ABSTRACT
β-defensin is a primary protein immune factor in channel catfish's (Ietalurus punetaus) resistance to pathogenic microorganisms. Its primary structure contains a signal peptide composed of 24 amino acid residues at the N-terminal and a mature peptide composed of 43 amino acid residues at the C-terminal. The mature peptide region is responsible for the biological activity of β-defensin. In the present study, a recombinant strain of Pichia pastoris that produces channel catfish β-defensin, was constructed to realize the biosynthesis of channel catfish β-defensin based on eukaryotic expression. First, the β-defensin gene "IPBD" was isolated from the skin of channel catfish by RT-PCR. After linking it with the expression vector pPICZA, pPICZA-IPBD was transferred into competent P. pastoris X-33 cells to obtain recombinant P. pastoris strains. The yeast transformants with multi-copy gene inserts were obtained by using the culture medium containing 1 000 μg/mL zeocin. Using BMM culture medium (without amino nitrogen culture medium) instead of BMMY culture medium (with amino nitrogen culture medium), the fermentation and culture conditions of the recombinant strain were optimized, and the optimal conditions for producing channel catfish β-defensin were determined as follows: the expression was induced for 96 h with 1.0% methanol at 28 °C , 250 r/min. Purified protein with molecular weight of 5.98 kDa was obtained by nickel affinity chromatography, and MALDI-TOF/TOF mass spectrometry proved that it was the expected recombinant IPBD. The antibacterial test results showed that the inhibitory rates of recombinant IPBD on Gram-positive Staphylococcus aureus and Listeria monocytogenes and Gram-negative Pseudomonas aeruginosa were 69.6%, 71.6% and 65.8%, respectively. This study provides a recombinant DNA technique for the development of small molecule natural antibacterial peptide from fish.
ABSTRACT
This study characterises morphologically Trichodina heterodentata Duncan, 1977 from channel catfish, Ictalurus punctatus (Rafinesque, 1818) in the State of Santa Catarina, Brazil. Body and gill smears were air-dried at room temperature, impregnated with silver nitrate and/or stained with Giemsa. Ten characteristics were selected to compare the present material with other morphological characterisations of T. heterodentata. Prevalence rate was 100 percent, mean intensity 89,333.70 (3,125 to 299,100 parasites per host). Trichodina heterodentata was considered medium-sized trichodinid with mean body diameter 59.4 ± 8.5 μm, denticulate ring 38.5 ± 4.5 μm, adhesive disc 60.2 ± 6.7 μm diameter and 24.4 ± 1.6 denticles. In relation to previous reports of T. heterodentata this material resembles in 90 percent of the analysed characters. This work confirms the biometrical variation that exists in the different populations of T. heterodentata. A list of hosts and comparative measurements of T. heterodentata are presented and the channel catfish is considered a new host.
Este estudo caracteriza morfologicamente Trichodina heterodentata Duncan, 1977 em bagre-do-canal, Ictalurus punctatus (Rafinesque, 1818) no Estado de Santa Catarina, Brasil. Esfregaços do corpo e brânquias foram secados à temperatura ambiente, impregnados com nitrato de Prata e/ou corados com Giemsa. Dez características foram selecionadas para comparar o presente material com as diferentes caracterizações morfológicas de T. heterodentata. A taxa de prevalência foi de 100 por cento, a intensidade média foi de 89.333.75 (3.125 a 299.100 parasitos por hospedeiro). Trichodina heterodentata foi considerado um tricodinídeo de tamanho médio com a média do diâmetro do corpo de 59,4 ± 8.5 μm, anel denticulado 38,5 ± 4,5 μm, disco adesivo 60,2 ± 6,7 μm de diâmetro e 24,4 ± 1,6 dentículos. Em relação a registros prévios de T. heterodentata, 90 por cento das características foram semelhantes. Este trabalho confirma a variação biométrica que existe em diferentes populações de T. heterodentata. Uma lista de hospedeiros e medidas comparativas de T. heterodentata são apresentadas e o bagre-do-canal considerado um novo hospedeiro.
Subject(s)
Animals , Ciliophora Infections/epidemiology , Fish Diseases/parasitology , Ictaluridae/parasitology , Oligohymenophorea/classification , Oligohymenophorea/isolation & purification , Brazil/epidemiology , Fish Diseases/epidemiology , PrevalenceABSTRACT
A novel method was established for the qualitative and quantitative determination of fatty acids in Channel Catfish muscle by gas chromatography-electron ionization-mass spectrometry (GC-EI-MS) after supercritical carbon dioxide fluid extraction (SFE-CO_2). The extraction parameters for the methodology were optimized). The optimal conditions were extraction pressure of 25 MPa at 45 ℃ and extraction time of 100 min at the rate of carbon dioxide 30 L/h. The fatty acids in the muscle oil were derived by boron-trifluoride method). The saponification time was 10 min, and the esterication time was 20 min. The obtained fatty acid methylesters were separated by gas chromatography using a HP-Innowax capillary column, and were detected by electron) ionization) mass spectrometry. Full scan mode and SIM mode were used for the qualitative and quantitative analysis), respectively. In the SIM mode, saturated fatty acids were determined with m/z 74, mono-unsaturated) fatty acids were determined with m/z 55, double-unsaturated fatty acids were determined with m/z 67, and polyunsaturated fatty acids were determined with m/z 79. The detection limits of 14 fatty acids were 2.2-20.0 μg/L(S/N=3)), and the quantitative limits were 7.39-59.85 μg/L(S/N=10). The recoveries fell in the range from 90.0% to 111.2%(n=4), and the relative standard deviation was between) 2.0% and 5.9%. This effective, sensitive and reproducible method can be used for the determination of fatty acids in Channel Catfish muscle sample.