| 摘要: |
| 采用 RT-PCR 技术克隆南极衣藻 GR 基因 ORF 全长 cDNA, 然后经酶切、连接等步骤构建其原核表达载体; 并对其表达的诱导时间、IPTG 浓度、温度进行了优化, 以期获得较大量的酶蛋白。结果表明, 将构建的原核表达载体 pET-GR 导入大肠杆菌 BL21, 可以高效表达融合蛋白; 且表达的蛋白均以包涵体的形式存在; 经 SDS-PAGE电泳结果显示, 获得的目的蛋白分子量为 52.2kDa, 符合预期分子量; GR 蛋白在大肠杆菌中诱导表达优化条件为, 1.0mmol/L 的 IPTG, 28℃诱导 3h。 这些结果为进一步深入研究该基因的特性与功能奠定了基础。 |
| 关键词: 南极衣藻Chlamydomonas sp. ICE-L, 谷胱甘肽还原酶, 原核表达, 诱导条件, 冰藻 |
| DOI：10.11693/hyhz201106011011 |
| 分类号: |
| 基金项目:国家自然科学基金资助项目, 40876102 号, 40876107 号 |
附件 |
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| PROKARYOTIC EXPRESSION AND ITS CONDITIONAL OPTIMIZATION OF GLUTATHIONE REDUCTASE GENE OF ANTARCTIC CHLAMYDOMONAS SP. ICE-L |
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LIU Ying1,2, DING Yu1,2, JIAN Ji-Chang1,2, WU Zao-He1,2, MIAO Jin-Lai3
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1.Guangdong Provincial Key Lab of Pathogenic Biology and Epidemiology for Aquatic Economic Animals;2.Fisheries College, Guangdong Ocean University;3.Key Lab of Marine Bioactive Substances, First Institute of Oceanography, State Oceanic Administration
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| Abstract: |
| Glutathione reductase (GR) is an important antioxidative enzyme in organisms. In order to construct prokaryotic expression vector and to study the recombinant gene expression in Escherichia coli, complete gene of Chlamydomonas sp. ICE-L GR ORF was cloned by the RT-PCR method followed by restriction enzymes cutting, connection. The induced time, IPTG concentration and temperature of prokaryotic expression were optimized using SDS-PAGE. The results showed that a recombinant of prokaryotic expression vector pET-GR was constructed successfully, and ICE GR protein in inclusion body can express effectively after the expression vector pET-GR was transformed to E. coli BL21. The SDS-PAGE results indicated that the molecular weight (52.2kDa) of the target protein was similar to the theoretic molecular weight. The optimal expression condition was set as 1.0mmol/L IPTG, induced temperature 28℃, and induced time 3 hours. These results are expected to lay a foundation for further studies on the properties and function of this gene. |
| Key words: Antarctic Chlamydomonas sp. ICE-L, Glutathione reductase (GR), Prokaryotic expression, Induced condition, Ice algae |
