翘嘴鳜髓过氧化物酶基因克隆及重链序列的原核表达

Cloning and prokaryotic expression of the myeloperoxidase gene in mandarin fish,Siniperca chuatsi

利用RT-PCR和RACE-PCR方法，从翘嘴鳜中克隆出髓过氧化物酶（myeloperoxidase，MPO）cDNA全长。其开放阅读框有2292个核苷酸，编码763个氨基酸的蛋白。虽然序列相似性分析发现该蛋白与哺乳动物MPO相似性为64％-65％，但分子质量大小相近。翘嘴鳜含有1个与人MPO相似的血红素基，1个保守的Ca^2＋结合位点，6个分子内二硫键，以及6个糖基化位点。由此推测翘嘴鳜MPO蛋白的空间结构及催化机制可能与哺乳动物的相似。将翘嘴鳜MPO的部分cDNA序列连接到pEF-32a-c（＋）载体上，成功构建了1个重组表达质粒pET-MPO，该质粒在大肠杆菌（DE3）中获得了高效表达。另外，利用重组蛋白N末端带有的6×His标签，通过Ni2-NTA柱纯化获得了翘嘴鳜MPO重组蛋白。

Myeloperoxidase （MPO） is widely distributed in phagocytes of vertebrates and mainly functions in catalyzing chloride and hydrogen peroxide to produce hypochlorous acid, a potent antimicrobial agent in polymorphonuclear neutrophils. Currently, little is known about MPO of fish and other lower vertebrates. In the present paper, the full-length cDNA of MPO was cloned from mandarin fish by utilizing reverse transcriptase-polymerase chain reaction and rapid amphfication of cDNA ends. The results showed that the full-length of mandarin MPO cDNA was 3 345 nucleotides （nt）, with an open reading frame of 2292 nt encoding 763 amino acids. Although amino acid sequence comparison of MPO of mandarin with those of mammals demonstrated that their similarity （64% - 65 % ） was not very high, their molecular weights were approximate the same. Like mammals, the MPO of mandarin fish also posesses one hemachrome group, one conserved banding calcium site, six intra-molecule disulfide bridges and six N-glycosylation sites, implying that it may function the same as its mammalian counterpart. Also, partial cDNA sequence of mandarin MPO was expressed in Escherichia coli rosetta-gami （DE3） using a recombinant expression plasmid pET-MPO, and the combinant protein was purified by the Ni2-NTA resin using its N-terminal His-Tag.