肽聚糖识别蛋白AaPGRP-LC参与埃及伊蚊的抗细菌免疫反应

摘要

[Aim] The objective of this study is to elucidate the roles of peptidoglycan recognition protein LC (PGRP-LC) in response to bacterial infection in Aedes aegypti.[Methods] The mRNA abundance of genes of antimicrobial peptides (AMPs) in mosquitoes at different time after infection of Enterobacter cloacae was measured by quantitative real-time PCR (qPCR).After interference of PGRP-LC by RNA interference (RNAi),the expression profiles of immune-related genes were detected by qPCR.The recombinant protein AaPGRP-LC was expressed by prokaryotic expression system and purified through a Ni2+-NTA agarose column.The quality of the purified recombinant protein was tested by Western blotting.[Results] Gram-negative E.cloacae bacteria could activate the expression of genes of AMPs in Ae.aegypti at 6 h post infection.After depletion of AaPGRP-LC,the transcription levels of genes of AMPs were significantly decreased during bacterial infection.The expression levels of immune-related genes involved in Toll and IMD pathways were down-regulated after interference of AaPGRP-LC.Western blotting of the purified recombinant protein AaPGRP-LC showed a clear and unique target band.[Conclusion] AaPGRP-LC modulates the transcriptional expression of genes of many essential AMPs and plays an important regulatory role in response to bacterial infection.It is involved in the regulation of IMD pathway and may also regulate the Toll pathway.The obtained recombinant AaPGRP-LC can be used in the follow-up study.%[目的]阐明在响应细菌感染的过程中,埃及伊蚊Aedes aegypti体内肽聚糖识别蛋白PGRP-LC(AaPGRP-LC)的功能.[方法]使用实时荧光定量PCR(qPCR)技术分析埃及伊蚊感染阴沟肠杆菌Enterobacter cloacae后不同时间抗菌肽(antimicrobial peptides,AMPs)基因的表达情况.结合RNA干扰技术和qPCR技术检测干扰AaPGRP-LC后免疫相关基因转录模式的变化.使用原核表达系统表达AaPGRP-LC,并通过Ni2+-NTA柱纯化,通过免疫印迹分析检测所纯化蛋白的质量.[结果]阴沟肠杆菌感染埃及伊蚊6h后埃及伊蚊体内抗菌肽基因的转录水平显著升高.干扰AaPGRP-LC并用阴沟肠杆菌感染后,埃及伊蚊体内重要抗菌肽基因的转录水平显著降低,同时埃及伊蚊免疫缺陷(immune-deficiency,IMD)通路和Toll信号(Toll-like receptors)通路的免疫相关基因的表达量也显著降低.纯化得到条带单一的AaPGRP-LC蛋白.[结论]AaPGRP-LC参与调控埃及伊蚊重要抗菌肽基因的转录,在埃及伊蚊响应细菌感染的过程中起到了重要的调控作用.AaPGRP-LC在参与调控IMD通路的同时,也可能参与了Toll信号通路的调控过程.从原核表达系统中获得的AaPGRP-LC重组蛋白可用于下一步的功能研究.