Recombinant expression of Crassostrea gigas defensin in Pichia pastoris and its antibacterial activity / 生物工程学报

Defensins are endogenous cationic antimicrobial peptides rich in arginine and cysteine residues. They are important immune factors resisting pathogenic bacteria infection for mollusks. The 43 amino acid residues near the carboxyl terminal for Crassostrea gigas defensin (CgD) form its mature peptide region, responsible for the biological activity of CgD. First, two target genes, CgDH⁺ (with 6×His-tag at 3' end) and CgDH- (without 6×His-tag at 3' end) were separated and amplified by RT-PCR with specific primers from Crassostrea gigas mantle. These two target genes were ligated to the expression vector pPICZαA to construct recombinant expression vectors, pPICZαA-CgDH⁺ and pPICZαA-CgDH-, which were transformed into competent Pichia pastoris X-33 cells by electroporation respectively. The recombinant target proteins, CgDH⁺ and CgDH-, were induced for 72 h with 1% methanol at 29 °C and 250 r/min. The recombinant CgDH⁺ (5.78 kDa) was purified by immobilized metal affinity chromatography (IMAC), and identified by MALDI-TOF-TOF analysis, demonstrating that it was the expected target protein. Based on the concentration of the purified product, the estimated yield of recombinant CgDH⁺ was 2.32 mg/L. Antimicrobial assay showed that the culture medium supernatant containing recombinant CgDH⁺ and recombinant CgDH-, respectively, had activities against Staphylococcus aureus and Pseudomonas aeruginosa, indicating that the existence of 6×His tag in the recombinant proteins do not affect their biological activities.

Evaluación del crecimiento y la sobrevivencia en cultivos de ostra japonesa desarrollados en Meanguera del Golfo, departamento de La Unión: en vínculo con CENDEPESCA / Growth assessment and survival of japanese oyster crops developed in Meanguera del Golfo, department of La Unión

En El Salvador, el cultivo de Ostra del Pacifico (Crassostrea gigas) es una actividad nueva, ya que en el país se desconocía la reproducción, el manejo de la semilla y el desarrollo del cultivo engorde en las zonas de cultivo. Es por ello que el Centro de Desarrollo de la Pesca y la Acuicultura (CENDEPESCA) juntamente con la cooperación técnica del Gobierno del Japón a través de la Agencia de Cooperación Internacional del Japón (JICA) ha implementado el "Proyecto para el Desarrollo de la Acuicultura de Moluscos en El Salvador" que ha introducido una nueva especie de ostra en El Salvador, la Ostra del Pacifico (Crassostrea gigas) con el objetivo de mejorar la calidad de vida de las comunidades de pescadores, que solamente se ha dedicado a la extracción de los recursos del medio natural, mas no al cultivo directamente a causa de la falta de recursos para la realización de la reproducción de los organismos, adquisición de la semilla e insumos para el desarrollo del cultivo. La Escuela Especializada en Ingeniería ITCA-FEPADE regional La Unión se unió a este esfuerzo y realizó una investigación en cooperación con CENDEPESCA, la cual inició con la siembra de 7,000 semillas en el sistema suspendido de Ostra japonesa, posterior a ello se elaboró un cronograma de trabajo para el control y se desarrollo del crecimiento del cultivo en los 6 meses de duración de la investigación,

In El Salvador, the cultivation of Pacific Oyster (Crassostrea gigas) is a new activity, due to, in the country, the reproduction, the handling of the seed and the development of the fattening crop in the cultivation areas were unknown. That is why the Center for the Development of Fisheries and Aquaculture (CENDEPESCA) together with the technical cooperation of the Government of Japan through the Japan International Cooperation Agency (JICA) has implemented the "Project for the Development of Aquaculture of Mollusks in El Salvador "that has introduced a new species of oyster in El Salvador, the Pacific Oyster (Crassostrea gigas) with the aim of improving the quality of life of the fishing communities, which has only been dedicated to the extraction of the resources of the natural environment, but not to the cultivation directly because of the lack of resources for the realization of the reproduction of the organisms, acquisition of the seed and inputs for the development of the crop. ITCA-FEPADE La Unión joined this effort and carried out an investigation in cooperation with CENDEPESCA, which began with the sowing of 7,000 seeds in the suspended system of Japanese Oyster, after which a schedule of work for the control and development of the growth of the crop in the 6 months of duration of the investigation,

Evaluación del crecimiento y la sobrevivencia de ostra japonesa (Cassostrea gigas) en cultivos desarrollados en Meanguera del Golfo, departamento de La Unión, El Salvador / evaluation of growth and survival of japanese oyster (Cassostrea gigas) in crops grown in Meanguera del Golfo, department of La Unión, El Salvador

La Escuela Especializada en Ingeniería ITCA-FEPADE Centro Regional MEGATEC La Unión, a través de la carrera "Técnico en Manejo Integrado de Recursos Costero Marinos, con especialidad en Acuicultura y Pesquería" y en coordinación con el Centro de Desa- rrollo de la Pesca y la Acuicultura (CENDEPESCA), se desarrolló el proyecto de investigación aplicada para determinar el crecimiento y sobre vivencia en cultivos de Ostra Japonesa, con la finalidad de buscar y proponer alternativas de cultivo a los productores acuícolas del municipio de Meanguera del Golfo, departamento de La Unión. Para la implementación de este proyecto de investigación, se realizó una siembra de 7,000 semillas de ostras, que fueron colocadas suspendidas en la columna de agua de un sistema de cultivo constituido por un Long Line y linternas de 5 pisos cada una. Al final del periodo de cultivo se cosecharon organismos con tamaño promedio de 5.5 cm, con una sobre vivencia del 7%. El mayor depredador encontrado fue el caracol (Cymatium wiegmanni) y el rango de temperaturas durante este periodo de cultivo fue de 30.1 y 30.5 °C.

The Specialized School in Engineering ITCA-FEPADE, regional Centre MEGATEC in La Unión, through its career "Technician in Integrated Control Coastal marine resources, with an aquaculture and fishery emphasis" and in conjunction with The Development of Fishing and Aquaculture Center (CENDEPESCA), implemented an applied research project to determine the growth and survival in Japanese oyster farming so as to find and propose farming alternatives to aquaculture producers from the municipality of Meanguera del Golfo, departamento de la Unión. To implement this research project, 7,000 oysters were farmed and placed hanging from the water column of a farming system made up of a Long line and flashlights of 5 levels each. By the end of the farming period, organisms with an average size of 5.5 cm were harvested with a 7% survival chance. The main oyster predator found was the (Cymatium wiegmanni) snail, and the temperature range during the farming period was between 30.1 and 30.5 °C.

Occurrence of potentially pathogenic Vibrio in oysters (Crassostrea gigas) and waters from bivalve mollusk cultivations in the South Bay of Santa Catarina

Introduction This research aimed to identify and quantify potentially pathogenic Vibrio from different cultivations of bivalve shellfish in the State of Santa Catarina, Brazil, and water regions in the South Bay, as well as correlate the incidence of these microorganisms with the physicochemical parameters of marine waters. Methods Between October 2008 and March 2009, 60 oyster and seawater samples were collected from six regions of bivalve mollusk cultivation, and these samples were submitted for Vibrio counts. Results Twenty-nine (48.3%) oyster samples were revealed to be contaminated with one or more Vibrio species. The Vibrio parahaemolyticus and Vibrio vulnificus counts in the samples ranged from < 0.5 log10 Most Probable Number (MPN) g–1 to 2.3 log10 MPN g–1 oyster and from < 0.5 log10 MPN g–1 to 2.1 log10 MPN g–1 oyster, respectively. Of the 60 seawater samples analyzed, 44 (73.3%) showed signs of contamination with one or more vibrio species. The counts of V. parahaemolyticus and V. vulnificus in the samples ranged from < 0.3 log10 MPN·100mL–1 to 1.7 log10MPN·100mL–1 seawater and from < 0.3 log10 MPN·100mL–1 to 2.0 log10 MPN·100mL–1 seawater, respectively. A positive correlation between V. vulnificus counts and the seawater temperature as well as a negative correlation between the V. parahaemolyticus counts and salinity were observed. Conclusions The results suggest the need to implement strategies to prevent vibrio diseases from being transmitted by the consumption of contaminated bivalve shellfish. .

Non-specific Defensive Factors of the Pacific Oyster Crassostrea gigas against Infection with Marteilioides chungmuensis: A Flow-Cytometric Study

In order to assess changes in the activity of immunecompetency present in Crassostrea gigas infected with Marteilioides chungmuensis (Protozoa), the total hemocyte counts (THC), hemocyte populations, hemocyte viability, and phagocytosis rate were measured in oysters using flow cytometry. THC were increased significantly in oysters infected with M. chungmuensis relative to the healthy appearing oysters (HAO) (P<0.05). Among the total hemocyte composition, granulocyte levels were significantly increased in infected oysters as compared with HAO (P<0.05). In addition, the hyalinocyte was reduced significantly (P<0.05). The hemocyte viability did not differ between infected oysters and HAO. However, the phagocytosis rate was significantly higher in infected oysters relative to HAO (P<0.05). The measurement of alterations in the activity of immunecompetency in oysters, which was conducted via flow cytometry in this study, might be a useful biomarker of the defense system for evaluating the effects of ovarian parasites of C. gigas.

Preparation and study on the physicochemical characteristics of glycoprotein from Crassostrea gigas / 中国海洋药物

Objective To study the preparation and the physicochemical characteristics of glycoprotein from Crassostrea gigas.Methods Oyster collected in Qingdao was taken as the experiment materials,and was extracted with H2O in low temperature.The extracts were purified using Sephacryl s-100 HR gel chromatography and HPLC.Three glycoprotein F22,F33,F42 were finally isolated from the oyster extracts.Results The physicochemical characteristics of glycoprotein were studied.The results showed that the pI value of F22 was 5.5,relative molecular mass of F22 was 34230Da,the protein contents of F22,F33,F42 were 34.1%,19.7% and 12.5%,the saccharide contents of F22,F33,F42 were 35.9%,28.8% and 40%.Conclusion The research result would be used for the study of bioactive component from Crassostrea gigas.

Study of isolation,preparation and structure identification of peptides from hydrolyzates of oyster / 中国海洋药物

Objective To study the preparation and the structure identification of peptides from hydrolyzates of oyster. Methods The oyster protein was hydrolyzed with trypsin. The hydrolysates were purified using Sephadex G25 gel chromatography,DEAE-Sepharose FF ion exchange chromatography,reversed-phase HPLC on C18 column. Results The peptide F32 was finally isolated from the oyster hydrolysates.The oyster protein was hydrolyzed with trypsin. IR spectrum showed the conformation of peptide chain was ?-helix.the amino acid sequence of F32 was determined by ESI-MS/MS. The sequence of F32 was as follows:Arg-Gln-Ile or Leu-Gly-Ala-Thr-Asn-Ala. Conclusion The research result would provide foundation for the structure-activity research of the peptide F32.