【作者】 王丹；

【导师】 赵春晖；

【作者基本信息】 辽宁师范大学 ， 细胞生物学， 2015， 硕士

【摘要】 七鳃鳗,现存最古老的无颌类脊椎动物之一,因其独特的进化地位和免疫系统获得了关注,成为研究脊椎动物先天免疫系统、抗氧化系统起源及进化的新型模式生物,因此七鳃鳗的先天免疫防御系统中必然存在一系列与解毒及参与水生动物氧化抗菌反应的相关蛋白。本论文首次从日本七鳃鳗(Lampetra japonica)白细胞中获得了乙醛脱氢酶9(aldehyde dehydrogenase 9,ALDH9)和谷胱甘肽过氧化物酶4(glutathione peroxidase4,GPx4)的同源基因,通过基因克隆、重组表达及生物信息学方法对基因进行分析预测,为更好的了解其活性奠定了基础。乙醛脱氢酶(ALDHs),能够将不同醛类氧化为相应的酸类,在内源性和外源性醛解毒过程中扮演了重要的角色。目前为止ALDH在哺乳动物及微生物中已有所研究,但其在鱼类及两栖类动物中研究较少。本论文首次克隆并原核表达了来自于日本七鳃鳗的ALDH9(Lj ALDH9)基因。LjALDH9 ORF区共1566 bp,编码521个氨基酸,理论分子量为55.68 kDa。LjALDH9蛋白含有信号肽区域(1-29)、Cys315活性位点及Aldedh结构域。除去信号肽区域其理论分子量为52.92 kDa,等电点为5.57。重组LjALDH9以包涵体形式存在,通过变性、复性及纯化对酶活性进行了测定,结果显示LjALDH9酶的最适反应pH为7.0,最适温度16-23°C,Mn2+为其激活剂。实时定量PCR表明LjALDH9在口腔腺中呈现高表达,证明了LjALDH9在七鳃鳗寄生生活阶段发挥着重要作用。谷胱甘肽过氧化物酶(GPx),是一类抗氧化酶,能够减少脂质氢过氧化物、氢过氧化物和有机氢过氧化物,维持组织正常的氧化还原水平,避免DNA、蛋白质和脂质等的氧化损伤。本论文首次克隆得到日本七鳃鳗GPx4基因(LjGPx4)及其基因组DNA序列。LjGPx4 ORF区全长477 bp,编码158个氨基酸,蛋白质分子质量预测为18.06 kDa,理论等电点为7.75。酶的活性位点为硒代半胱氨酸(33 TGA),由终止密码子编码。LjGPx4完整基因组序列共6541bp,包含六个外显子及五个内含子结构。脂多糖(LPS)体内刺激七鳃鳗后发现,LjGPx4 mRNA在白细胞中表达升高,说明LjGPx4参与抗菌免疫应答,为抗氧化剂酶在无颌类脊椎动物的先天免疫反应中发挥至关重要的作用提供了实验依据。更多还原

【Abstract】 Lampreys are one of the oldest class jawless vertebrates, gained attention with its unique evolutionary status and unique immune system, become the new model when studying the origin and evolution of vertebrate innate immune system and antioxidant system. In this paper, we report for the first time the molecular cloning and characterization of aldehyde dehydrogenase 9(ALDH9) and glutathione peroxidase 4(GPx4) from Japanese lamprey(Lampetra japonica) leukocytes and we analyze the genes by using gene cloning, recombination and bioinformatics methods, the researches laying the groundwork for better understanding protein properties and activity.Aldehyde dehydrogenases(ALDHs), which oxidize aldehyde to corresponding acids, play a major role in the detoxification of various endogenous and exogenous aldehydes. To date, ALDH has been studied in mammals and microorganisms, but it has not been studied in fish and amphibians. In this study, we report for the first time the molecular cloning and prokaryotic expression of aldehyde dehydrogenase 9(LjALDH9) from Lampetra japonica. The open reading frame of LjALDH9 was 1566 bp, encoding 521 amino acids with a predicted molecular mass of 55.68 kDa. LjALDH9 protein had a signal peptide(1-29) and Aldedh domain with the active site Cys315. The predicted molecular mass of LjALDH9 without the signal peptide was 52.92 kDa and the theoretical isoelectric point was 5.57. The recombinant protein was existing in the form of inclusion bodies, it was successfully assayed enzyme activity through denaturing, refolding and purification, the result showed that the most suitable reaction conditions were pH 7.0, 16-23°C and Mn2+ as the activator. Real-time quantitative PCR revealed that LjALDH9 was highly expressed in the buccal gland, it provides theoretical proof that LjALDH9 plays an important role in the parasitic life phase of lamprey.The antioxidant enzyme, glutathione peroxidase(GPx), is capable of reducing complex lipid hydroperoxides, hydrogen peroxide and organic hydroperoxides which can maintain normal ROX level to avoid the oxidative damage of DNA, protein and lipid. In this study, we report for the first time the molecular cloning of glutathione peroxidase 4(LjGPx4) genomic DNA sequences from Lampetra japonica. The open reading frame of LjGPx4 was 477 bp, encoding 158 amino acids with a predicted molecular mass of 18.06 kDa and the theoretical isoelectric point was 7.75. The active site of the enzyme contains a selenocysteine encoded by a TGA termination codon at position 33. The complete genomic sequence was 6541 bp and revealed a six exon-five intron structure. Furthermore, upon in vivo stimulation of lampreys with lipopolysaccharide(LPS), LjGPx4 mRNA was upregulated in leukocytes, showed its involvement in the antibacterial immune response of lamprey, provide evidence for the evolutionary existence of this antioxidant enzyme playing a vital role in innate immune responses in jawless vertebrates.更多还原