Molecular characterization and expression of a crustin-like gene from Chinese mitten crab, Eriocheir sinensis.
Crustin is a cysteine-rich antibacterial peptide which widely distributes within decapod crustaceans. In the present study, the cDNA encoding crustin-like peptide (designated CrusEs) was cloned from Chinese mitten crab Eriocheir sinensis by using rapid amplification of cDNA ends (RACE) approaches and expressed sequence tag analysis. The full-length cDNA of CrusEs was of 796bp, containing a 5' untranslated region (UTR) of 214bp, a 3' UTR of 267bp with a poly(A) tail, and an open reading frame (ORF) of 315bp encoding a polypeptide of 104 amino acids including a putative signal peptide of 21 amino acids. A WAP domain and the consensus framework existing in class I crustins were both identified in CrusEs, suggesting that CrusEs is a new member of type I crustins. Quantitative real-time RT-PCR was employed to examine the expression of CrusEs, and its mRNA transcript was mainly detected in haemocytes and gill. The CrusEs mRNA transcript in haemocytes was down-regulated after the challenge with Gram-positive bacteria Micrococcus luteus, while Gram-negative bacteria Listonella anguillarum did not induce significant variation of CrusEs mRNA. The mature peptide of CrusEs was cloned into pET-21a(+) with a C-terminal hexa-histidine tag fused in-frame, and expressed in Escherichia coli, and the recombinant CrusEs inhibited the growth of Gram-positive bacteria with low MIC. The results indicate that CrusEs is a potent antibacterial protein against Gram-positive bacteria infection, and it may play an important role in innate immune response of E. sinensis.
Mu
Changkao
C
The Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, Qingdao, China.
Zheng
Peilin
P
Zhao
Jianmin
J
Wang
Lingling
L
Zhang
Huan
H
Qiu
Limei
L
Gai
Yunchao
Y
Song
Linsheng
L
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D023181:Antimicrobial Cationic Peptides / Q000235:genetics* / Q000302:isolation & purification / Q000494:pharmacology; D001483:Base Sequence; D003386:Brachyura / Q000235:genetics* / Q000276:immunology / Q000382:microbiology; D020224:Expressed Sequence Tags; D006094:Gram-Positive Bacteria / Q000254:growth & development; D008826:Microbial Sensitivity Tests; D008969:Molecular Sequence Data; D010802:Phylogeny; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D019105:Random Amplified Polymorphic DNA Technique; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA
D023181:Antimicrobial Cationic Peptides; D012333:RNA, Messenger; D011994:Recombinant Proteins