学术
黄鳍棘鲷血清IgM的纯化及兔抗血清的制备
刘振兴[1,2] 张殿昌[1] 苏天凤[1] 姜巨峰[1] 龚世园[2] 江世贵[1]
[1]中国水产科学研究院南海水产研究所,广东广州510300 [2]华中农业大学水产学院,湖北武汉430070
摘 要:
分别采用Sepharose-6B凝胶过滤层析、Phenyl Sepharose FF疏水作用层析和rProtein A Sepharose亲和层析技术纯化黄鳍棘鲷（Acanthopagrus latus）血清IgM，并将这3种层析纯化方法进行了比较。纯化产物进行SDS—PAGE，电泳结果扫描后经Band Scan5．0软件分析显示，单独使用Sepharose-6B凝胶过滤层析或Phenyl Sepharose FF疏水作用层析得到的IgM纯度只有56％左右；二者联合使用纯度可以达到69％，而rProtein A Sepharose亲和层析一步就可以达到89％的纯度；纯化得到的黄鳍棘鲷血清IgM的重链和轻链分子量分别为73．6kD和26．5kD。本研究还以rProtein A Sepharose亲和层析纯化的黄鳍棘鲷血清IgM为抗原，制备其兔抗血清，采用Western Blot和双向琼脂扩散检测抗血清免疫学活性，并用间接ELISA检测其效价达到1：25600，为进一步开展黄鳍棘鲷免疫学方面的研究奠定了基础。[著者文摘]
文章出处:
《中国水产科学》-2008年1期 -129-135页
栏目信息:
分 类 号:
文献标识码:
A
文章编号:
1005-8737-（2008）01-0129-07
Purification of serum IgM in Acanthopagrus latus and preparation of rabbit sera anti-IgM
LIU Zhen-xing, ZHANG Dian-chang, SU Tian-feng, GONG Shi-yuan, JIANG Shi-gui （1. South China Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Guangzhou 510300, China; 2.Huazhong Agricultural University, Wuhan 430070, China）
Abstract:
The serum IgM of Acanthopagrus latus was purified by using Sepharose-6B gel filtration chromatography, Phenyl Sepharose FF hydrophobic interaction chromatography and rProtein A Sepharose affinity chromatography. The purified IgM obtained through the three methods was then compared. By the methods of Sepharose-6B gel filtration chromatography and Phenyl Sepharose FF hydrophobic interaction chromatography the IgM was obtained at the purity of only 56%. While through combination of Sepharose-6B gel filtration chromatography and Phenyl Sepharose FF hydrophobic interaction chromatography, we obtained a bit higher purity at about 69%. The purity reached approximately 89% when using rProtein A Sepharose affinity chromatography. However, the rProtein A Sepharose affinity chromatography suffered relatively high cost. Sepharose-6B gel filtration chromatography and Phenyl Sepharose FF hydrophobic interaction chromatography could be good choices for the purification of the IgM in terms of cost and performance. It was revealed that molecular weights of the heavy- and light- chains oflgM were 73.6 and 26.5kD, respectively. The IgM purified by rProtein A Sepharose affinity, chromatography was used to prepare rabbit anti-IgM sera. The antibody activity of anti-sera was tested by Western Blot and double agar diffusion. Indirect ELISA revealed that the anti-serum titer reached 1 ： 25 600. It is considered that the purified IgM and developed anti-sera provide a foundation for further studies in relation to fish immune response. [Journal of Fishery Sciences of China, 2008, 15 （1） ： 129-135 ][著者文摘]
Key words:
Acanthopagrus latus; immunoglobulin; purification; rabbit sera anti-IgM; ELISA
基金资助:
国家科技基础平台项目资助（2005DKA30470）.
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