翘嘴鳜NADPH氧化酶2个催化亚基cDNA的克隆和表达特征

cDNA Cloning and Expression Characterization of Two Catalytic Subunits of NADPH Oxidase from Mandarin Fish Siniperca chuatsi

吞噬细胞NADPH氧化酶能生成用于清除病原微生物的活性氧(reactive oxygen species,ROS),在机体的防御体系中起着非常重要的作用.本文利用RT-PCR结合RACE-PCR的方法,克隆到翘嘴鳜NADPH氧化酶的催化亚基gp91phox和p22phox的cDNA全长.并研究两者在正常的翘嘴鳜和注射了柱状黄杆菌灭活菌苗(FKG4)的翘嘴鳜组织中的表达模式.结果表明,gp91phox基因cDNA序列全长2 037 nt,开放阅读框长度为1 698 nt,翻译成565个氨基酸;p22phox基因cDNA序列全长1 296 nt,开放阅读框561 nt,翻译成186个氨基酸.将这2个亚基推导的氨基酸序列与人的对应亚基相比,相似性分别为68.7%和60.8%,且具有相似的结构域和功能域,说明翘嘴鳜与人的NADPH氧化酶具有相似的功能活性.半定量PCR分析显示,在翘嘴鳜血液、脑、心脏、肾、肝、脾、胸腺等11种组织中均能检测到gp91phox和p22phox的基因表达.经FKG4免疫后,gp91phox在翘嘴鳜血液、头肾和脾3种组织中的表达量显著上升,p22phox在头肾和脾2种组织中的表达量显著上升.由此推断,NADPH氧化酶可能参与了机体的抗菌免疫应答.

The phagocyte NADPH oxidase plays a crucial role in host defense against invading microorganisms by catalyzing the formation of reactive oxygen species,which is the precursor of a variety of microbicidal oxidants such as hydrogen peroxide （H_2O_2）.In the present study,full-length cDNAs of two catalytic subunits of NADPH oxidase,including gp91phox,p22phox,were cloned from head kidney of mandarin fish utilizing the reverse transcription-PCR and rapid amplification of cDNA ends.Sequence analysis showed that the clones of mandarin fish gp91phox and p22phox contained open reading frames of 1698 bp,561 bp encoding for proteins of 565 and 186 amino acids,respectively.Comparison of the deduced amino acid sequences showed that mandarin gp91phox and p22phox sequences shared 68.7% and 60.8% identity with those of human components,respectively.The functional domains were highly conserved in mandarin fish NADPH oxidase subunits,although their sequences showed low homology with those of mammals.These results suggest that mandarin fish NADPH oxidase components possess functional activities similar to those of human.Semi-quantitative RT-PCR analyses showed that gp91phox and p22phox genes were expressed in various tissues of unstimulated fish.Stimulating the fish with formalin killed Flavobacterium columnare G4 significantly up-regulated the expression of mandarin gp91phox in head kidney,spleen,blood, and p22phox in head kidney and spleen,which indicated that mandarin NADPH oxidase was involved in the immune responses against bacteria.