病毒诱导牙鲆上调表达基因的筛选及牙鲆色素上皮衍生因子的特征分析
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摘要
牙鲆(Paralichthys olivaceus)是我国已经形成规模经济效益的几种重要海水养殖鱼类之一。随着养殖集约化程度的提高,近年来频繁遭受病毒性疾病的侵袭并导致巨大经济损失。因此,深入研究牙鲆免疫系统的分子机制将为牙鲆疾病特别是病毒病的预防提供理论依据。
本研究选用紫外灭活的具有双链RNA基因组的草鱼出血病病毒(grass carp hemorrhage virus,GCHV)对牙鲆胚胎细胞(flounder embryonic cells, FEC)进行诱导,运用抑制性差减杂交技术(suppression subtractive hybridization, SSH),成功构建了dsRNA病毒诱导的牙鲆胚胎细胞基因差异表达的差减cDNA文库。从差减cDNA文库中,通过PCR和斑点杂交两轮筛选,共得到有效ESTs为148个。所得序列通过生物信息学分析表明,这些EST代表的相应基因的功能包括细胞分裂、信号传导、细胞结构、细胞防御、蛋白表达和代谢等几个方面。
在上调表达的EST中,有三个为色素上皮衍生因子(pigment epithelium- derived factor, PEDF)的同源基因(PoPEDF),该基因属于丝氨酸蛋白酶抑制剂(serine protease inhibitor, serpin)超家族中无抑制活性的成员之一,编码蛋白具有抗血管增生、神经营养和保护功能。但关于该基因与病毒诱导研究还未见报道。通过RACE-PCR方法,从SMART cDNA文库得到了PoPEDF基因的全长cDNA共1803bp,开放阅读框长1212bp,编码403个氨基酸。序列同源性比较、蛋白结构分析、基因组DNA的外显子与内含子组合及系统进化分析等都表明PoPEDF为哺乳动物PEDF的同源基因。
PoPEDF与GFP共转染细胞及免疫组化结果表明,牙鲆PEDF是一种分泌蛋白,合成后的PEDF主要分布于细胞质而不在细胞核。连续观察发现表达的PEDF逐渐向细胞外分泌。
在哺乳动物中,PEDF是一个多功能蛋白,其功能和调节机制非常复杂。以牙鲆和牙鲆胚胎细胞为材料,用不同的诱导剂诱导,对牙鲆PEDF的功能特性作了初步探讨。结果显示,PEDF对ATRA的诱导变化不明显;低葡萄糖浓度可以诱导PEDF的上调表达,随着葡萄糖浓度的升高,PEDF的表达逐渐降低;缺氧时,PEDF先上调表达,随后再下调表达;随着细胞培养时间的增加,
Japanese flounder (Paralichthys olivaceus) is one of important marine aquaculture fishes, and has been formed a large-scale economic benefit in China. However, the increasing cultivating density causes frequent breakout of virus disease in flounder, which has leaded to tremendous economic losses. In order to provide theoretic basis on defending viral diseases in flounder, it is significant and urgent to further study the molecular mechanism underlying flounder antiviral immune responses.
In this study, a subtractive cDNA library was successfully constructed with mRNAs made from Japanese flounder embryonic cells (FEC) treated with UV-inactivated grass carp hemorrhage virus (GCHV) and control cells using suppression subtractive hybridization (SSH). PCR and dot blotting obtained a total of 148 ESTs through screening of subtractive cDNA library. Sequencing and bioinformative analyses reveal that the genes represented by these ESTs can be divided into seven groups, including cell division, cell signaling/cell communication, cell structure/motility, cell/organism defense, gene/protein expression and metabolism and so on.
Among 148 ESTs, three are homologous to mammalian pigment epithelium-derived factor (PEDF) genes, indicating that they represent a flounder PEDF homolog (named Paralichthys olivaceus PEDF, PoPEDF). PEDF is a
本研究选用紫外灭活的具有双链RNA基因组的草鱼出血病病毒(grass carp hemorrhage virus,GCHV)对牙鲆胚胎细胞(flounder embryonic cells, FEC)进行诱导,运用抑制性差减杂交技术(suppression subtractive hybridization, SSH),成功构建了dsRNA病毒诱导的牙鲆胚胎细胞基因差异表达的差减cDNA文库。从差减cDNA文库中,通过PCR和斑点杂交两轮筛选,共得到有效ESTs为148个。所得序列通过生物信息学分析表明,这些EST代表的相应基因的功能包括细胞分裂、信号传导、细胞结构、细胞防御、蛋白表达和代谢等几个方面。
在上调表达的EST中,有三个为色素上皮衍生因子(pigment epithelium- derived factor, PEDF)的同源基因(PoPEDF),该基因属于丝氨酸蛋白酶抑制剂(serine protease inhibitor, serpin)超家族中无抑制活性的成员之一,编码蛋白具有抗血管增生、神经营养和保护功能。但关于该基因与病毒诱导研究还未见报道。通过RACE-PCR方法,从SMART cDNA文库得到了PoPEDF基因的全长cDNA共1803bp,开放阅读框长1212bp,编码403个氨基酸。序列同源性比较、蛋白结构分析、基因组DNA的外显子与内含子组合及系统进化分析等都表明PoPEDF为哺乳动物PEDF的同源基因。
PoPEDF与GFP共转染细胞及免疫组化结果表明,牙鲆PEDF是一种分泌蛋白,合成后的PEDF主要分布于细胞质而不在细胞核。连续观察发现表达的PEDF逐渐向细胞外分泌。
在哺乳动物中,PEDF是一个多功能蛋白,其功能和调节机制非常复杂。以牙鲆和牙鲆胚胎细胞为材料,用不同的诱导剂诱导,对牙鲆PEDF的功能特性作了初步探讨。结果显示,PEDF对ATRA的诱导变化不明显;低葡萄糖浓度可以诱导PEDF的上调表达,随着葡萄糖浓度的升高,PEDF的表达逐渐降低;缺氧时,PEDF先上调表达,随后再下调表达;随着细胞培养时间的增加,
Japanese flounder (Paralichthys olivaceus) is one of important marine aquaculture fishes, and has been formed a large-scale economic benefit in China. However, the increasing cultivating density causes frequent breakout of virus disease in flounder, which has leaded to tremendous economic losses. In order to provide theoretic basis on defending viral diseases in flounder, it is significant and urgent to further study the molecular mechanism underlying flounder antiviral immune responses.
In this study, a subtractive cDNA library was successfully constructed with mRNAs made from Japanese flounder embryonic cells (FEC) treated with UV-inactivated grass carp hemorrhage virus (GCHV) and control cells using suppression subtractive hybridization (SSH). PCR and dot blotting obtained a total of 148 ESTs through screening of subtractive cDNA library. Sequencing and bioinformative analyses reveal that the genes represented by these ESTs can be divided into seven groups, including cell division, cell signaling/cell communication, cell structure/motility, cell/organism defense, gene/protein expression and metabolism and so on.
Among 148 ESTs, three are homologous to mammalian pigment epithelium-derived factor (PEDF) genes, indicating that they represent a flounder PEDF homolog (named Paralichthys olivaceus PEDF, PoPEDF). PEDF is a
引文
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