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中华鳖MHCⅡα基因cDNA的克隆及组织表达分析
童正飞1, 葛玲瑞1,2,3, 胡亚洲1,3, 杨涛1, 涂开发1, 王佩1, 周先文1, 王晓清1,3, 熊钢2,3
1.湖南农业大学动物科学技术学院 湖南 长沙 410128;2.湖南生物机电职业技术学院 湖南 长沙 410127;3.湖南省水产产业技术体系 湖南 长沙 410128
摘要:
为了探究中华鳖(Pelodiscus sinensis)的主要组织相容性复合体(major histocompatibility complex, MHC)的结构和功能,本研究利用RACE技术成功克隆获得中华鳖MHCⅡα基因的cDNA全长序列,其长度为1296 bp,开放阅读框(ORF)为807 bp,共编码268个氨基酸,分为信号肽序列、Ⅱ类组织相容性抗原α结构域、IGc1结构域及跨膜结构域4个功能结构域。通过NJ法构建的系统进化树分析显示,中华鳖与西部锦龟(Chrysemys picta bellii)亲缘较近,而与哺乳类、鸟类及鱼类亲缘关系较远。荧光定量PCR结果显示,MHCⅡα mRNA在中华鳖8个组织中均有表达,在脾、心、肝及肠组织中表达水平较高,在肌肉组织中表达量最低。感染嗜水气单胞菌(Aeromonas hydrophila) 12 h后,中华鳖MHCⅡα mRNA在肝和肠组织中的表达显著上调;感染1 d时,在脾组织中的表达显著上调;感染1 d及5 d时,在肾组织中的表达显著上调。研究表明,中华鳖MHCⅡα基因参与了中华鳖免疫反应。
关键词:  中华鳖  MHCⅡα  序列分析  基因表达
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Cloning and expression analysis of MHCⅡα cDNA in Pelodiscus sinensis
TONG Zhengfei1, GE Lingrui1,2,3, HU Yazhou1,3, YANG Tao1, TU Kaifa1, WANG Pei1, ZHOU Xianwen1, WANG Xiaoqing1,3, XIONG Gang2,3
1.College of Animal Science and Technology, Hunan Agricultural University, Changsha, Hunan 410128, China;2.Hunan Biological and Electromechanical Polytechnic, Changsha, Hunan 410127, China;3.Technology System of Aquatic Industry in Hunan Province, Changsha, Hunan 410128, China
Abstract:
To study the structure and function of MHC (major histocompatibility complex) in Pelodiscus sinensis, we obtained the full-length cDNA of MHCⅡα using RACE-PCR (rapid amplification of cDNA ends PCR) technology. The resulting sequence was 1296 bp in length, including an ORF (open reading frame) region of 807 bp. The peptide-encoded MHCⅡα gene could be divided into four parts, including the signal peptide, MHCⅡ alpha domain, IGc1 domain, and transmembrane region. A neighbor-joining tree showed that the MHCⅡα gene from P. sinensis and Chrysemys picta bellii cluster into one cluster. The P. sinensis gene had a close genetic-relationship with C. picta bellii and a farther genetic-relationship with mammals, aves, and fish. Using qRT-PCR in all 8 tissues, the highest expression of the MHCⅡα gene was observed in the spleen, heart, liver, and intestine, while the lowest level was found in muscle. Meanwhile, MHCⅡα mRNA expression levels were significantly up-regulated in the liver and intestine (after 12 h), the spleen (on the 1st day), and the kidney (on 1st and 5th day) after being infected with Aeromonas hydrophila. The liver, spleen, intestine, and kidney are closely related to immunity, which indicates that this gene has important effects on the immune response.
Key words:  Pelodiscus sinensis  MHCⅡα  Sequence analysis  Gene expression