嗜水产气单胞菌诱导的草鱼免疫球蛋白分析

摘要

取草鱼经嗜水产气单胞菌疫苗免疫，获免疫血清分两组，一组样品经饱和硫酸铵分级沉淀、透析和电泳分析，另一组经Sephadex G-200柱层析分离及电泳分析。各分离样品经特异性凝集活性测定，发现沉淀分离组样品中抗体凝集活性最强的主要为35%饱和硫酸铵沉淀的组分，而柱层析分离组样品的凝集抗体主要集中在第一个洗脱峰里。在电泳图谱中，各免疫组均比对照组增加了一条大分子B带，分子量为309 kD左右，后者经变性电泳分离表现为两条区带，相对分子量分别为52 kD和26 kD。因而推测它们是单体IgG解离的重链和轻链，而聚集在B带的是IgG的聚合形式IgM。%Grass carp (Ctenopharyngoden idellus) is an economically important fresh wa t er fish in China. Although a significant amount of effort has been devoted to st udy the development of vaccines against infectious diseases in this fish, little was known about the biological features of its immunoglobulin. rnWe induced the immune response in Grass carp with the vaccine consisting of inactivated bacterial Aeromonas hydrohila. The increased level of immuno glob ulin in response to the vaccination challenge was observed. The immunological an d biochemical features of the immunoglobulin protein was characterized. rnWe used oneyear fish C. idellus of 100 to 150 grams of body weight . Eac h fish in the experimental group received A. hydrophila vaccine (strain TPS 30 one billion inactivated bacteria per ml) at a dose of 0.5 ml. Vaccine solution w as injected into the fish muscle under the back fins. Controlled group of fish r eceived no treatment, but were raised for the same period of time. Sever and 14 days after the first injection, blood was collected from the tail arteries and s era was separated. The activity and specificity of immunoglobulin in fish sera w ere tested with standard agglutination assays conventionally. Immunoglobulin pr oteins was isolated from fish sera either by the fractionation of saturated ammo nium sulfate or by Sephadex G200 gel column chromatography and by high pH disc ontinuous polyacrylamide gel electrophoresis. rnAntigenspecific agglutination testing revealed that the highest antibody titer was present in the fraction precipitated with 35% saturated ammonium sulfate, or in the first elution peak of chromatography from the first collected tube. The results by electrophoresis indicated that the components having antigenspecifi c agglutination were mainly located on the B band of the electrophoretic gel. Aft er being treated with 2mercaptoethanol, the agglutination acvtivity for compon ents dropped greatly. rnBy comparison with nonimmunized control group, electrophoresis under nonredu ci ng conditions identified an extra Bband with Mr=309 kD in the immunized sample s. When this Bband was isolated and subjected to electrophoresis under denaturin g conditions, two separate polypeptides were detected with Mr of 52 kD and 26 kD, respectively, which should be identical of the molecular weights of fish IgG he avy and light chains. Therefore, we conclude that the A. hydrophila induced B bind likely represents a fish IgM class polymerized with four IgG molecules. 