斑点叉尾(鱼回)血清免疫球蛋白纯化及其结构分析

伊光辉; 林天龙; 熊邦喜; 刘晓东; 杨金先

斑点叉尾(鱼回)血清免疫球蛋白纯化及其结构分析

1.
福建省农业科学院畜牧兽医研究所, 福建, 福州, 350013;
2.
华中农业大学水产学院, 湖北, 武汉, 430070

基金项目:

国家863课题(2003AA622050)

详细信息

通讯作者:
林天龙(1955-),男,研究员。主要从事鱼类免疫学、病原学研究(E-mai:lint@,pub3.fz.fj.cn).

中图分类号: Q7
计量
- 文章访问数: 2080
- HTML全文浏览量: 69
- PDF下载量: 499
- 被引次数: 0
出版历程
- 收稿日期: 2003-10-15
- 修回日期: 2004-06-30
- 刊出日期: 2005-06-15

Purification and preliminary structure analysis of serum immunoglobulin in channel catfish (Ictalurus punctatus)

1.
Animal Husbandry and Veterinary Medicine Institute, Fujian Academy of Agricultural Sciences, Fuzhou, Fujian, 350013, China;
2.
Fisheries College, Huazhong Agricultural University, Wuhan, Hubei 430070, China

摘要

摘要: 通过饱和硫酸铵分步盐析结合柱层析纯化温和气单胞菌免疫的斑点叉尾(鱼回)血清免疫球蛋白(Ig),结果表明,斑点叉尾(鱼回)血清Ig主要分布在35%-55%的硫酸铵沉淀区间;而在Sepharose-4B凝胶柱和DEAE-52阴离子交换柱层析纯化的Ig均出现在第1个蛋白质峰。SDS-PAGE分析表明,纯化后的血清Ig纯度提高。通过变性还原、变性非还原和非变性非还原3种条件下的免疫印迹(Western blot)试验对血清Ig的结构进行初步分析,发现SDS变性还原条件下血清Ig重链分子量为72 kD,轻链有3条.其分子量分别为21 kD、23.5 kD和26 kD;在变性非还原条件下血清Ig则出现多种结构形式,主要条带的分子量分别为760 kD、525 kD、330 kD和230 kD;而在非变性非还原条件下Ig仅出现一种结构形式,分子量约为870 kD。
- 斑点叉尾(鱼回) /
- 血清免疫球蛋白 /
- 纯化 /
- 结构分析
Abstract: Serum immunoglobulin (Ig) from channel catfish (Ictalurus punctatus) immunized with Aeromonas so-bria was purified with ammonium sulfate precipitation followed by column chromatography.The results showed that serum Ig was mainly precipitated at 35%-55% ammonium sulfate saturation.Further purification by Sepharose-4B gel filtration and DEAE-52 anion exchange chromatography showed that the Ig from both of the columns all presented in the first protein peak.SDS-PAGE demonstrated that serum Ig was more pure after each step of purification.Structure analysis of the catfish serum Ig was 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 molecular weight of the heavy chain was 72 kD, and the three distinct light chains were 21 kD, 23.5 kD, 26 kD respectively.The results suggested the heterogeneity were presented in channel catfish light chains.The serum Ig only had a molecular weight about 870 kD under non-reduced and non-denatured conditions.
- Ictalurus punctatus /
- Immunoglobulin /
- Purification /
- Structure analysis

HTML全文

参考文献(6)

[1]	Amemjya C T, Litmen G W. Complete nucleotide sequence of an immunoglobulin heavy-chain gene and analysis of immunogobulin gene organization in a primative teleost species [J]. Proc Natl Acad Sci USA, 1990, 87: 811-815.
[2]	Havarstein L S, Assjord P M, Ness S, et al. Purification and partial characterization of an IgM-like serum immunoglobulin from Atlantic salmon (Salmo salar) [J]. Dev Comp Immunol,1988, 12: 773-785.
[3]	Harlow, David L. Antibodies-A laboratory manual [M]. Cold Spring Harbor Laboratory press, 1988.
[4]	Sanchez C, Dominguez J. Trout immunoglobulin populations differing in light chains revealed by monoclonal antibodies [J].Molecular Immunology, 1991, 28 (11): 1271-1277.
[5]	张永安,聂品.鳜血清免疫球蛋白的分离纯化及其亚单位分子量的测定[J].水生生物学报,1998,22(2):192-194.
[6]	Bourmand C A F, Romestand B, Bouix G. Isolation and partial characterization of IgM-like seabass (Dicentrarchus labrax L.1758) immunoglobulins [J]. Aquaculture, 1995, 132: 53-58.