草鱼血清IgM蛋白的纯化及抗血清的制备

丁炜东, 曹丽萍, 曹哲明

丁炜东, 曹丽萍, 曹哲明. 草鱼血清IgM蛋白的纯化及抗血清的制备[J]. 水生生物学报, 2010, 34(1): 164-169.
引用本文: 丁炜东, 曹丽萍, 曹哲明. 草鱼血清IgM蛋白的纯化及抗血清的制备[J]. 水生生物学报, 2010, 34(1): 164-169.
DING Wei-Dong, CAO Li-Ping, CAO Zhe-Ming. PURIFICATION OF SERUM IGM FROM GRASS CARP (CIENOYHARYNGODONI DELLUS) AND PREPARATION OF RABBIT SERA ANTI-IGM[J]. ACTA HYDROBIOLOGICA SINICA, 2010, 34(1): 164-169.
Citation: DING Wei-Dong, CAO Li-Ping, CAO Zhe-Ming. PURIFICATION OF SERUM IGM FROM GRASS CARP (CIENOYHARYNGODONI DELLUS) AND PREPARATION OF RABBIT SERA ANTI-IGM[J]. ACTA HYDROBIOLOGICA SINICA, 2010, 34(1): 164-169.

草鱼血清IgM蛋白的纯化及抗血清的制备

基金项目: 

国家自然科学基金项目(30471343)

中央级公益性科研院所基本科研业务费专项资金(中国水产科学研究院淡水渔业研究中心)项目(2007JBFC07,2009JBFC01)

中国水产科学研究院热带亚热带鱼类选育与养殖重点开放实验室开放课题(2008-Y-004)资助

PURIFICATION OF SERUM IGM FROM GRASS CARP (CIENOYHARYNGODONI DELLUS) AND PREPARATION OF RABBIT SERA ANTI-IGM

  • 摘要: 采用盐析法、重组蛋白A(HiTrapr Protein A Sepharose)亲和层析法分离纯化草鱼血清中的IgM,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并制备兔抗IgM抗血清。结果表明:33%硫酸铵溶液可以沉淀血清中大部分蛋白,但电泳条带仍较多,其中含有78kD和28kD的条带,因此仅可作为免疫球蛋白粗提的方法;而rProtein A亲和层析法所提蛋白则仅有上述重链(78kD)和轻链(28kD)。Western-blot显示,鼠抗人Ig抗体可与78kD及28kD条带发生发应。rProtein A亲和法提纯蛋白的纯度较高,但含量较低,条带较淡,仅可作为实验室小量提纯草鱼IgM的有效方法。将提纯的蛋白免疫实验兔后可制得效价高达1:25600的兔抗鱼IgM血清,并测得血清蛋白总量和IgM含量分别为25.87mg和4.5mg,IgM占血清蛋白总量的17.39%。本实验所采用的蛋白A亲和层析法提取草鱼血清IgM可以方便、快捷地获得高纯度的产物,适合在实验室中纯化鱼类IgM。同时本研究所制备的兔抗草鱼IgM血清也为今后的相关研究工作打下基础。
    Abstract: Immunoglobulins are the primary humoral component of the acquired immune system. In order to know the ImmunoglobulinM (IgM) of grass carp (Cienoyharyngodoni dellus), two different isolating methods were applied to find the best way to purify serum IgM in the present study. The purity and molecular weight of the serum IgM were determined, and then the rabbit polyclonal antisera were prepared with the purified IgM. In this study, blood was collected with a syringe from the caudal sinus of 30 fish, and allowed to clot at 20 for 20 ℃ min and at 4℃ for 30 min. Serum was obtained by centrifugation at 500 g to remove cells and at 15000 g to remove particulate matter, and stored frozen at ?80℃. Firstly, 33% ammonium sulfate precipitation was used to purify the serum, the result showed that most proteins in grass carp were precipitated and could not get high purity IgM, so it could only be a crude method. At the same time, HiTrap rProteinA Sepharose affinity was performed at 10℃ to purify IgM. The partial characteristics of purified proteins were then analyzed and compared by SDS-PAGE and Western-blot. The results revealed that grass carp serum immunoglobulin purified by HiTrap rProtein A affinity chromatography had only two bands of 78 kD and 28 kD, the same two bands as those in serum. Western-blot analysis showed that the mouse anti-human Ig antibody can recognize bands of 78 kD and 28 kD. Sera anti-IgM of grass carp had been prepared by repeatedly immunized New Zealand rabbits with purified IgM. and the titers of anti-sera obtained up to 1:25600 examined by indirect ELISA. Total serum protein and the concentration of IgM of the normal sera of grass carp were determined by using the Coomassie blue dye binding method of Bradford and sandwich ELISA, the values being 25.87 mg/mL and 4.5 mg/mL, respectively. The percentage of IgM in the total serum protein was 17.39%. Measurements of IgM levels in serum from several fish species have been surveyed and the results point to a range between 0.25 and 23.5 mg/mL. The studies also pointed to considerable individual variations in serum IgM levels among fish, as occurs with other fish immune activities. These changes may be related to size and/or age, environmental conditions or disease status. It must be emphasized, therefore, that the serum IgM values obtained in the present work represent the mean of at least thirty healthy and non-immunized individuals of similar size kept under same conditions. In present work, the 13mg amount of purified IgM from 10 mL serum was not larger than other fish because the grass carp was not immunized with antigen such as BSA in the research. In conclude, the results indicated that protein A-sepharose affinity chromatography was feasible in purification of IgM of grass crap. The polyclonal antibody obtained could be used in correlative studies in future.
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出版历程
  • 收稿日期:  2008-04-15
  • 修回日期:  2009-01-31
  • 发布日期:  2010-01-24

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