Cloning and Expression of a cDNA Coding for Catalase from Zebrafish (Danio rerio)
Cloning and Expression of a cDNA Coding for Catalase from Zebrafish (Danio rerio)
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DOI：
10.1021/jf990838
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发表时间：
2000-06-01
影响因子：
6.1
通讯作者：
Shaw, JF
中科院分区：
文献类型：
--
作者：
Ken, CF;Lin, CT;Shaw, JF
采用cDNA末端快速扩增-聚合酶链反应（RACE-PCR）技术，从斑马鱼（Danio rerio）mRNA中扩增出编码过氧化氢酶的全长cDNA。核苷酸序列分析表明，该cDNA克隆包含一个完整的开放阅读框架，编码526个氨基酸残基，分子量为59 654 Da，推导的氨基酸序列与猪过氧化氢酶序列高度相似（86.9%）、小鼠（85.8%）、大鼠（85%）、人（83.7%）、果蝇（75.6%）、线虫（71.1%）和酵母（58.6%）。二级结构的氨基酸残基明显保守，因为它们存在于其他哺乳动物物种中。将斑马鱼过氧化氢酶基因导入原核表达载体pET-20 b（+）中，转化大肠杆菌BL 21（DE 3）pLysS，获得表达载体pET-20 b（+）。表达了一个60 kDa的活性过氧化氢酶蛋白，并通过考马斯亮蓝染色以及聚丙烯酰胺凝胶电泳活性染色进行检测。
A full-length complementary DNA (cDNA) clone encoding a catalase was amplified by the rapid amplication of cDNA ends-polymerase chain reaction (RACE-PCR) technique from zebrafish (Danio rerio) mRNA. Nucleotide sequence analysis of this cDNA clone revealed that it comprised a complete open reading frame coding for 526 amino acid residues and that it had a molecular mass of 59 654 Da. The deduced amino acid sequences showed high similarity with the sequences of catalase from swine (86.9%), mouse (85.8%), rat (85%), human (83.7%), fruit fly (75.6%), nematode (71.1%), and yeast (58.6%). The amino acid residues for secondary structures are apparently conserved as they are present in other mammal species. Furthermore, the coding region of zebrafish catalase was introduced into an expression vector, pET-20b(+), and transformed into Escherichia coli expression host BL21(DE3)pLysS. A 60-kDa active catalase protein was expressed and detected by Coomassie blue staining as well as activity staining on polyacrylamide gel followed electrophoresis.