›› 2010, Vol. 53 ›› Issue (2): 131-138.doi:

• 研究论文 • 上一篇    下一篇


陈杨, 凌尔军   

  • 出版日期:2010-03-25 发布日期:2010-02-20
  • 通讯作者: 凌尔军

Molecular cloning and functional characterization of PGRP-LC1 in Anopheles stephensi (Diptera: Culicidae)

CHEN Yang, LING Er-Jun   

  • Online:2010-03-25 Published:2010-02-20

摘要: 天生免疫系统是昆虫抵御外界病原入侵的主要方式。目前研究发现, Imd信号通路与按蚊感染柏氏疟原虫Plasmodium berghei的强度密切相关, 而PGRP-LC1是Imd信号通路最上游的受体之一。为了研究斯氏按蚊Anopheles stephensi肽聚糖识别蛋白PGRP-LC1, 采用RT-PCR并结合RACE技术克隆斯氏按蚊PGRP-LC1基因, 通过序列比较分析, 得到两条cDNA序列, 其开放阅读框分别为1 365 bp和1 290 bp, 3′非编码区为320 bp, 5′非编码区为240 bp。将两条cDNA分别命名为AsPGRP-LC1a(GenBank注册号 GU214232)和AsPGRP-LC1b(GenBank注册号GU214233)。AsPGRP-LC1a编码454个氨基酸, 分子量约为49.07 kDa;AsPGRP-LC1b编码429个氨基酸, 分子量约为46.3 kDa。AsPGRP-LC1bAsPGRP-LC1a少一个长度为75 bp的外显子, 该外显子在冈比亚按蚊Anopheles gambiae PGRP-LC1基因的某些可变剪切形式中也有发现。分别将两个斯氏按蚊PGRP-LC1基因在冈比亚按蚊细胞系L3-5和斯氏按蚊细胞系MSQ43中过量表达, 通过双荧光素酶检测系统检测抗菌肽的表达情况, 结果显示克隆得到的PGRP-LC1基因在两种细胞系中均能够启动Imd信号通路, 为进一步研究斯氏按蚊的Imd信号通路提供了依据。

关键词: 按蚊, 斯氏按蚊, 疟疾, 肽聚糖识别蛋白, Imd信号通路, 克隆, 过量表达, 抗菌肽

Abstract: Insect defense against pathogen invasion mainly rely on innate immunity system. Recent research showed that Imd pathway is involved in limiting the number of Plasmodium berghei oocysts developing in mosquito midgut, and PGRP-LC1 is one of the receptors of Imd pathway. In order to investigate PGRP-LC1 in Anopheles stephensi, we cloned the PGRP-LC1 gene through a combination of reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) approaches. Two cDNA fragments, with the open reading frame (ORF) of 1 365 bp and 1 290 bp respectively, were obtained. The 3′untranslated region (UTR) is 320 bp, and the 5′ UTR 240 bp. We named the two sequences as AsPGRP-LC1a (GenBank accession no. GU214232) and AsPGRP-LC1b (GenBank accession no. GU214233), respectively. AsPGRP-LC1a encodes a polypeptide of 454 amino acids with a predicted molecular weight of 49.07 kDa. AsPGRP-LC1b encodes a polypeptide of 429 amino acids with a predicted molecular weight of 46.3 kDa. AsPGRP-LC1b contains one exon less than AsPGRP-LC1a. The exon is 75 bp and present in some of the alternative splicing isoforms of Anopheles gambiae PGRP-LC1 too. The two PGRP-LC1 genes were over-expressed in both A. gambiae cell line L3-5 and A. stephensi cell line MSQ43. The expression profile of antimicrobial peptides was monitored by dual luciferase assay system, and the results showed that the PGRP-LC1 we cloned could activate Imd pathway in both cell lines, which provided basis for further investigations of Imd pathway in A. stephensi.

Key words: Anopheline mosquito, Anopheles stephensi, malaria, peptidoglycan recognition protein, Imd pathway, cloning, over-expression, antimicrobial peptide