小菜蛾PGRP-S2基因的克隆表达及功能分析

Cloning,expression and functional analysis of PGRP-S2 gene in Plutella xylostella

肽聚糖识别蛋白(peptidoglycan recognition protein,PGRP)对于昆虫来说是一种高度保守的病原识别蛋白。为阐明PGRP-S2在小菜蛾Plutella xylostella抵抗病原微生物过程中的作用,本研究结合RT-PCR和RACE技术克隆得到小菜蛾PGRP-S2基因的cDNA全长序列,命名为PGRP-S2(GenBank登录号:MG570190)。生物信息学分析结果表明,PGRP-S2的开放阅读框为588 bp,编码195个氨基酸;蛋白质预测分子量为21.46 kDa,理论等电点为8.46;编码蛋白具有PGRP超家族保守结构域和酰胺酶结构域,是典型的肽聚糖识别蛋白,包含一条信号肽,不存在跨膜结构;同源序列比对和系统进化树分析表明PGRP-S2与家蚕Bombyx mori的BmPGRP-S 1进化距离最近。利用大肠杆菌Escherichia coli BL21(DE3)高效表达重组蛋白PxPGRP-S2,利用倒置显微镜及平板涂布观察重组蛋白对大肠杆菌E.coli和金黄色葡萄球菌Staphylococcus aureus的作用,结果表明PxPGRP-S2蛋白能够与两种细菌发生结合并凝集细菌,但不具备直接杀菌功能。本研究为进一步研究基于PGRP-S2介导的小菜蛾免疫防御反应提供基础。

Peptidoglycan recognition protein(PGRP)is a highly conserved pathogen recognition protein for insects.In order to elucidate the role of PGRP-S2 in the resistance of P.xylostlla to pathogenic microorganisms,the full-length cDNA of PGRP-S 2 gene of P.xylostlla was obtained by RT-PCR and RACE,and named as PGRP-S 2(GenBank accession:MG570190).According to bioinformatics analysis,with an open reading frame of 588 bp,PGRP-S 2 encodes 195 amino acids,the predicted molecular weight of the protein is about 21.46 kDa,and the theoretical isoelectric point is 8.46.The encoded protein has a conserved domain of PGRP superfamily and amidase domain,which are typical of peptidoglycan recognition proteins,and contains a signal peptide without transmembrane structure;homologous sequence alignment and phylogenetic tree analysis showed that PGRP-S 2 was closest to BmPGRP-S 1 of Bombyx mori.The recombinant protein PxPGRP-S2 was highly expressed in Escherichia coli BL21(DE3).The interaction between the recombinant protein PxPGRP-S2 with E.coli and Staphylococcus aureus was observed by an inverted microscope and plate coating.The results showed that PxPGRP-S2 protein could be bind to both bacteria and agglutinate bacteria,but not could kill them directly.The study would lay a foundation for the prevention and further study for the PGRP-S2-mediated immune defense responses in P.xylostella.