| 摘要: |
| 精氨酸激酶是调节能量代谢的重要酶类, 在无脊椎动物体内能量平衡过程中起重要作用。利用PCR扩增技术, 从白肛海地瓜的cDNA文库中克隆得到精氨酸激酶(arginine kinase)基因, 其全长为2139bp, 包括5′非翻译区(5’-UTR)序列92bp, 3′端非翻译区(3’-UTR)序列934bp和1113bp的ORF, 编码371个氨基酸, 预测分子量大小和等电点分别为42.32kDa和7.19。构建了精氨酸激酶原核表达质粒并转化表达菌株, 经IPTG诱导表达后, 获得了与预期的分子量大小一致的表达产物。利用该目的蛋白制备了白肛海地瓜精氨酸激酶的特异性多克隆抗体, 经Western blot证明该蛋白为精氨酸激酶。 |
| 关键词: 白肛海地瓜, 精氨酸激酶, 原核表达, 多克隆抗体 |
| DOI：10.11693/hyhz201204004004 |
| 分类号: |
| 基金项目:国家星火计划项目, 2010GA701063 号；浙江省重点社会发展项目, 2009C03017-4 号；宁波市科技局国际合作项目, 2008C500265号 |
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| CLONING AND OVEREXPRESSION OF ACAUDINA LEUCOPROCTA ARGININE KINASE |
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ZHOU Jun1, LI Cheng-Hua1, LI Ye1, LIU Yan1, JIN Chun-Hua1, XIA Chang-Ge1, LI Tai-Wu1,2, SU Xiu-Rong1
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1.School of Marine Sciences, Ningbo University;2.Ningbo City College of Vocational Technology
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| Abstract: |
| Arginine kinase is a phosphotransferase that plays a critical role in maintaining equilibrium of invertebrate energy metabolism. We isolated the gene encoding arginine kinase from Acaudina leucoprocta. The full-length of cDNA is 2139bp long, including 92bp of 5′untranslated region (5′-UTR) sequence, 934bp of 3' untranslated region (3′-UTR) sequence and 1113bp of open reading frame. It encodes 319 amino acids, the predicted molecular mass and isoelectric point are 42.32kDa and 7.19, respectively. The resulting product was then cloned into the expression vector and induced with IPTG. The overexpressed protein was with the expected size. Polyclonal antibody was prepared and the specificity of the antibody was confirmed by Western blot analysis. |
| Key words: Acaudina leucoprocta, Arginine kinase, Prokaryotic expression, Polyclonal antibody |
