摘要:
As a family of carbohydrate binding proteins with Ca2+, C-type lectins play important roles in the first line of innate immune defense.In this research, a novel C-type lectin gene (LvLc1, GenBank Accession Number:KY937940) was cloned from Litopenaeus vannamei according to the data of shrimp transcriptome in our lab.The full-length cDNA consists of 1251 bp with an 891 bp open reading frame, encoding 296 amino acids.The deduced amino acid sequence contains a putative signal peptide of 19 amino acids.It also contains one carbohydrate recognition domains/C-type lectin-like domains (CRD).The potential carbohydrate-binding motif (QPD) presented in the CRD ofLvLc1 may support its ability to bind galactose-type sugars.The deduced amino acid sequence of LvLc1 showed high identity with mannose-binding lectins of arthropod Procambarus clarkii and Pacifastacus leniusculus.It could be deduced thatLvLc1 is a novel member of C-type lectin superfamily.The recombinant target protein (rLvLc1) was expressed by prokaryotic expression system.The LC-ESI-MS analysis showed that the peptide fragments of rLvLc1 were identical with the corresponding sequence of L.vannamei C-type lectin.rLvLc1 had agglutinating activity against main pathogens (G+, G– and fungi) in aquaculture in a calcium-dependent manner.The agglutinating activity can be inhibited by multiple carbohydrates, such as galactose, mannose and lipopolysaccharide.These results suggest thatLvLc1, as a Ca2+ dependent carbohydrate-recognition protein, is one of the important PRRs.It might play a crucial role in the innate immunity of the shrimp and it is expected to be applied to disease control.%C型凝集素是一种依赖于Ca2+而发挥功能的糖蛋白,在一线的固有免疫防御过程中发挥着重要作用.围绕对虾C型凝集素开展深入研究,不仅可以丰富无脊椎动物固有免疫学内容,还有望将其开发为具有免疫增强效果的活性饵料,应用于对虾的健康养殖.本实验根据实验室前期转录组信息提示克隆获得了凡纳滨对虾一种新的C型凝集素基因(LvLc1,GenBank注册号:KY937940).生物信息学分析显示LvLc1基因的开放阅读框全长891 bp,编码296个氨基酸,该基因编码的蛋白质含有一个保守的糖识别结构域(carbohydrate recognition domain,CRD),该结构域中具有潜在的半乳糖结合位点(QPD motif),进化发生分析显示LvLc1与来自节肢动物的甘露糖结合凝集素家族成员聚类在一起.对LvLc1基因的CRD结构域进行了原核重组表达与蛋白活性分析研究,结果显示:重组目的蛋白(rLvLc1)在Ca2+存在的条件下,对多种病原菌(G+、G–和真菌)具有凝集作用,其凝集活性可被半乳糖、甘露糖、脂多糖等多种病原相关分子模式所抑制.研究表明,LvLc1作为C-型凝集素家族一个新成员,可能通过重要的模式识别受体作用,参与机体应答病原微生物侵染的防御过程.