和Western blot中发现血清Ig出现多种结构形式，分子量分别为870KD、760KD、525KD、330KD利230KD。更多还原

【Abstract】 Serum immunoglobulin (Ig) from normal and Aeromonas sobria (A.s) immunized channel catfish (Ictalurus punctatus) were each purified by using ammonium sulfate precipitation followed by sepharose-4B gel filtration and DEAE-52 anion exchange chromatography. The purified Ig fractions were defined by enzyme-linked immunosorbent assay (ELISA) with monoclonal antibody (MAF-13) before being pooled and accessed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). The results showed that normal channel catfish serum Ig were mainly present in the range of 35% - 45% ammonium sulfate precipitation, however, the serum Ig of immunized channel catfish were present in the range of 35%-55% ammonium sulfale precipitation. The immunoglobulin appeared in the first protein peak both in the Sepharose-4B gel filtration and DEAE-52 anion exchange chromatography. SDS-PAGE results showed that serum Ig had been further purified by each step of purification. The purified Ig were used as antigen to produce rabbit antiserum. Polyclonal and monoclonal antibodies both reacted with serum of European eel (Anguilla anguilla) and Japanese eel (Anguilla Japonica) in ELISA. Structure analysis of serum Ig were performed by Western blot under denatured and reduced, denatured but non-reduced, non-denatured and non-reduced conditions using monoclonal and polyclonal antibodies as probes. The results showed that the denatured and reduced serum Ig of normal channel catfish had two different heavy (H) chains with molecular weight about 72 0 00 and 55 000 respectively. For the immunized channel catfish, however, the molecular weight of heavy chain was predominantly determined to be 72 000, and three distinct light (L) chains were found with molecular weight of 21 000, 23 500, 26 000 respectively. The naive serum Ig was estimated to have a molecular weight about 900KD using 3%~12% gradient PAGE and western blot under non-reduced and non-denatured conditions. This polymeric Ig could dissociated into several subpopulations with molecular weight about 870 000,760 000,525 000,330 000 and 230 000 when analyzed in the present of denaturing solvent (SDS).更多还原