Zhang Qingli, Li Fuhua, Wang Bing, Zhang Jiquan, Liu Yichen, Zhou Qian, Xiang Jianhai
Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, 7 Nanhai Road, Qingdao, Shandong 266071, PR China.
Dev Comp Immunol. 2007;31(5):429-40. doi: 10.1016/j.dci.2006.08.005. Epub 2006 Oct 6.
Manganese superoxide dismutase (MnSOD) plays an important role in crustacean immune defense reaction by eliminating oxidative stress. Knowledge on MnSOD at molecular level allows us to understand its regulatory mechanism in crustacean immune system. A novel mitochondrial manganese superoxide dismutase (mMnSOD) was cloned from hepatopancreas of Chinese shrimp Fenneropenaeus chinensis by 3' and 5' rapid amplification of cDNA ends (RACE) PCR. The full-length cDNA consists of 1185bp with a 660bp open reading frame, encoding 220 amino acids. The deduced amino acid sequence contains a putative signal peptide of 20 amino acids. Sequence comparison showed that the mMnSOD of F. chinensis shares 88% and 82% identity with that of giant freshwater prawn Macrobrachium rosenbergii and blue crab Callinectes sapidus, respectively. mMnSOD transcripts were detected in hepatopancreas, hemocytes, lymphoid organ, intestine, ovary, muscle and gill by Northern blotting. RT-PCR analysis indicated that mMnSOD showed different expression profiles in shrimp hemocytes and hepatopancreas after artificial infection with white spot syndrome virus (WSSV). In addition, a fusion protein containing mMnSOD was produced in vitro. LC-ESI-MS analysis showed that two peptide fragments (-GDVNTVISLAPALK- and -NVRPDYVNAIWK-) of the recombinant protein were identical to the corresponding sequence of M. rosenbergii mMnSOD, and the enzyme activity of the refolded recombinant protein was also measured.
锰超氧化物歧化酶（MnSOD）通过消除氧化应激在甲壳类动物免疫防御反应中发挥重要作用。在分子水平上对MnSOD的了解使我们能够理解其在甲壳类动物免疫系统中的调节机制。通过3'和5' cDNA末端快速扩增（RACE）PCR从中国对虾凡纳滨对虾的肝胰腺中克隆出一种新型线粒体锰超氧化物歧化酶（mMnSOD）。全长cDNA由1185bp组成，开放阅读框为660bp，编码220个氨基酸。推导的氨基酸序列包含一个20个氨基酸的假定信号肽。序列比较表明，凡纳滨对虾的mMnSOD与罗氏沼虾和青蟹的mMnSOD分别具有88%和82%的同一性。通过Northern印迹法在肝胰腺、血细胞、淋巴器官、肠道、卵巢、肌肉和鳃中检测到mMnSOD转录本。RT-PCR分析表明，人工感染白斑综合征病毒（WSSV）后，mMnSOD在对虾血细胞和肝胰腺中表现出不同的表达谱。此外，体外产生了一种含有mMnSOD的融合蛋白。LC-ESI-MS分析表明，重组蛋白的两个肽段（-GDVNTVISLAPALK-和-NVRPDYVNAIWK-）与罗氏沼虾mMnSOD的相应序列相同，并且还测定了复性重组蛋白的酶活性。
