中华绒螯蟹甲壳肽EsCrus3和EsCrus4表达模式与免疫功能

EXPRESSION PATTERN AND IMMUNE FUNCTION ANALYSIS OF ESCRUS3AND ESCRUS4 FROM CHINESE MITTEN CRAB(ERIOCHEIR SINENSIS)

研究从中华绒螯蟹(Eriocheir sinensis)中克隆并鉴定了两个Ⅰ型甲壳肽(Crustin)基因,分别命名为EsCrus3和EsCrus4。随后通过生物信息学、基因表达分析、蛋白表达与纯化、免疫功能验证等研究手段,分析了这2个新型抗菌肽的表达、结构和功能。EsCrus3的cDNA序列全长567 bp,开放阅读框(Open Reading Frame,ORF)为351 bp,编码116个氨基酸(Amino acid,aa);EsCrus4序列全长743 bp,ORF区288 bp,编码95个aa。两者都含有信号肽、富半胱氨酸的结构域(Cysteine-rich region,CRR)和WAP(Whey acidic protein)结构域。EsCrus3和EsCrus4氨基酸序列的一致性仅为30.77%,并且进化分析显示EsCrus3和EsCrus4分别位于两个具有分类意义的分支上,提示两者生物学功能可能存在差异。EsCrus3和EsCrus4均在卵巢中高度表达且具有相似的表达水平,在金黄色葡萄球菌(Staphyloccocus aureus)和嗜水气单胞菌(Aeromonas hydrophila)刺激时呈上调表达趋势,其中EsCrus3响应速度更快,在刺激2h时上调表达,EsCrus4则在刺激后12—24h时显著上调。重组蛋白EsCrus3和EsCrus4对革兰氏阳性菌、革兰氏阴性菌和真菌均具有不同程度的结合活性,并且对不同微生物多糖结合活性也存在明显差异。EsCrus3对革兰氏阳性菌的肽聚糖(PGN)和脂磷壁酸(LTA)具有很高结合活性,对革兰氏阴性菌的脂多糖(LPS)和真菌β-葡聚糖也有一定结合活性,而EsCrus4只对PGN具有显著结合活性。上述研究结果表明EsCrus3响应病原刺激更迅速、与病原多糖结合能力更强,提示其在卵巢的免疫防御体系中发挥更重要作用。此外,EsCrus3和EsCrus4序列和功能的差异为新型药物的挖掘提供了基础数据。

Antibacterial peptides(AMPs) are the main effector molecules of the innate immune system of invertebrates,and their understanding can aid in discovering new drugs and preventing diseases.In the present study,two type ⅠCrustin genes were cloned and identified from the mitten crab Eriocheir sinensis,and they were named as Escrus3 and Escrus4 following the nomenclature formed in this field.The cDNA sequence of EsCrus3 was 567 bp,with an Open Reading Frame(ORF) of 351 bp that encode 116 amino acids(aa).EsCrus4 sequence was 743 bp in length with an ORF of 288 bp that encode a putative protein of 95 aa.The domain structure of these two amino acid sequences was further predicted,and they were found to have a N-terminal signal peptide,a C-terminal WAP(Whey acid protein) domain,and a cysteine-rich region(CRR).An evolutionary tree was constructed based on the BLASTP results and the reported Crustin sequences.The results showed that Es Crus3 had the closest evolutionary relationship with Scylla tranquebarica St Crus(AFI56572),while Es Crus4 displayed the closest evolutionary relationship with Portunus trituberculus Pt Crus2(AFU61578).The two molecules were located at different branches with taxonomic significance,suggesting that they might have different biological functions.The tissue distribution showed that both EsCrus3 and EsCrus4were highly expressed in the ovary with a similar expression level,while the expression level in other tissues was very low or even not detected.When challenged by Staphylococcus aureus or Aeromonas hydrophila,both EsCrus3 and EsCrus4 were up-regulated.EsCrus3 responded faster and significantly up-regulated at 2h after bacterial challenges,while EsCrus4 was significantly up-regulated at 12—24h after bacterial challenges,suggesting that these two genes may play an important role in the antibacterial immune system of the mitten crab.Recombinant proteins Es Crus3 and Es Crus4 were obtained by expression in E.coli system and purification by affinity column.The results of the microbebinding assay showed that the recombinant proteins Es Crus3 and Es Crus4 exhibited different binding activities to gram-positive bacteria,gram-negative bacteria and fungi,and their binding activities to polysaccharides were significantly different as well.Es Crus3 showed high binding activities to peptidoglycan(PGN) and lipoteichoic acid(LTA) of gram-positive bacteria,and low binding activities to lipopolysaccharide(LPS) of gram-negative bacteria and β-Glucan of fungal;Es Crus4 only has a significant binding activity to PGN.The above research results show that Es Crus3 responds faster to bacterial challenges and has stronger binding abilities to polysaccharide components of pathogens,suggesting that it plays a more important role in the immune defense system of ovary.In addition,the differences in the expression and function of Es Crus3 and Es Crus4 also provide basic data for the mining of new drugs.