| 鳜皮肤黏液IgM样蛋白的纯化 |
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| 引用本文： | 罗 霞,吴淑勤.鳜皮肤黏液IgM样蛋白的纯化[J].水产学报,2007,31(6):726-730. |
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| 作者姓名： | 罗 霞 吴淑勤 |
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| 作者单位： | 1. 中国水产科学研究院珠江水产研究所,广东,广州,510380;广东海洋大学水产学院,广东,湛江,524088
2. 中国水产科学研究院珠江水产研究所,广东,广州,510380 |
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| 基金项目： | 国家科技支撑计划;广东省科技厅科技计划;广东省自然科学基金 |
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| 摘 要： | 对经嗜水气单胞菌全菌疫苗浸泡免疫后的鳜皮肤黏液中的免疫球蛋白采用盐析法、重组蛋白A(HiTrap rProteinA Sepharose)亲和层析法及鼠抗鳜血清IgM单克隆抗体偶联的Sepharose 4B亲和层析法分离纯化,并通过SDS-PAGE及Western-blot技术对纯化蛋白的部分特性进行分析比较。结果表明:50%硫酸铵溶液可以沉淀黏液中大部分蛋白,条带仍较多,约十几条,其中含有72 ku和29 ku的条带,因此仅可作为免疫球蛋白粗提的方法;Sepharose 4B亲和层析法所提取鳜黏液Ig经SDS-PAGE检测,只含有72 ku和29 ku 2个条带(初步认为鳜黏液Ig的重链和轻链),与鳜血清IgM的重、轻链分子量相同;rProteinA亲和层析法所提蛋白除具有上述重链(72 ku)和轻链(29 ku)外,还含有43 ku的蛋白带(可能为鳜黏液Ig另外一种形式的重链)。Western-blot显示,兔抗鳜Ig多克隆抗体可与72 ku及43 ku条带发生发应。两种亲和法所提蛋白纯度较高,但含量较低,条带较淡,仅可作为实验室小量提纯鳜黏液Ig的有效方法。
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| 关 键 词： | 鳜 皮肤黏液免疫球蛋白 盐析 亲和层析 |
| 文章编号： | 1000-0615（2007）06-0726-05 |
| 收稿时间： | 7/3/2007 12:00:00 AM |
| 修稿时间： | 7/3/2007 12:00:00 AM |
Purification of skin mucus IgM-like proteins in Siniperca chuatsi |
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| LUOXia and.Purification of skin mucus IgM-like proteins in Siniperca chuatsi[J].Journal of Fisheries of China,2007,31(6):726-730. |
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| Authors: | LUOXia and |
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| Institution: | 1. Pearl River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Gnangzhou 510380, China ; 2. Fisheries College, Gnangdong Ocean University, Zhanjiang 524088, China |
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| Abstract: | Skin mucus immunoglobulin was purified by the methods of ammonium sulfate precipitation, HiTrap rProteinA Sepharose affinity chromatography and Sepharose 4B linked with mouse monoclonal antibody against serum IgM affinity chromatography from Siniperca chuatsi immersed with inactivated Aeromonas hydrophila(Ah)strain GYK1.And partial characteristics of purified proteins were analyzed and compared by SDS-PAGE and Western-blot.The results revealed that most proteins in mucus were precipitated by 50% ammonium sulfate solution,so it can only be a crude method.Determined by SDS- PAGE,Siniperca chuatsi skin mucus immunoglobulin purified by Sepharose 4B affinity chromatography has only two bands of 72 ku and 29 ku,the same two bands as those in serum.Besides the heavy chain(72 ku) and the light chain(29 ku)mentioned above,the immunoglobulin purified by HiTrap rProteinA,has a 43 ku protein band,which may be another kind of heavy chain.Western-blot analysis showed that the rabbit polyclonal antibody can recognize bands of 72 ku and 43 ku.The experiment also proved that both the methods of HiTrap rProtein A and Sepharose 4B affinity chromatography were effective to purify proteins in small quantity,whereas the contents were relatively low. |
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| Keywords: | Siniperca chuatsi skin mucus immunoglobulin ammonium sulfate precipitation affinity chromatography |
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