The grass carp (Ctenopharyngodon idella) collectin gene was cloned from mixed liver and kidney cDNA library. The full length sequence of grass carp collectin was 1128 bp, contained a 5' untranslated region of 229 bp and a 3' untranslated region of 104 bp. The open reading frame of grass carp collectin was 795 bp which could code a 264 amino acids polypeptide, including a terminal codon. Phylogenetic analyses showed that grass carp collectin shared the highest homology with that of zebrafish (Danio rerio). To understand the function of grass carp collectin, we expressed and purified the recombinant protein (P(CRD)) that comprised carbohydrate recognition domain (CRD). Agglutination of Aeromonas hydrophila and Staphylococcus aureus etc. and sugars inhibition experiments showed that: galactose, glucose, mannose and maltose could inhibit the agglutination of Aeromonas hydrophila. Maltose could lower the agglutination of Staphylococcus aureus, whereas peptidoglycan and glucose inhibited it well. In addition, the activity of grass carp collectin could not dependent on Ca(2+).
Amino Acid Sequence ; Animals ; Base Sequence ; Carps ; genetics ; Cloning, Molecular ; Collectins ; genetics ; physiology ; Fish Proteins ; genetics ; physiology ; Molecular Sequence Data ; Phylogeny ; Zebrafish ; genetics

To express Pleurocidin in Escherichia coli and to enhance the secretory efficiency of the fusion protein, the gene encoding Pleurocidin was ligated with Cherry DNA sequence via blunt-end ligation. Then this fusion gene was cloned into pET22b (+) vector and the recombinant plasmid was transformed into E. coli BL21 (DE3). Lactose was used to induce expression of fusion protein. The recombinant plasmid pET22b (+) -CP was successfully constructed and high-level expression of fusion protein was induced with lactose. Statistics showed that addition of glycine after 16 h of induction significantly enhanced the secretory efficiency of the fusion protein. After hydrolysis of the fusion protein by diluted hydrochloric acid and some further purification steps, r-Pleurocidin was obtained with antibacterial activity against E. coli DH5α and Bacillus subtilis BS168. In conclusion, the fusion protein was expressed in E. coli and biologically active r-Pleurocidin was obtained after hydrochloric acid cleavage and purification.
Animals ; Cloning, Molecular ; Escherichia coli ; metabolism ; Fish Proteins ; biosynthesis ; Flounder ; Recombinant Fusion Proteins ; biosynthesis

Fuantai-03(FAT-03), isolated from the Dasyatis akajei, has a strong antiangiogenic activity. The recombinant Fuantai-03 (GST/rFAT-03) fusion protein can be obtained with the DNA recombination technology. In this study, expression conditions of GST/rFAT-03 were optimized by response surface experimental design method. The constructed engineering bacteria containing GST/rFAT-03 plasmid was induced by isopropy-beta-D-thiogalactosid (IPTG), the GST affinity column was used for isolation and purification, and then the effects of different culture time, IPTG concentration, induction temperature and induction time on the amount of soluble GST/rFAT-03 fusion protein were compared. The culture time for optimal expression was 6.13 h, IPTG concentration was 0.36 mmol/L, induction temperature was 19.71 degrees C, and induction time was 13.60 h. The amount of soluble GST/rFAT-03 fusion protein was 7.57 mg/L under above mentioned expression conditions. The results also showed that rFAT-03 significantly inhibited angiogenesis in chicken chorioallantoic membrane in a dose-dependent manner. Moreover, the soluble form of the target protein is useful for further work on purification and on studying its biological function.
Angiogenesis Inhibitors ; biosynthesis ; genetics ; Animals ; Chickens ; Chorioallantoic Membrane ; blood supply ; Escherichia coli ; genetics ; metabolism ; Fish Proteins ; biosynthesis ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; Skates (Fish)

Interferon response is the first line of host defense against virus infection. Recent years have witnessed tremendous progress in understanding of fish innate response to virus infection, especially in fish interferon antiviral response. A line of fish genes involved in interferon antiviral response have been identified and functional studies further reveal that fish possess an IFN antiviral system similar to mammals. However, fish virus-induced interferon genes contain introns similar to mammalian type III interferon genes although they encode proteins similar to type I interferons, which makes it hard to understand the evolution of vertebrate interferon genes directly resulting in a debate on nomenclature of fish interferon genes. Actually, fish display some unique mechanisms underlying interferon antiviral response. This review documents the recent progress on fish interferon response and its molecular mechanism.
Animals ; Fish Diseases ; immunology ; virology ; Fish Proteins ; genetics ; metabolism ; Fishes ; immunology ; virology ; Gene Expression Regulation ; Interferons ; genetics ; immunology ; STAT1 Transcription Factor ; metabolism ; Virus Diseases ; immunology ; veterinary

Animals ; Carps ; genetics ; Cloning, Molecular ; Fish Proteins ; genetics ; Hydroxylation ; Hypoxia-Inducible Factor 1, alpha Subunit ; genetics ; Lakes

A mutant strain ΔVcrV was constructed by using homologous recombination method for investigating the function of the VcrV gene in Vibrio alginolyticus type Ⅲ secretion system. The genetic stability of ΔVcrV was detected by PCR, and the biological characteristics between the mutant and the wild type strains were compared. ΔVcrV muntat had no significant changes in growth rate and autoagglutination compared with the wild type strain, but the ability to form biofilms was reduced, and the LD₅₀ was increased by 16.5 times. The swimming and swarming motility of the mutant strain ΔVcrV were significantly enhanced, while cell adhesion was significantly reduced than the wild strain (P < 0.01). The tolerance of ΔVcrV mutant to H₂O₂ and NaCl was decreased. Compared with that of the wild type strain, the sensitivity of ΔVcrV mutant to cefuroxime, medimycin and clindamycin was increased, but to amikacin and polymxin B was decreased. The reactive oxygen species (ROS) content of ΔVcrV mutant was significantly decreased (P < 0.01), and the indexes of proline, peptidoglycan, β-lactamase, catalase, superoxide dismutase and glutathione peroxidase of ΔVcrV mutant were significantly increased than that of the wild type strain (P < 0.01). The biological characteristics of ΔVcrV mutant indicated that VcrV gene was involved in pathogenicity and various biological functions of V. alginolyticus type Ⅲ secretion system.
Animals ; Bacterial Proteins/metabolism* ; Fish Diseases ; Hydrogen Peroxide/metabolism* ; Type III Secretion Systems ; Vibrio Infections ; Vibrio alginolyticus/genetics*

Hepcidin are small cationic peptides with antibacterial activity expressed mainly in the liver of living organisms, and they play important roles in the host's immune response against microbial invasion and regulation of iron metabolism. Thus, they are considered to be good substitutes for traditional antibiotics. It is a good choice that the antimicrobial peptides are prepared by recombinant DNA expression. In the present study, two hepcidin mature peptide cDNAs from channel catfish (Ictalurus punctatus) (mCH) and tilapia (Oreochromis niloticus) (mTH) were connected by SOE-PCR in order to obtain more recombinant hepcidin with broad antimicrobial spectrum, and EcoR I and Not I sites were added to 5'- and 3'- ends of the fragment, respectively. The recombinant eukaryotic expression vector "pPIC9K-mCH-mTH" was successfully constructed, and transformed into Pichia pastoris GS115. The transformants containing multicopy gene insertion were selected by using different concentrations of G418 and other specific mediums, and identified by PCR for yeast genomic DNA. Expression was induced by adding 1% methanol at 30 degrees C for different times. Tricine-SDS-PAGE analysis demonstrated that the most appropriate expression time was 72 h, at which a high expression yield (77 mg/L) for the target protein was exhibited. The highly purified target protein was obtained from the fermentation supernatant by SP-Sepharose cation exchange chromatography. Bacteriostatic activity assay demonstrated that the fermentation supernatant containing the target protein and purified recombinant target protein had bacteriostatic activities against gram-positive and gram-negative bacterium. The present result provides the important initial value for industrial production of hepcidin antimicrobial peptide.
Animals ; Electrophoresis, Polyacrylamide Gel ; Fish Proteins ; biosynthesis ; genetics ; Fishes ; genetics ; Gram-Negative Bacteria ; Gram-Positive Bacteria ; Hepcidins ; biosynthesis ; genetics ; Pichia ; Polymerase Chain Reaction ; Recombinant Proteins ; biosynthesis

Vibrios are universal conditioned-pathogenic bacteria in marine culture environment, and the outbreak of vibrio disease resulted in a serious damage to aquaculture. Considering that vibrio disease in aquatic species, especially fishes, usually originated from mixed infection of different species (serotypes or subspecies) of vibrios, it is important to select the potential cross-protective protein antigens as candidates of polyvalent or combined vaccines. In present research, several strains of vibrios were isolated from infected large yellow croaker (Pseudosciaena crocea) and subsequently identified as six strains of V. harveyi, one V. parahaemolyticus and one V. alginolyticus by physiological, biochemical and molecular biological methods. Their outer membrane proteins (OMPs) were extracted and the SDS-PAGE and Western blotting results show that three immuno-blots with common molecular weight presented at approximate 45 kDa, 35 kDa and 22 kDa on their OMP electrophoretogram, indicating the existence of antigens with cross-protection in their OMPs. With the aids of combination of two-dimensional electrophoresis (2-D) and Western blotting and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), a deduced porin (GenBank Accession No. ZP_01260407) from V. alginolyticus and a maltoporin precursor (GenBank Accession No. NP_801154) from V. parahaemolyticus were able to react with polyclonal antibody to whole V. harveyi, suggesting these two proteins could act as the cross-protective antigens and the vaccines prepared with these porins would be probable to bring cross protection to three different vibrios.
Animals ; Antigens, Bacterial ; immunology ; Bacterial Outer Membrane Proteins ; immunology ; Cross Reactions ; Fish Diseases ; microbiology ; Perciformes ; microbiology ; Vibrio ; classification ; immunology ; isolation & purification ; pathogenicity ; Vibrio Infections ; microbiology

The sequence variation of medicinal fish of Culter (Pisces: Cyprinidae) was analyzed by using cytochrome c oxidase subunit I (COI) sequencing collected from different regions of the Yangtze River basin, and we examine whether barcoding of COI can be used to discriminate medicinal fish of Culter. The AT content in the COI region of medicinal fish of Culter was higher than that of GC, which was similar with other species of Cypriniformes. Ninty-six percent of nucleotide changes were observed at the 3rd codon position of COI sequence, but the amino acid compositions translated by COI sequences of all Culter fish stayed the same. It is suggested that most synonymous mutations might occur at the 3rd position. The average Kimura-2-parameter (K2P) distance within-species was lower than 1%, and the K2P distance of pairwise-species was 10 times as much as that of within-species. The phylogenetic tree estimated by Neighbour-joining method indicated that species within genera invariably clustered, and generally so did individuals within species. Individuals from operational taxonomic units designated as different Culter species, supporting morphological evidence for each of these being separate species. It is suggested that the COI barcoding can be used to identify medicinal fish species of Culter.
Animals ; China ; Cyprinidae ; classification ; genetics ; DNA Barcoding, Taxonomic ; DNA, Mitochondrial ; genetics ; Electron Transport Complex IV ; genetics ; Fish Proteins ; genetics ; Medicine, Chinese Traditional ; Molecular Sequence Data ; Phylogeny

Cystatin, which widely distributed in both tissues and body fluids of animal and plant, was a superfamily of cysteine proteinase inhibitors. It could form activity-inhibitor complexes with cysteine proteinases to inhibit the hydrolytic activity of proteinases. Cystatin played important roles not only in the inhibition of the proteolytic degradation of fish muscle, but also in biological defense systems against invaders. To explore the functions of fish cystatin and the potential values in fish disease prevention and cure, as well as seafood processing, the recombinant yeast strains which could express Chinese sturgeon cystatin were constructed. First, the cystatin cDNA of Chinese sturgeon, which had been PCR modified, was subcloned into yeast integrated vector pPICZaA. After extracted and purified, the recombinant plasmids were linearized by Sac I. The yeast Pichia pastoris GS115 strain was transformed by use of the Lithium Chloride transformation method, and the recombinant cystatin yeast strains got. After 0.5% methanol induction, SDS-PAGE analysis of the culture supernatant indicated that the yield of recombinant cystatin was about 215mg x L(-1) with the percentage about 73.6%. The recombinant cystatin was purified through Q-Sepharose anion-exchange chromatography, and the purity reached about 94.2%. The inhibitory activity of recombinant cystatin was measured by inhibiting the proteinase activity of papain. The results showed that about 1 microg recombinant cystatin could inhibit the activity of 15 microg papain. Heat stability assay results showed that there was a decrease in inhibitory activity of cystatin with the increasing of temperature. When solution of recombinant cystatin was kept at 70 degrees C for 5min, the inhibitory activity reduced fast. While the recombinant cystatin was heated to 90 degrees C for 5min, the inhibitory activity of recombinant cystatin was undetected. The inhibitory activity for recombinant Chinese sturgeon cystatin was higher than that of CPI (cysteine proteinase inhibitor) from seeds of corn, that about 1 microg purified CIP could inhibited the activity of 0.278 microg papain. But the heat stability of recombinant cystatin is lower than that of the corn CPI. The expression level and the activity of recombinant cystatin from yeast Pichia pastoris were higher than those from E. coli. Moreover, recombinant cystatin from Pichia pastoris was easier to separate and purify. This paper reported that recombinant fish cystatin was produced in a highly efficient expression system based on the methylotrophic yeast, further work will focus on the function of recombinant Chinese sturgeon cystatin to resist fish disease and explore the value of cystatin as a food additive to inhibit cysteine proteinases during surimi processing.
Animals ; Cystatins ; genetics ; metabolism ; pharmacology ; Cysteine Proteinase Inhibitors ; genetics ; metabolism ; pharmacology ; Enzyme Activation ; drug effects ; Fish Proteins ; genetics ; metabolism ; Pichia ; genetics ; metabolism ; Polymerase Chain Reaction ; Protein Stability ; Temperature