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大竹蛏β-整合素基因SgβInt的特性分析和重组表达
刘佳乐1,2, 韦秀梅3, 杨顶珑4, 童 潼1, 刘相全3
1.广西壮族自治区海洋研究所 广西海洋生物技术重点实验室 北海 536000;2.上海海洋大学水产与生命学院 上海 201306;3.山东省海洋资源与环境研究院 山东省海洋生态修复重点实验室 烟台 264006;4.山东省海洋资源与环境研究院 山东省海洋生态修复重点实验室 烟台 264007
摘要:
整合素作为一种细胞粘附因子,在细胞粘附、迁移、增殖、凋亡和吞噬等过程中发挥重要作用。本研究利用分子克隆技术获得了大竹蛏(Solen grandis) β-整合素基因(SgβInt)的cDNA全长,分析了其编码氨基酸序列的进化特点,并以SWISS-MODEL预测了氨基酸序列的三级结构。SgβInt基因序列全长为1168 bp,5′和3′非编码区(UTR)分别为61 bp和18 bp,开放阅读框(ORF)为1089 bp,编码362个氨基酸,理论等电点约为4.98,分子量为30.0 kDa。通过荧光定量PCR法检测了SgβInt在健康大竹蛏各组织中以及大竹蛏受到脂多糖、肽聚糖和葡聚糖等微生物多糖刺激后的表达。结果显示,SgβInt在血细胞、鳃、肝胰腺、性腺、肌肉和外套膜等组织中均可表达,在鳃中的表达量最高;脂多糖、肽聚糖和葡聚糖都可以诱导SgβInt表达量上调,SgβInt的表达量分别在脂多糖和葡聚糖刺激后的3 h和48 h达到最高;肽聚糖刺激后SgβInt表达量上调幅度最大,在6 h时达到最高,证实SgβInt参与大竹蛏抵御外源微生物的免疫应答。利用基因重组技术构建了SgβInt的重组表达载体,为进一步分析软体动物整合素的功能、揭示大竹蛏先天性免疫机制奠定了基础。
关键词:  大竹蛏  整合素  免疫应答  荧光定量PCR
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Recombinant Expression and Characterization of the β-Integrin Gene of Solen grandis
LIU Jiale,WEI Xiumei,YANG Dinglong,TONG Tong,LIU Xiangquan
1.Guangxi Institute of Oceanology, Key Laboratory of Marine Biotechnology of Guangxi, Beihai 536000;2.College of Fisheries and Science, Shanghai Ocean University, Shanghai 201306;3.Key Laboratory of Marine Ecological restoration, Shandong Marine Resource and Environment Research Institute, Yantai 264006
Abstract:
Integrins are heterodimeric cell surface receptors that consist of α and β subunits to regulate cell adhesion, migration, proliferation, apoptosis and phagocytosis. The present study identified the β-integrin gene from Solen grandis (SgβInt) and analyzed the characterization of its encoded protein. The phylogenetic tree was constructed by the neighbor-joining method and the three-dimensional structure was predicted with SWISS-MODEL. The full-length cDNA of SgβInt was 1168 bp, containing a 61 bp 5´UTR, 18 bp 3´UTR and an open reading frame (ORF) of 1089 bp that encodes a polypeptide of 362 amino acids with an estimated molecular mass of 30.0 kDa. The encoded protein of SgβInt shared significant similarities with that in Capra hircus and Mus musculus. The phylogenetic tree indicated that SgβInt had a close genetic relationship with Crassostrea gigas to form a mollusk branch. The three-dimensional structure of SgβInt consisted of one α-helice and nine β-sheets as a member of integrin superfamily. SgβInt expressed in all tested tissues, including mantle, gill, hemocyte, gonad, muscle and hepatopancreas, with the highest expression in gill which was 487.5 times higher than gonad. SgβInt was induced by all the three pathogen associated molecular patterns (PAMPs) including LPS, PGN and glucan with the peak level at 3 and 48 h post LPS and glucan stimulation, respectively. SgβInt expression reached the maximal level at 6 h post PGN stimulation with 53.5-fold induction. All the results revealed that SgβInt might regulate the immune response of S. grandis to microorganism. This study shed new light on the research of β-integrin in mollusk and immune defense mechanism of S. grandis.
Key words:  Solen grandis  Integrin  Immune response  Real-time PCR