Expression and immune function characteristics of peptidoglycan recognition protein gene in disk abalone (Haliotis discus hannai)
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摘要: 为研究皱纹盘鲍（Haliotis discus hannai）肽聚糖识别蛋白HdhPGRP-SC2-like的免疫功能特性， 利用RACE技术克隆了HdhPGRP-SC2-like基因的全长cDNA序列，采用实时荧光定量PCR（qPCR）分析了HdhPGRP-SC2-like基因的组织分布特征，通过原核表达获得了重组蛋白rHdhPGRP-SC2-like， 并对其酰胺酶活性及免疫特性进行了验证。结果表明：HdhPGRP-SC2-like基因的cDNA全长为938 bp，开放阅读框为780 bp，编码260个氨基酸，包含保守的PGRP结构域和Ami_2结构域；HdhPGRP-SC2-like属于短型PGRP，与软体动物近缘物种PGRP具有高度相似性；qPCR分析显示，HdhPGRP-SC2-like mRNA在皱纹盘鲍各组织中均有表达，其中，鳃中表达量最高，外套膜、消化腺中表达量次之，腹足和血细胞中表达量较低；进一步构建原核表达载体pET-28a（+）-HdhPGRP-SC2-like，经诱导表达、分离纯化获得重组蛋白rHdhPGRP-SC2-like，该蛋白具有肽聚糖结合活性，且在Zn2+存在的情况下显示出酰胺酶活性。研究表明，HdhPGRP-SC2-like能够结合细菌肽聚糖成分，在机体抵御入侵细菌等免疫防御中发挥重要作用。Abstract: In order to investigate the immune function of the peptidoglycan recognition protein HdhPGRP-SC2-like gene in disk abalone (Haliotis discus hannai), the full-length cDNA sequence of HdhPGRP-SC2-like gene was cloned by RACE technology, and tissue distribution of HdhPGRP-SC2-like gene was analyzed in the disk abalone by real-time fluorescent quantitative PCR(qPCR). The recombinant protein rHdhPGRP-SC2-like was obtained by prokaryotic expression, and its amidase activity and immune characteristics were verified. The results showed that the cDNA of the HdhPGRP-SC2-like gene was 938 bp in length, with an open reading frame of 780 bp, encoding 260 amino acids and containing the conserved PGRP and Ami_2 structural domains. HdhPGRP-SC2-like gene as a short type PGRP had high similarity with the closely related species PGRP of mollusks. qPCR analysis showed that HdhPGRP-SC2-like mRNA was expressed in all tissues of the disk abalone, with the maximal expression level in gill, a certain amount of expression level in mantle and digestive gland, and the minimal content in foot and hemocyte. The prokaryotic expression vector pET-28a(+)-HdhPGRP-SC2-like was further constructed, and the recombinant protein rHdhPGRP-SC2-like gene was obtained by induction of expression, isolation, and purification. The recombinant protein had peptidoglycan binding activity and showed amidase activity in the presence of Zn2+. The finding indicated that HdhPGRP-SC2-like gene binded bacterial peptidoglycan components and played an important role in the immune defence against invading bacteria.
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