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.
The Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, Qingdao, China.
Changkao
C
Mu
Peilin
P
Zheng
Jianmin
J
Zhao
Lingling
L
Wang
Huan
H
Zhang
Limei
L
Qiu
Yunchao
Y
Gai
Linsheng
L
Song
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