摘要
采用饱和硫酸铵分步盐析结合Sephacryl S-300凝胶过滤层析及DEAE Sepharose离子层析法,对牙鲆皮肤黏液中的免疫球蛋白(Ig)进行了分离纯化,并通过SDS-PAGE及Western-blotting技术对纯化蛋白的部分特性进行了分析。结果表明,30%、50%饱和硫酸铵分步盐析可以去除牙鲆黏液中除Ig外的很多杂蛋白,得到粗提黏液Ig;再经Sephacryl S-300纯化,Ig纯度较高,其中含有72和26 ku的条带;DEAE Sepharose层析法可进一步纯化Sephacryl S-300柱层析的产物,所提取黏液Ig经SDS-PAGE检测,只含有72和26 ku两个条带,初步认为是牙鲆黏液Ig的重链和轻链。Western-blotting结果显示,抗牙鲆血清Ig单克隆抗体可与黏液Ig重链72 ku条带发生发应。
Teleost fish have a unique physical barrier composed of mucus and skin which act as the first line of defence.It has been shown that lymphocytes are present in the epidermis of fish,and the cutaneous mucus of several fish species contains immunoglobulin（Ig）.As fish mucosal composition was complicated and the content of Ig was low,it is difficult to obtain high purity mucus Ig.In this study,skin mucus Ig of Japanese flounder（Paralichthys olivaceus）was purified by the combination of saturated ammonium sulfate precipitation,Sephacryl S-300 gel filtration chromatography and DEAE Sepharose chromatography,and partial characteristics of purified proteins were analyzed by SDS-PAGE and Western-blot.According to SDS-PAGE,many other proteins were removed from mucus by precipitation of 30% and 50% saturated ammonium sulfate solution,and Ig that was crudely extracted could reach a high purity by further purification using Sephacryl S-300.After being finally purified by DEAE Sepharose chromatography,skin mucus Ig has only two bands of 72 and 26 ku,initially presumed as the heavy chain and light chain of Japanese flounder mucus Ig.Western-blot analysis showed that the monoclonal antibody produced from serum Ig could recognize the heavy chain of mucus Ig（72 ku）.
出处
《水产学报》
CAS
CSCD
北大核心
2011年第4期614-619,共6页
Journal of Fisheries of China
基金
国家自然科学基金项目(30771648,31072232)
关键词
牙鲆
肤黏液免疫球蛋白
盐析
凝胶过滤层析
离子交换层析
Paralichthys olivaceus
mucus immunoglobulin
ammonium sulfate precipitation
gel filtration chromatography
DEAE Sepharose chromatography
