| 摘要: |
| 利用 PCR 扩增技术, 从泥蚶的 cDNA 文库中克隆出了精氨酸激酶(arginine kinase)基因, 并进行了原核表达。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。 结果表明, 泥蚶的精氨酸激酶全长 1601bp, 包括 5′非翻译区(5′-UTR)序列 332bp, 3′非翻译区(3′-UTR)序列 246bp、1023bp 的开放阅读框序列(ORF), 编码 340 个氨基酸, 具有精氨酸激酶典型的酶活性部位氨基酸序列(CPTNLGT)。构建了原核表达质粒 AK-pET-28a(+)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泥蚶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。
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| 关键词: 泥蚶, 精氨酸激酶, 基因克隆与表达, 多克隆抗体 |
| DOI：10.11693/hyhz201101018018 |
| 分类号: |
| 基金项目:国家自然科学基金资助项目, 30771665 号; 长江学者和创新团队发展计划资助项目, 2007—2009; 浙江省科技厅重点科研社会发展项目, 2005C23083 号 |
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| CLONING AND OVEREXPRESSION OF TEGILLARCA GRANOSA ARGININE KINASE |
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LI Song-Wei1, LI Ye1, LI Cheng-Hua1, LI Tai-Wu1,2, SU Xiu-Rong1
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1.Faculty of Life Science and Biotechnology, Ningbo University;2.Ningbo City College of Vocational Technology
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| Abstract: |
| In vertebrate, arginine kinase is a phosphotransferase that plays a critical role in maintaining equilibrium in energy metabolism. We isolated full-length cDNA of arginine kinase from Tegillarca granosa. It is 1601 bp long, including 203 bp of 5' untranslated region (5'-UTR) sequence and 438 bp of 3' untranslated region (3'-UTR) sequence. It encodes 319 amino acids with arginine kinase activity in part of the amino acid sequence (CPTNLGT). And the resulting product was then cloned into the expression vector. The prokaryotic expression plasmid AK-Pet-28a(+) containing the intact ORF was constructed and the target protein was induced with IPTG. The overexpressed protein was with the expected size. This study provided information further built on the foundation in understanding the function of arginine kinase in energy metabolism and host defense.
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| Key words: Tegillarca granosa, Arginine kinase, Genic cloning and expression, Polyclonal antibody |
