Identification and characterization of lamprey nucleolin-like gene
Identification and characterization of lamprey nucleolin-like gene
复制标题
七鳃鳗核蛋白样基因的鉴定与表征
DOI：
10.1093/abbs/gmy150
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发表时间：
2018
期刊：
影响因子：
--
通讯作者：
Xu Yang
中科院分区：
文献类型：
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作者：
Zhao Huan;Tian Yang;Li Qingwei;Xu Yang
核仁素于 1973 年首次被描述为参与核糖体生物发生以及 DNA 和 RNA 代谢的蛋白质。它是核仁中众所周知的主要非核糖体蛋白，主要分布在核仁的致密纤维状和颗粒状区域。根据其在 2D 凝胶上的迁移率，它最初被命名为 C23 [1]。核仁素在核仁中最丰富，但它也位于其他区域，例如细胞质和质膜。由于其广泛的定位和机制，核蛋白功能的研究尤其困难。从生化角度来看，核仁素调节 DNA 和 RNA 代谢的许多方面，包括核仁染色质的组织、核糖体 DNA (rDNA) 转录的控制和核糖体前包装 [2]。最近的研究进一步证明核仁素参与细胞凋亡、成核、信号转导、复制、诱导染色质解聚、调节转录过程和胞质分裂等[3]。此外，核仁素在哺乳动物的免疫反应中起着至关重要的作用。它影响病毒感染的多个方面，包括利用宿主细胞产生病毒蛋白、病毒与宿主细胞的结合以及病毒遗传物质的插入[4, 5]。七鳃鳗和盲鳗是一些最古老脊椎动物的幸存代表。七鳃鳗是迄今为止唯一现存的无颌类[6]。作为原始脊椎动物的一员，七鳃鳗不仅具有先天性免疫反应系统，还表现出适应性免疫反应系统[7]。七鳃鳗具有独特的适应性免疫系统和可变的淋巴细胞受体。这种独特的进化位置使七鳃鳗成为了解适应性免疫出现的理想模型[8]。与对无脊椎动物核仁素的广泛研究相反，人们对核仁素在无颌七鳃鳗中的生物活性和生理作用知之甚少。在这项研究中，我们首次描述了七鳃鳗中核仁素同系物（称为类核仁素）的鉴定和克隆。核仁素样可能是哺乳动物核仁素的对应物，哺乳动物核仁素以其在免疫反应中的重要作用而闻名。还研究了Poly(I:C)攻击后核仁素样cDNA和蛋白的表达谱，以验证其在免疫反应中的作用。 2015年12月从黑龙江通江段（中国通江）获得成年七鳃鳗（长度：36.4~58.4 cm，重量：112~274.5 g）。外周血采集于 尾部皮下窦，通过Ficolle-Paque梯度离心进一步富集七鳃鳗淋巴细胞。然后，收集淋巴细胞并在含有10%胎牛血清（FBS）的RPMI 1640培养基中培养。为了去除死淋巴细胞，使用淋巴细胞分离试剂盒（Solarbio，北京，中国）获得活细胞。上述所有动物实验程序均经大连医科大学动物福利与研究伦理委员会批准（许可证号：SYXK2004-0029）。基于我们实验室使用七鳃鳗淋巴细胞构建的 cDNA 文库的表达序列标签 (EST) 分析，使用基本局部比对搜索工具 (BLAST) 发现了与核仁素同源的单个 EST 序列。使用3'-RACE核心套件（TaKaRa，大连，中国）和5'-RACE SMARTerTM RACE cDNA扩增试剂盒（Clontech，山景城，美国）以及补充表S1中的3'-RACE和5'-RACE引物扩增全长cDNA。所有 PCR 产品...
Nucleolin was first described in 1973 as a protein involved in ribosome biogenesis and in DNA and RNA metabolism. It is a wellknown major non-ribosomal protein in the nucleolus, and is mainly distributed in the dense fibrillar and granular regions of the nucleolus. It was initially named as C23 according to its mobility on a 2D gel [1]. Nucleolin is most abundant in the nucleolus, however it also locates in other regions, such as the cytoplasm and plasma membrane. Study of nucleolin functions is especially difficult because of its broad range of localization and mechanisms. Biochemically, nucleolin regulates many aspects of DNA and RNA metabolism, including organization of nucleolar chromatin, control of ribosomal DNA (rDNA) transcription, and pre-ribosome packaging [2]. Recent researches have further proved that nucleolin participates in apoptosis, nucleogenesis, signal transduction, replication, induction of chromatin decondensation, modulating transcriptional processes, and cytokinesis [3]. Moreover, nucleolin plays a crucial role in the immune response in mammals. It affects multiple aspects of viral infection, including the utilization of the host cell to produce viral proteins, association of the virus with the host cell, and insertion of viral genetic material [4, 5]. Lamprey and hagfish are surviving representatives of some of the most ancient vertebrates. Lampreys are so far the only existing ones of the class Agnatha [6]. As a member of primitive vertebrates, lampreys not only have innate immune response systems but also exhibit adaptive immune response systems [7]. Unique adaptive immune systems and variable lymphocyte receptors are found in lampreys. This unique evolutionary position makes lampreys an ideal model for understanding the appearance of adaptive immunity [8]. In contrast to extensive studies of nucleolin invertebrates, little is known about the biological activities and physiological roles of nucleolin in jawless lampreys. In this study, for the first time, we described the identification and cloning of a homolog of nucleolin (named nucleolin-like) in the lamprey. Nucleolin-like could be the counterpart of mammalian nucleolin, which is known for its important roles in the immune response. The expression profiles of nucleolin-like cDNA and protein after challenge with Poly (I: C) were also investigated to verify its roles in the immune response.Adult lampreys (lengths: 36.4~ 58.4 cm, weights: 112~ 274.5 g) were obtained from the Tongjiang section of the Heilongjiang River (Tongjiang, China) in December of 2015. The peripheral blood was collected from the caudal subcutaneous sinus, and the lamprey lymphocytes were further enriched by Ficolle-Paque gradient centrifugation. Then, the lymphocytes were collected and cultured in RPMI 1640 medium containing 10% fetal calf serum (FBS). To get rid of dead lymphocytes, Lymphocyte Separation Kit (Solarbio, Beijing, China) was used to obtain the living cells. All animal experimental procedures mentioned above were approved by the Animal Welfare and Research Ethics Committee of the Institute of Dalian Medical University (Permit Number: SYXK2004–0029). Based on the expressed sequence tag (EST) analysis of the cDNA library that was constructed using lamprey lymphocytes in our laboratory, a single EST sequence homologous to nucleolin was found using Basic Local Alignment Search Tool (BLAST). Full-length cDNA was amplified using the 3′-RACE Core Set Kit (TaKaRa, Dalian, China) and the 5′-RACE SMARTerTM RACE cDNA Amplification Kit (Clontech, Mountain View, USA) with the 3′-RACE and 5′-RACE primers in Supplementary Table S1. All of the PCR products …