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呼吸爆发过程可以产生大量的活性氧,核因子E2相关因子2(Nrf2)在此过程中起着重要作用。本文克隆和分析了草鱼的Nrf2基因,随后制备了Nrf2蛋白的多克隆抗体,研究了其和呼吸爆发的关系。草鱼Nrf2基因cDNA长度为1994 bp,开放阅读框为1782 bp,编码593个氨基酸(aa)。氨基酸序列比对发现,草鱼Nrf2基因与鲤鱼的同源性最高,为87%;草鱼Nrf2基因含有6个进化过程中保守的Neh(Nrf2-Epoxy chloropropane(ECH)homology)区。实时定量PCR(qRT-PCR)和蛋白印迹法(WB)检测结果表明,Nrf2基因在检测的草鱼8个组织中均有表达。Nrf2激活剂叔丁基对苯二酚(t BHQ)处理草鱼肾细胞(CIK)后,CIK细胞总抗氧化力显著上调,产生的活性氧下调,Nrf2及其下游的HO-1和GST基因的mRNA表达上调。WB和免疫荧光(IF)检测结果表明,tBHQ处理CIK后,Nrf2的蛋白表达也上调,并伴随着入细胞核现象。综上结果表明,保守的草鱼Nrf2基因在机体中广泛表达,能通过上调自身及其下游抗氧化基因的表达下调细胞活性氧的产生,从而参与调控呼吸爆发过程。
Respiratory burst process can produce a large number of reactive oxygen species, nuclear factor E2-related factor 2 (Nrf2) plays an important role in this process. In this paper, the Nrf2 gene of grass carp was cloned and analyzed. Subsequently, a polyclonal antibody against Nrf2 protein was prepared and its relationship with respiratory burst was studied. Grass carp Nrf2 gene cDNA length of 1994 bp, open reading frame of 1782 bp, encoding 593 amino acids (aa). The amino acid sequence alignment showed that Nrf2 gene of grass carp had the highest homology with that of common carp, which was 87%. Nrf2 gene of grass carp contained 6 Neh (Nrf2-Epoxy chloropropane (ECH) homology) regions which were conserved during evolution. Real-time quantitative PCR (qRT-PCR) and western blot (WB) results showed that Nrf2 gene was expressed in 8 tissues of grass carp. The total antioxidant capacity of CIK cells was significantly up-regulated after Nrf2 activator t-butyl hydroquinone (t BHQ) treatment, and the production of reactive oxygen species was down-regulated. The levels of Nrf2 and its downstream HO-1 and GST genes Upregulation of mRNA expression. WB and immunofluorescence (IF) test results showed that, after tBHQ treatment of CIK, Nrf2 protein expression is also up-regulated, accompanied by into the nucleus. In summary, the conserved grass carp Nrf2 gene is widely expressed in the body and can regulate the process of respiratory burst by up-regulating the expression of antioxidant genes and down-regulating the production of reactive oxygen species.