| 摘要: |
| 采用SDS 聚丙烯酰胺凝胶电泳方法, 对锯缘青蟹肌肉中可溶性蛋白质进行分离, 以过敏患者阳性血清和正常人阴性血清作对照, 通过Western blot 鉴定出分子量为14.3kDa 的蛋白为锯缘青蟹的特异性过敏原。然后用Sephadex G75 凝胶过滤层析和DEAE-52 离子交换层析纯化出该过敏原,再经MALDI-TOF/TOF-MS 检测, 利用Blastz 在线分析。结果表明, 锯缘青蟹特异性过敏原为血蓝蛋白亚基, 经Clustal W 分析该蛋白的多肽片段序列与可口美青蟹、邓杰内斯蟹、加州龙虾、艾氏真蟹的血蓝蛋白亚基序列有很高的同源性, 经Prosite 数据库检索后发现肽段1 和肽段2 有酪蛋白激酶Ⅱ磷酸化位点和蛋白激酶C 磷酸化位点, 是过敏原参与机体反应的重要活性位点。
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| 关键词: 锯缘青蟹过敏原, SDS-PAGE, 蛋白免疫印迹, 层析, 质谱分析 |
| DOI：10.11693/hyhz200901010010 |
| 分类号: |
| 基金项目:浙江省重大科研项目, 2004C12029 号; 浙江省教育厅重点项目, 20051701 号; 浙江省重中之重学科资助项目, XK0613040 号 |
附件 |
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| THE SPECIFIC ALLERGENS IN CRAB SCYLLA SERRATA |
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SONG Yi-Yin1, LIN Lei2, SU Xiu-Rong1, WANG Chun-Lin1, ZHANG Chun-Dan1
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1.Key Laboratory of Applied Technology of Marine Biology, Ministry of Education, Faculty of Life Science and Biotechnology, Ningbo University;2.Affiliated Hospital of Medical School, Ningbo University
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| Abstract: |
| To study the specific allergen in crab Scylla serrata, soluble protein from the muscle of S. serrata was extracted, and then separated by SDS-PAGE. With the positive and negative sera of anaphylactic patients, the special allergen was identified with the Western Blot, showing that the 14.3kDa was the specific allergen. The protein of 14.3kDa was then analyzed by gel filtration in ion exchange chromatography, and the MALDI-TOF/TOF-MS search on the Internet. The results indicate that the protein was hemocyanin subunit. Compared the peptide framents from S. serrata and the hemocyanin subunit from Callinectes sapidus, Cancer magister, Panulirus interruptus and Carcinus aestuarii by Clustal W analysis, the protein of allergen from S. serrata was very similar to the hemocyanin subunit. Furthermore, prosite search showed that the peptide fragments contained casein kinase Ⅱ phosphorylation site and protein kinase C phosphorylation site. Both were the important active sites involved in allergic reaction.
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| Key words: Specific allergen of Scylla serrata, SDS-PAGE, Western Blot, Chromatography, Mass spectrometry |
