三角帆蚌短型肽聚糖识别蛋白S3基因克隆及表达分析

CLONING AND EXPRESSION ANALYSIS OF SHORT TYPE PEPTIDOGLYCAN RECOGNITION PROTEIN S3 GENE FROM HYRIOPSIS CUMINGII

肽聚糖识别蛋白(PGRPs)是机体先天免疫系统中的重要模式识别受体,研究对三角帆蚌的一种短型肽聚糖识别蛋白进行了克隆与表达分析。研究采用5′、3′RACE技术,对高通量转录组测序所得三角帆蚌PGRPS3基因(hc PGRPS3)片段分别进行了5′,3′末端克隆,经拼接得到hc PGRPS3 c DNA全长序列。采用多种分子生物学软件对hc PGRPS3 c DNA全长序列进行了特征分析,实时荧光定量(q RT-PCR)检测了其组织分布,及经肽聚糖(Peptidoglycan,PGN)和脂多糖(Lipopolysaccharide,LPS)刺激后其在肝胰腺中的基因表达变化。hc PGRPS3 c DNA全长1438 bp,开放阅读框为858 bp,编码285个氨基酸,预测分子量大小为32.3 ku,p H 7.0时的理论等点为7.98。序列中不存在信号肽与跨膜结构。氨基酸序列保守性分析表明,其他物种短型PGRP中,以夏威夷短尾鱿PGRP4与hc PGRPS3同源性最高(51%)。q RT-PCR检测结果显示,hc PGRPS3在性腺及肝胰腺中表达水平较高。经PGN或LPS刺激后,肝胰腺中hc PGRPS3表达水平显著上调,表明hc PGRPS3可能在机体抗菌免疫反应中发挥重要作用。

The present study cloned and sequenced Hyriopsis cumingii PGRPS3 （hcPGRPS3） and measured its expree- sion in multiple tissues. Results showed that the hcPGRPS3 cDNA sequence was 1438 bp in length, consisting of a 858 bp open reading frame （ ORF） encoding 285 amino acid residues with 32.3 ku of predicted molecular weight and 7.98 of the theoretical isoelectric point, which have no signal petptide and transmembrane helices but have two latent N-glycosylation sites N40PT and N92YS. Multiple alignment analysis revealed that hcPGRPS3 shareed the highest identity with Euprymna scolopes PGRP4 （51%）. hcPGRPS3 expressed in all examined organs of healthy H. cumingii with the low level in gill, heart and mantle and high level in gonad and hepatopancreas. Lipopolysaccharide and Pepti-doglycan obviously up-regulated the expression of hcPGRPS3 in hepatopancreas, suggesting that hcPGRPS3 may play an important role in H. cumingii hepatopancreas innate immunity system against bacterial challenged.