Structure, organization and expression of common carp (Cyprinus carpio L.) NKEF-B gene
Structure, organization and expression of common carp (Cyprinus carpio L.) NKEF-B gene
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鲤鱼NKEF-B基因的结构、组织和表达
DOI：
10.1016/j.fsi.2008.10.013
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发表时间：
2009-02-01
影响因子：
4.7
通讯作者：
Guo, Qiong-Lin
中科院分区：
文献类型：
--
作者：
Huang, Rong;Gao, Long-Ying;Guo, Qiong-Lin
自然杀伤（NK）细胞增强因子（NKEF）属于新定义的过氧化物还氧蛋白（Prx）家族。其功能是增强NK细胞的细胞毒性，保护DNA和蛋白质免受氧化损伤。本研究从胸腺cDNA文库中分离出鲤鱼NKEF-B的部分cDNA序列。随后，分别用3′和5′RACE法获得了鲤鱼NKEF-B的全长cDNA。鲤鱼NKEF-B全长1022 bp，由73 bp的5'端非翻译区（UTR）、3555 bp的t端非翻译区和594 bp的开放阅读框组成，编码一个197个氨基酸的蛋白。鲤鱼NKEF-B含有2个一致的VCP基序和3个一致的半胱氨酸基序（Cys-51、Cys-70和Cys-172）。序列比较表明，推导出的鲤鱼NKEF-B氨基酸序列与其他同源种属的氨基酸序列总体相似度为74-96%。系统发育分析表明，鲤鱼NKEF-B与其他已知硬骨鱼NKEF-B形成一个簇。然后，通过PCR得到鲤鱼NKEF-B基因组DNA，全长5.1 k，包含6个外显子和5个内含子。实时RT-PCR结果显示，在未感染条件下，NKEF-B基因主要在肾脏和头肾中检测到。而svcv感染条件下，NKEF-B基因在血细胞、鳃、肠和脾脏中的表达量显著升高，在肝脏中表达量维持不变，在肾脏和头肾中表达量显著降低。最后构建rNKEF-B并在大肠杆菌中表达。利用鲤鱼rNKEF-B抗体，进一步免疫组化研究表明，NKEF-B阳性细胞主要是鳃和肠中的部分红细胞和少数上皮细胞，在svcv感染条件下，这些阳性细胞或其细胞质中的阳性产物主要在鲤鱼鳃和脾脏切片中增加。本研究结果将有助于了解NKEF-B在硬骨鱼先天免疫中的作用。(C) 2008 Elsevier Ltd版权所有。
Natural killer (NK) cell enhancing factor (NKEF) belongs to the newly defined peroxiredoxin (Prx) family. Its functions are to enhance NK cell cytotoxicity and to protect DNA and proteins from oxidative damage. In this study, a partial cDNA sequence of carp NKEF-B was isolated from thymus cDNA library. Subsequently, the full-length cDNA of carp NKEF-B was obtained by means of 3' and 5' RACE, respectively. The full-length cDNA of carp NKEF-B was 1022 bp, consisting of a 73 bp 5'-terminal untranslated region (UTR), a 355 bp T-terminal UTR, and a 594 bp open reading frame coding for a protein of 197 amino acids. Carp NKEF-B contained two consensus Val-Cys-Pro (VCP) motifs and three consensus cysteine (Cys-51, Cys-70 and Cys-172) residues. Sequence comparison showed that the deduced amino acid sequence of carp NKEF-B had an overall similarity of 74-96% to that of other species homologues. Phylogenetic analysis revealed that carp NKEF-B forms a cluster with other known teleost NKEF-Bs. Then, by PCR we obtained a 5.1 -k long genomic DNA of carp NKEF-B containing six exons and five introns. Realtime RT-PCR results showed that carp NKEF-B gene was predominantly detected in kidney and head kidney under un-infected conditions. Whereas under SVCV-infection condition, the expression of NKEF-B gene was significantly increased in blood cells, gill, intestine and spleen, but maintained in liver, and decreased significantly in kidney and head kidney. Finally, the rNKEF-B was constructed and expressed in Escherichia coli. By using an antibody against carp rNKEF-B, immunohistochemical study further indicated that NKEF-B positive cells are mainly some RBCs and a few epithelial cells in gill and intestine, and that under SVCV-infection condition, these positive cells or positive products in their cytoplasm were mainly increased in gill and spleen sections of carp. The results obtained in the present study will help to understand the function of NKEF-B in teleost innate immunity. (C) 2008 Elsevier Ltd. All rights reserved.