Duojiao Ni

Molecular cloning, genomic organization and functional analysis of an anti-lipopolysaccharide factor from Chinese mitten crab Eriocheir sinensis

Anti-lipopolysaccharide factor (ALF) represents one kind of basic proteins, which binds and neutralizes LPS and exhibits strong antibacterial activity against Gram-negative R-type bacteria. The ALF gene of Chinese mitten crab Eriocheir sinensis (Milne Edwards, 1853) (denoted as EsALF) was identified from haemocytes by expressed sequence tag (EST) and PCR approaches. The full-length cDNA of EsALF consisted of 700 nucleotides with a canonical polyadenylation signal-sequence AATAAA, a polyA tail, and an open-reading frame of 363bp encoding 120 amino acids. The high similarity of EsALF-deduced amino acid sequence shared with the ALFs from other species indicated that EsALF should be a member of ALF family. The mRNA expression of EsALF in the tissues of heart, gonad, gill, haemocytes, eyestalk and muscle was examined by Northern blot analysis and mRNA transcripts of EsALF were mainly detected in haemocytes, heart and gonad. The temporal expression of EsALF in haemocytes after Vibrio anguillarum challenge was recorded by quantitative real-time RT-PCR. The relative expression level of EsALF was up-regulated rapidly at 2 h post-injection and reached 3-fold to that in blank group. After a drastic decrease to the original level from 4 to 8 h, the expression level increased again and reached 4-fold to that in the blank group at 12 h post-injection. The genomic DNA sequence of EsALF gene consists of 1174 bp containing three exons and two introns. The coding sequence of the EsALF mature peptide was cloned and expressed in Escherichia coli BL21(DE3)-pLysS to further elucidate its biological functions. The purified recombinant product showed bactericidal activity against both Gram-positive (G+) and Gram-negative (G-) bacteria, which demonstrated that the rEsALF was a broad-spectrum antibacterial peptide. All these results indicated that EsALF was an acute-phase protein involved in the immune responses of Chinese mitten crab, and provided a potential therapeutic agent for disease control in aquaculture.

Molecular cloning, characterization and expression of heat shock protein 90 gene in the haemocytes of bay scallop Argopecten irradians

Heat shock protein 90 (HSP90) is a highly conserved molecular chaperone that plays key roles in the folding, maintenance of structural integrity and regulation of a subset of cytosolic proteins. In the present study, the cDNA of Argopecten irradians HSP90 (designated AiHSP90) was cloned by the combination of homology cloning and rapid amplification of cDNA ends (RACE) approaches. The full-length cDNA of AiHSP90 was of 2669 bp, including an open reading frame (ORF) of 2175 bp encoding a polypeptide of 724 amino acids with predicted molecular weight of 83.08 kDa and theoretical isoelectric point of 4.81. BLAST analysis revealed that AiHSP90 shared high similarity with other known HSP90s, and the five conserved amino acid blocks defined as HSP90 protein family signatures were also identified in AiHSP90, which indicated that AiHSP90 should be a cytosolic member of the HSP90 family. Fluorescent real-time quantitative PCR was employed to examine the expression pattern of AiHSP90 mRNA in haemocytes of scallops challenged by Gram-negative bacteria Vibrio anguillarum and Gram-positive bacteria Micrococcus luteus. In both bacterial challenged groups, the relative expression level of AiHSP90 transcript was up-regulated and reached maximal level at 9h after injection, and then dropped progressively to the original level at about 48 h post challenge. The results indicated that AiHSP90 was potentially involved in the immune responses against bacteria challenge in scallop A. irradian.

Alteration of metallothionein mRNA in bay scallop Argopecten irradians under cadmium exposure and bacteria challenge.

Metallothionein (MT) is a superfamily of cysteine-rich proteins contributing to metal metabolism, detoxification of heavy metals, and immune response such as protecting against ionizing radiation and antioxidant defense. A metallothionein (designated AiMT2) gene was identified and cloned from bay scallop, Argopecten irradians. The full length cDNA of AiMT2 consisted of an open reading frame (ORF) of 333 bp encoding a protein of 110 amino acids, with nine characteristic Cys-X-Cys, five Cys-X-X-Cys, five Cys-X-X-X-Cys and two Cys-Cys motif arrangements and a conserved structural pattern Cys-x-Cys-x(3)-Cys-Tyr-x(3)-Cys-x-Cys-x(3)-Cys-x-Cys-Arg at the C-terminus. The cloned AiMT showed about 50% identity in the deduced amino acid sequence with previously published MT sequences of mussels and oysters. The conserved structural pattern and the close phylogenetic relationship of AiMT2 shared with MTs from other mollusc especially bivalves indicated that AiMT2 was a new member of molluscan MT family. The mRNA transcripts in hemolymph of AiMT2 under cadmium (Cd) exposure and bacteria challenge were examined by real-time RT-PCR. The mRNA expression of AiMT2 was up-regulated to 3.99-fold at 2 h after Listonella anguillarum challenge, and increased drastically to 66.12-fold and 126.96-fold at 16 and 32 h post-challenge respectively. Cadmium ion exposure could induce the expression of AiMT2, and the expression level increased 2.56-fold and 6.91-fold in hemolymph respectively after a 10-day exposure of 100 microg L(- 1) and 200 microg L(- 1) CdCl(2). The sensitivity of AiMT2 to bacteria challenge and cadmium stress indicated it was a new Cd-dependent MT in bay scallop and also regulated by an immune challenge. The changes in the expression of AiMT2 could be used as an indicator of exposure to metals in pollution monitoring programs and oxidative stress, and bay scallop as a potential sentinel organism for the cadmium contamination in aquatic environment.

The phenoloxidase activity and antibacterial function of a tyrosinase from scallop Chlamys farreri

Tyrosinase (TYR), also known as monophenol monooxygenase, is a ubiquitous binuclear copper-containing enzyme which catalyzes the hydroxylation of phenols to catechols and the oxidation of catechols to quinones. In the present study, the cDNA of a tyrosinase (CfTYR) was identified from scallop Chlamys farreri, which encoded a polypeptide of 486 amino acids. The CfTYR mRNA transcripts were expressed in all the tested tissues, including haemocytes, adductor muscle, kidney, hepatopancreas, gill, gonad and mantle, with the highest level in mantle. The expression level of CfTYR mRNA in haemocytes decreased significantly during 3-6 h after LPS stimulation, and reached the lowest level at 6 h (0.05-fold, P < 0.05). Then, it began to increase at 12 h (0.32-fold, P > 0.05), and reached the highest level at 24 h (2.91-fold, P < 0.05). At 3 h after LPS stimulation, the phenoloxidase activity catalyzing L-dopa and dopamine in haemolymph increased significantly to 53.13 and 40.36 U mg(-1) respectively, but it decreased to 10.82 U mg(-1) and even undetectable level after CfTYR activity was inhibited. Furthermore, the antibacterial activity of haemolymph against Escherichia coli was also increased significantly at 3 h after LPS stimulation, but it decreased significantly when the haemolymph was treated by TYR inhibitor. The recombinant protein of the mature CfTYR peptide expressed in the in vitro Glycoprotein Expression Kit displayed phenoloxidase activity of 64.36 ± 5.51 U mg(-1) in the present of trypsinase and Cu(2+). These results collectively suggested that CfTYR was a homologue of tyrosinase in scallop C. farreri with the copper-dependence phenoloxidase activity, and it could be induced after immune stimulation and mediate immune response for the elimination of invasive pathogens in scallop.