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