遗传 ›› 2008, Vol. 30 ›› Issue (8): 1043-1050.doi: 10.3724/SP.J.1005.2008.01043

• 研究报告 • 上一篇    下一篇

细菌攻毒杂色鲍血淋巴细胞抑制性差减杂交文库构建及巨噬细胞表达蛋白cDNA的克隆与差异表达

任洪林1, 2; 徐丹丹1; 乔琨1; 蔡灵1; 黄伟滨1; 张鼐1; 王克坚1   

  1. 1. 近海海洋环境科学国家重点实验室 厦门大学海洋与环境学院, 厦门 361005;
    2. 人兽共患病教育部重点实验室 吉林大学人兽共患病研究所, 长春 130062
  • 收稿日期:2008-01-02 修回日期:2008-04-04 出版日期:2008-08-10 发布日期:2008-08-10
  • 通讯作者: 王克坚

Construction of SSH library from haemocyte of variously colored abalone challenged with bacteria and differential expression analysis of macrophage expressed protein

REN Hong-Lin1,2; XU Dan-Dan1; QIAO Kun1; CAI Ling1; HUANG Wei-Bin1; ZHANG Nai1; WANG Ke-Jian1   

  1. 1. State Key Laboratory of Marine Environmental Science, College of Oceanography and Environmental Science, Xiamen University, Xiamen 361005, China;
    2. Key Laboratory of Zoonosis, Ministry of Education, Institute of Zoonosis, Jilin University, Changchun 130062, China
  • Received:2008-01-02 Revised:2008-04-04 Online:2008-08-10 Published:2008-08-10
  • Contact: WANG Ke-Jian

摘要: 以雌性杂色鲍为对象, 以大肠杆菌、副溶血弧菌、溶壁微球菌、表皮葡萄球菌、金黄色葡萄球菌的混悬液做为攻毒菌, 利用抑制性差减杂交(SSH)技术构建细菌攻毒的杂色鲍血淋巴细胞SSH cDNA文库。随机挑取生长菌落110个克隆子, 进行菌液PCR鉴定, 计算文库重组率为98.18%, 文库容量为1.37×106 pfu。将重组子测序, 经BLAST一致性搜索比对分析, 有一重组片段含有穿孔素(Perforin)保守结构域, 为巨噬细胞表达蛋白(MEP)类穿孔素部分cDNA序列, 片段大小为1 551 bp, 连续编码517个氨基酸残基, 申请GenBank登录号为EU272049。经半定量PCR和荧光定量PCR差异显示分析, 发现在细菌感染状态下MEP基因在血淋巴细胞中存在明显的上调表达现象。

关键词: 血淋巴细胞, 杂色鲍, 巨噬细胞表达蛋白, 抑制性差减杂交, 细菌

Abstract: Abalones are considered to be the most precious delicacy from the sea, and become very important commercial seafood in aquaculture worldwide. Variously colored abalone (Haliotis diversicolor Reeve, 1846) has been widely cultured on the southeast coast for more than twenty years. However, abalone culture frequently suffers from bacterial infection and mass mortality of reared abalones causes serious economic losses. Unfortunately, knowledge of the defense mechanism in this animal is still lacking. In this study, using suppression subtractive hybridization (SSH) technology, a forward SSH library was constructed from haemocytes of H. diversicolor, with the content of 1.37×106 pfu and the recombinant rate of 98.18%. After the recombinant plasmids were sequenced, partial cDNA of macrophage expressed protein (MEP) was recognized based on BLAST searches in NCBI, with the size of 1 551 bp, and continuously encoding 517 amino acids. Semi-quantitative PCR and quantitative real-time PCR results showed that MEP cDNA was distinctly up-regulated in haemocytes of the bacterial-challenged group compared to the unchallenged group. The gene information obtained from this library will provide new insights into the immune mechanism of H. diversicolor and facilitate future study of target genes involved in the response to invading microorganisms.