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ABSTRACT: Edwardsiella tarda is an important cause of haemorrhagic septicaemia in fish and also of gastro- and extra-intestinal infections in humans. We have recently demonstrated that the PhoP-PhoQ two-component regulatory system plays important roles in both virulence and stress tolerance in E. tarda. In this study the proteomes of the wild-type and phop mutant strains were compared to define components of the PhoP regulon in E. tarda EIB202. Overall, 18 proteins whose expression levels exhibited a 2-fold or greater change were identified; 13 of these proteins were found to require the presence of PhoP for full expression, while 5 were expressed at a higher level in the phoP mutant background. Identified proteins were representing diverse functional categories including energy production, amino acid metabolism, and oxidative stress defense. Quantitative real-time PCR (qRT-PCR) analysis of the mRNA levels for the identified proteins confirmed the proteomics data. Interestingly, β subunit of the F1F0 ATP synthase (AtpD) playing an important role in growth and virulence of E. tarda, was listed as one of the proteins whose expression was greatly dependent on PhoP. The F1F0 ATP synthase was encoded in a gene cluster (atpIBEFHAGDC) and the 9 genes were transcribed as an operon. PhoP positively regulated the transcription of the 9 ATP synthase genes and exerted this effect through direct binding to the promoter of atpI. Overall, the results provide new insights into the PhoP regulon and unravel a novel role for PhoP in the regulation of the F1F0 ATP synthase.
Microbiology 05/2013; · 3.06 Impact Factor
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ABSTRACT: Effects of different light conditions on development, growth, and secondary metabolism of three marine-derived filamentous fungi were investigated. Darkness irritated sexual development of Aspergillus glaucus HB1-19, while white, red, and blue lights improved its asexual behavior. The red and blue lights improved asexual stroma formation of Xylaria sp. (no. 2508), but the darkness and white light inhibited it. Differently, development of Halorosellinia sp. (no. 1403) turned out to be insensitive to any tested light irradiation. Upon the experimental data, no regularity was observed linking development with secondary metabolism. However, fungal growth showed inversely correlation with productions of major bioactive compounds (aspergiolide A, 1403C, and xyloketal B) from various strains. The results indicated that aspergiolide A biosynthesis favored blue light illumination, while 1403C and xyloketal B preferred red light irradiation. With the favorite light sensing conditions, productions of aspergiolide A, 1403C, and xyloketal B were enhanced by 32.9, 21.9, and 30.8 % compared with those in the dark, respectively. The phylogenetic analysis comparing the light-responding proteins of A. glaucus HB 1-19 with those in other systems indicated that A. glaucus HB 1-19 was closely related to Aspergillus spp. especially A. nidulans in spite of its role of marine-derived fungus. It indicated that marine fungi might conserve its light response system when adapting the marine environment. This work also offers useful information for process optimization involving light regulation on growth and metabolism for drug candidate production from light-sensitive marine fungi.
Folia Microbiologica 04/2013; · 0.68 Impact Factor
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ABSTRACT: Edwardsiella tarda is a Gram-negative, facultative aerobic pathogen which infects multifarious hosts including fish, amphibians, and human beings. A twin-arginine translocation (Tat) gene cluster important for high-salt tolerance in E. tarda was previously identified. Here the genetic structure and pleiotropic roles of the Tat system in physiological adaptation of the bacterium were further characterized. Functional analysis indicated that tatD was not required for Tat export process and tatE might be an allelic gene of tatA in the bacterium. The results showed that disruption in Tat system did not affect morphology and biofilm formation in E. tarda, but affected motility, hemagglutination, cell aggregation and infection of eukaryotic cells (e.g., macrophage J774a). Comparative proteomics analysis of subcellular proteins using two-dimensional gel electrophoresis and a qualitative shotgun protein sequencing method were implemented to identify proteins differentially expressed in E. tarda EIB202 vs. ∆tatABCD. The results revealed a large repertoire of differentially expressed proteins (n=61), sheding light on Tat system associated with the virulence and stress-associated processes in E. tarda. © 2013 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.
FEMS Microbiology Letters 03/2013; · 2.04 Impact Factor
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ABSTRACT: BACKGROUND: There is a continuous demanding for tightly regulated prokaryotic expression systems, which allow functional synthesis of toxic proteins in Escherichia coli for bioscience or biotechnology application. However, most of the current promoter options either are tightly repressed only with low protein production levels, or produce substantial protein but lacking of the necessary repression to avoid mutations initiated by leaky expression in the absence of inducer. The aim of this study was to develop a tightly regulated, relatively high-efficient expression vector in E. coli based on the principle of iron uptake system. RESULTS: By using GFP as reporter, PfhuA with the highest relative fluorescence units, but leaky expression, was screened from 23 iron-regulated promoter candidates. PfhuA was repressed by ferric uptake regulator (Fur)-Fe2+ complex binding to Fur box locating at the promoter sequence. Otherwise, PfhuA was activated without Fur-Fe2+ binding in the absence of iron. In order to improve the tightness of PfhuA regulation for toxic gene expression, Fur box in promoter sequence and fur expression were refined through five different approaches. Eventually, through substituting E. coli consensus Fur box for original one of PfhuA, the induction ratio of modified PfhuA (named PfhuA1) was improved from 3 to 101. Under the control of PfhuA1, strong toxic gene E was successfully expressed in high, middle, low copy-number vectors, and other two toxic proteins, Gef and MazF were functionally synthesized without E. coli death before induction. CONCLUSIONS: The features of easy control, tight regulation and relatively high efficiency were combined in the newly engineered PfhuA1. Under this promoter, the toxic genes E, gef and mazF were functionally expressed in E. coli induced by iron chelator in a tightly controllable way. This study provides a tightly regulated expression system that might enable the stable cloning, and functional synthesis of toxic proteins for their function study, bacterial programmed cell death in biological containment system and bacterial vector vaccine development.
BMC Biotechnology 03/2013; 13(1):25. · 2.35 Impact Factor
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ABSTRACT: Edwardsiella tarda is an enteric Gram-negative invasive intracellular pathogen, which causes enteric septicemia in fish. It could be potentially used to develop a recombinant attenuated E. tarda vaccine for the aquaculture industry. Because live vaccine strains can potentially be released into the environment upon vaccination, medical and environmental safety issues must be considered. Deletion of the asdB gene in E. tarda resulted in a diaminopimelic acid (DAP)-dependent mutant. The wild type asdB gene was inserted in place of the antibiotic-resistance gene in the plasmid, and the resultant non-antibiotic resistant vector was transformed into the attenuated and DAP-dependent E. tarda vaccine strain (WEDΔasdB) to obtain a balanced-lethal system for heterologous antigen expression. The balanced-lethal expression system was further optimized by comparing plasmid replicons with different Shine-Dalgarno sequences and start codons for the asdB gene. Utilizing the optimized balanced-lethal expression system, the protective antigen gene gapA34 from the fish pathogen Aeromonas hydrophila LSA34 was expressed in the attenuated E. tarda to generate the multivalent vaccine candidate WEDΔasdB/pUTta4DGap. This vaccine was shown to evoke an effective immune response against both E. tarda and A. hydrophila LSA34 by vaccinating turbot via a simple immersion route. This multivalent E. tarda vector vaccine has great potential for broad applications in aquaculture.
Fish & Shellfish Immunology 02/2013; · 3.32 Impact Factor
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ABSTRACT: Despite the importance and success of vaccine immunization against bacterial diseases in fish, little is known about the molecular mechanisms of vaccine-induced immune protection in teleost fish. In this study, the live attenuated Edwardsiella tarda vaccine strain WED, which has been shown to evoke efficacious protection against edwardsiellosis and ascites diseases in fish, was extensively evaluated for multiple parameters in a 5-week immunization and challenge experiment in zebrafish. The parameters evaluated included the immunologic potency (relative percent survival, RPS), the specific IgM antibody titers and the expression profiles of multiple immune-related gene markers at multiple time points following immunization and challenge. During the 4-week immunization phase, the toll-like receptor (TLR) 5 signaling pathway, the MHC-I antigen processing pathway and cytotoxic T lymphocyte (CTL) responses were activated in succession. In contrast, the MHC-II antigen processing pathway and the markers of CD4+ T lymphocyte activation were down-regulated, and IgM transcription and specific IgM antibody titers were not significantly induced following immunization. During the 1-week challenge phase, the induction of MHC-I and CTL responses and the inhibition of MHC-II and CD4+ T cell responses were similarly observed in immunized zebrafish following challenge with wild E. tarda. With the 5-week immunization and challenge model, our data suggest the basic mechanism that underlying the long-lasting protective immunity elicited by WED in zebrafish. This mechanism involved the induction of the TLR-5 signaling pathway, the MHC-I antigen processing pathway and CTL effector function, and CTL function seems play a major role in the protection against E. tarda infection in zebrafish.
Developmental and comparative immunology 02/2013; · 3.29 Impact Factor
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ABSTRACT: In the design of recombinant bacterial vector vaccine, heterogeneous antigen is displayed on the outer membrane of the vector strain to evoke polyvalent immunological protection. Thus, the expression of heterogeneous antigen in cells and its display on the outer membrane are of great concern for vaccine preparation. In our previous work, a multivalent bacterial vector vaccine MVAV6203A-1 was constructed by displaying the protective antigen GAPDH from Aeromonas hydrophila on the surface of an attenuated Vibrio anguillarum MVAV6203. In this work, a new fermentation medium was designed by a four-step method to improve the cell growth and antigen display of V. anguillarum MVAV6203A-1. First, suitable carbon and nitrogen sources were selected by a component swapping method. Second, the initial concentrations of carbon and nitrogen sources were determined by orthogonal design. Then three main factors to significantly affect cell growth and antigen expression were screened by a Plackett-Burman design. Finally, the three main factors were meticulously optimized by response surface methodology. Based on this medium, a fed-batch fermentation process was established in a 5-L bioreactor, and the dry cell weight, the antigen expression in cells, and its display on outer membrane reached 5.98 g/L, 2.82 mg/g DCW, and 0.119 mg/g DCW, respectively.
Preparative Biochemistry & Biotechnology 01/2013; 43(1):79-94. · 0.47 Impact Factor
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ABSTRACT: To investigate the N-glycosylation characteristics of recombinant human erythropoietin (rhEPO) produced by an industrial Chinese hamster ovary (CHO) cell line that is currently used in a large scale manufacturing process, we cultured this cell strain in static mode. The produced rhEPO in the culture supernatant was analyzed using isoelectric focusing (IEF) and Ricinus communis agglutinin-I (RCA-I) lectin precipitation. The lactate dehydrogenase (LDH) and sialidase activity in the serum-free supernatant were assayed as well. The analyses revealed that this cell strain could produce rhEPO with high sialic acid content, but during prolonged culture, cell viability decreased with time whilst the activity of sialidase present in the supernatant increased. The loss in rhEPO quality was due to a decrease in terminal sialic acid on the N-glycans, caused by sialidase degradation. The methods and findings in this paper serve as basis for further investigation of industrial production process.
Sheng wu gong cheng xue bao = Chinese journal of biotechnology 12/2012; 28(12):1492-9.
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ABSTRACT: Bacterial twin-arginine translocation (Tat) system contributes to translocate folded proteins to the periplasm and plays pleiotropic roles in physiological fitness. Here, we showed that the fish pathogen Edwardsiella tarda Tat pathway was functional and was essential for H(2)S production and hemolytic activity. E. tarda Tat mutant was more susceptible to diverse stresses such as high temperature, SDS, ethanol, and high-salt conditions. However, E. tarda Tat mutant displayed marginal in vivo virulence attenuation in fish models. Comparative proteomics analysis using two-dimensional gel electrophoresis (2-DGE) and matrix-assisted laser desorption/ionization time-of-flight/time-of-flight tandem mass spectrometry were performed to identify proteins undergoing changes in expression levels under high-salt conditons when the Tat pathway was mutilated. Of the 96 differently expressed proteins on the 2-DGE map, 15 proteins were successfully identified with a MASCOT score >45 (p < 0.05) and fold change higher than 2. These significantly differentially expressed proteins were functionally related to basal metabolism and the biosynthesis of proteins and macromolecules. The results of plate counting further confirmed that the Tat mutant was high-salt-sensitive, indicating that Tat mutant merits as a novel salt-sensitive biological containment system for live attenuated vaccine (LAV) in marine fish vaccinology. To test this, we deleted the type III secretion system genes and cured endogenous plasmid pEIB202 to construct a LAV candidate in the context of Tat abrogation in E. tarda. The results indicated that the LAV candidate was highly attenuated when injected intraperitoneally and elicited significant protection against challenge of wild-type E. tarda in turbot while being rapidly eliminated in seawater.
Applied Microbiology and Biotechnology 10/2012; · 3.42 Impact Factor
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ABSTRACT: Edwardsiella tarda is a Gram-negative pathogen which causes systemic infection in turbot. The increasing frequency of edwardsiellosis in turbot farming has stressed the need to understand the immune responses of fish, for the further development of prevention and control strategies. As a broad spectrum protective antigen, a recombinant glyceraldehyde-3-phosphate dehydrogenase (GAPDH) of E. tarda EIB202 has been proven to present remarkable protection against E. tarda, Aeromonas hydrophila, Vibrio anguillarum, Vibrio alginolyticus and Vibrio harvei in zebrafish. Here, the protection and immune responses of turbot vaccinated with this antigen were studied. Fish vaccinated with recombinant GAPDH via intraperitoneal injection exhibited a low cumulative mortality when challenged with E. tarda EIB202, while high levels of specific antibodies and enhanced bactericidal activities of the immunized sera were observed. In addition, significantly increased transcription levels of four immune-related genes including IL-1β, MHC Iα and IIα, and IgM in the liver, spleen and kidney tissues of vaccinated fish showed that both humoral and cellular immune responses were soundly aroused in the vaccinated fish. Moreover, the IgM antibodies induced by recombinant GAPDH exhibited obvious cross-reactions with the other four pathogens. These results suggested that recombinant GAPDH could present effective protective immunity not only against E. tarda but also other extracellular pathogens, and would be a potential vaccine candidate against polymicrobial infections in the aquaculture industry.
Veterinary Immunology and Immunopathology 09/2012; · 2.08 Impact Factor
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ABSTRACT: Vibrio alginolyticus, a Gram-negative marine bacterium, has brought about severe economic damage to the mariculture industry by causing vibriosis in various fish species. We are intrigued in the regulation of the pathogenesis in this bacterium. Here, we reported a complex regulatory connection among the newly defined type VI secretion system (T6SS), quorum sensing (QS), and 3',5'-cyclic diguanylic acid (c-di-GMP) signal through the phosphatase PppA encoded in the T6SS gene cluster of V. alginolyticus. Whole-genome transcriptome analysis revealed various regulatory targets of PppA including the T6SS substrate hemolysin coregulated protein (Hcp), quorum sensing regulator LuxR, exotoxin alkaline serine protease (Asp), flagellar proteins, as well as proteins involved in polysaccharide biosynthesis and transport. Western blot analysis showed PppA served as a negative regulator of the expression and secretion of Hcp1. Mutation of pppA resulted in an increased level of the intracellular second messenger c-di-GMP and a decreased expression of the QS regulator LuxR as well as exotoxin Asp. Complementation of intact pppA gene in ΔpppA mutant restored the production of c-di-GMP, LuxR, and Asp to the wild-type level. Phenotypic studies suggested that PppA takes part in the modulation of biofilm formation, motility, and cell aggregation. These results demonstrated new roles of PppA in controlling virulence factors and pleiotropic phenotypes and contributed to our understanding of the regulation of pathogenesis in V. alginolyticus.
Veterinary Microbiology 09/2012; · 3.33 Impact Factor
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ABSTRACT: PURPOSE OF WORK: The biosynthetic pathway of a new antitumor compound, haloroquinone, is elucidated to facilitate metabolic regulation for product accumulation and modification to produce new bioactive structural analogues of the compound. The biosynthetic origin of a novel promising protein kinase B inhibitor and anti-tumor compound, haloroquinone, from a marine-derived fungus, Halorosellinia sp. was clarified. The origin of carbon skeleton of haloroquinone was elucidated by feeding experiments with [2-(13)C]malonate and [1,2,3-(13)C(3)]malonate followed by (13)C-NMR analysis of the isolated compounds: 15 carbon atoms were derived from malonate, of which eight were from the methylene group and seven from the carboxyl group. The remaining one is probably obtained by O-methylation. Haloroquinone is thus synthesized via a polyketide pathway using malonyl-CoA as both the starter unit and the extender unit.
Biotechnology Letters 07/2012; 34(11):2119-24. · 1.68 Impact Factor
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ABSTRACT: Zebrafish (Danio rerio) is a prominent vertebrate model of human development and pathogenic disease and has recently been utilized to study teleost immune responses to infectious agents threatening the aquaculture industry. In this work, to clarify the host immune mechanisms underlying the protective effects of a putative vaccine and improve its immunogenicity in the future efforts, high-throughput RNA sequencing technology was used to investigate the immunization-related gene expression patterns of zebrafish immunized with Edwardsiella tarda live attenuated vaccine.
Average reads of 18.13 million and 14.27 million were obtained from livers of zebrafish immunized with phosphate buffered saline (mock) and E. tarda vaccine (WED), respectively. The reads were annotated with the Ensembl zebrafish database before differential expressed genes sequencing (DESeq) comparative analysis, which identified 4565 significantly differentially expressed genes (2186 up-regulated and 2379 down-regulated in WED; p<0.05). Among those, functional classifications were found in the Gene Ontology database for 3891 and in the Kyoto Encyclopedia of Genes and Genomes database for 3467. Several pathways involved in acute phase response, complement activation, immune/defense response, and antigen processing and presentation were remarkably affected at the early stage of WED immunization. Further qPCR analysis confirmed that the genes encoding the factors involved in major histocompatibility complex (MHC)-I processing pathway were up-regulated, while those involved in MHC-II pathway were down-regulated.
These data provided insights into the molecular mechanisms underlying zebrafish immune response to WED immunization and might aid future studies to develop a highly immunogenic vaccine against gram-negative bacteria in teleosts.
BMC Genomics 07/2012; 13:319. · 4.07 Impact Factor
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ABSTRACT: Edwarsiella tarda is highly resistant to the action of cationic antimicrobial peptides (CAMPs). However, the mechanism underlying CAMP resistance is not clear. The enzyme UDP-glucose dehydrogenase (Ugd) that converts UDP-glucose into UDP-glucuronic acid may be important for this resistance. In this study, a ugd gene was identified in E. tarda and its functional role was analyzed using an in-frame deletion mutant Δugd and the complemented strain ugd+. The lipopolysaccharide (LPS) produced by Δugd consisted of a truncated core oligosaccharide (OS) with no O-antigen attached. The ugd mutant was extremely sensitive to CAMPs, presumably because of alterations in LPS structure. The mutant also exhibited enhanced autoaggregation and biofilm formation and reduced hemolytic activity. Using different infection models we found that Δugd was impaired in survival within macrophages and displayed significantly attenuated virulence and an impaired ability to persist within the host. The expression of ugd was induced by polymyxin B and under the control of PhoP and RcsB, two response regulators of the bacterial two-component systems that we identified previously. Moreover, vaccination of turbot (Scophthalmus maximus) with Δugd by intraperitoneal injection elicited significant protection against the wild-type E. tarda strain, suggesting that Δugd may be promising as a potential vaccine candidate against edwardsiellosis.
Veterinary Microbiology 06/2012; · 3.33 Impact Factor
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ABSTRACT: The aims of this study were to reveal the roles of the gene locus qseEGF in the pathogenesis of Edwardsiella tarda.
Genome sequencing of fish pathogen E. tarda EIB202 reveals that the gene locus qseEGF, which encodes a novel two-component system QseEF, were located in E. tarda. The transcription of qseE, qseF and qseG was firstly characterized to be cotranscribed by reverse-transcribed PCR (RT-PCR). The mutant strains ΔqseE, ΔqseF and ΔqseG were constructed with in-frame deletion strategy. Compared with the wild type, all of the mutants showed attenuated virulence and impaired intracellular survival capabilities. Deletion in qseE, qseF and qseG resulted in different effects on hemolysin production in E. tarda. qRT-PCR results indicated that QseEF played a role in regulation of secretion systems, which in turn affected the virulence of E. tarda.
The results manifested that QseEF system affected the virulence in E. tarda EIB202 by controlling the secretion system and hemolysin production. QseE, QseG and QseF in E. tarda serve for the physiological fitness and pathogenesis related to the bacterial survival in macrophage and in vivo of fish. SIGNIFICANCE AND IMPACT: The present results suggested that the important role of two-component system QseEF in regulation of E. tarda pathogenesis and its potential for attenuated live vaccine construction.
Letters in Applied Microbiology 06/2012; 55(2):91-8. · 1.62 Impact Factor
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ABSTRACT: Filamentous fungi from the marine environment have shown great potential as cell factories for the production of pharmacologically active metabolites, but extremely low frequency of homologous recombination brings difficulty to further molecular biology studies. To bypass this problem and develop a highly efficient gene targeting system in marine-derived filamentous fungus Aspergillus glaucus, LigD, a homolog of Neurospora crassa Mus-53 which is considered to play a significant role in nonhomologous end joining (NHEJ), was coloned and deleted, and frequency of targeted gene replacement (TGR) increased dramatically from <2% to 85% in comparison with that in the wild type, when containing 1000 bp of homologous flanking sequence. Such results strongly indicate that AgLigD is indeed involved in the repair of NHEJ in A. glaucus and functions in this pathway. Furthermore, the AgLigD-defective mutant has no discernible differences with wild type regarding sensitivity to mutagens and UV, growth characteristics and transformation frequency. The AgligD-deficient transformant, as the first NHEJ-defective mutant in the field of marine-derived filamentous fungus, will help in expediting studies of molecular biology of marine-derived microorganisms.
Journal of applied genetics 05/2012; 53(3):355-62. · 1.66 Impact Factor
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ABSTRACT: Edwardsiella tarda is a gram-negative bacterium and a causative agent of edwardsiellosis, resulting to severe loss of the aquaculture industry. In this study, based on the reverse vaccinology, sixteen flagellar proteins were selected from highly pathogenic E. tarda EIB202 genome information and in silico analyzed as potential vaccine candidates. Among them, ten recombinant proteins were highly expressed in Escherichia coli and successfully purified. The immunoprotective potentials of these purified recombinant proteins were evaluated in zebrafish model. And recombinant FlgD and FliD were found to lead to a high relative percent survival (RPS, about 70%) against E. tarda EIB202. Furthermore, FlgD required in flagellum hook assembly brought about the similar immune protection in turbot. The immune responses of zebrafish and turbot to recombinant FlgD were also investigated, and the results indicated that its high protection was mainly involved in cellular mediated immune response, corresponding to the intracellular pathogenicity of E. tarda.
Vaccine 04/2012; 30(26):3849-56. · 3.77 Impact Factor
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ABSTRACT: Endostatin, a 20 kDa C-terminal fragment of collagen XVIII, is a specific inhibitor of endothelial cell proliferation and
angiogenesis. In the present study, we produced soluble and biologically active recombinant human endostatin (rhEndostatin)
in Escherichia coli by expressing via fusion with solubility-promoting peptides and optimizing the expression conditions. The rhEndostatin was expressed via fusion with glutathione S-transferase (GST) and NusA protein, respectively. It revealed that NusA protein enhanced the production
of soluble rhEndostatin; but GST didn’t. By optimizing the expression conditions, the production of soluble NusA-rhEndostatin
fusion protein was about 50% of total cellular proteins and about 90% of the products appeared in the cellular supernatant
fraction. The soluble NusA-rhEndostatin fusion protein was purified by one-step hydrophobic interaction chromatography and
NusA was removed by thrombin. Then rhEndostatin was purified by affinity chromatography and gel filtration chromatography.
As a result, a simple and economical purification procedure for rhEndostatin isolation was obtained. The biological activity
of the rhEndostatin was demonstrated in vitro using a human vascular endothelial cells (HuVECs) proliferation assay. Our study provides a feasible and convenient approach
to produce soluble and biologically active rhEndostatin.
KeywordsHuman endostatin-soluble expression-
Escherichia coli
-purification procedure
Biotechnology and Bioprocess Engineering 04/2012; 15(2):229-235. · 1.28 Impact Factor
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ABSTRACT: A fish vaccine candidate, live attenuated Vibrio anguillarum, which can protect fish from vibriosis, was established in our laboratory. In this study, the protective immunological mechanism of live attenuated V. anguillarum was investigated in zebrafish as a model animal. After bath-vaccinated with the live attenuated strain, zebrafish were challenged with wild pathogenic strain to test the immunoprotection of the live attenuated strain. As the results, specific antibody response of fish against V. anguillarum was found to gradually increase during 28 days post-vaccination, and remarkable protection was showed with a high relative protection survival (RPS) of about 90%. Moreover, the vaccination changed the expressions of several immune-related genes in the spleens and livers of zebrafish. Among them, the expressions of pro-inflammatory factors such as IL-1 and IL-8 were tenderly up-regulated with about 3-4 fold in 1-7 days post-vaccination, while MHC II rose to a peak level of 4-fold in 7th day post-vaccination. These results gave some important messages about the mechanism of specific protection induced by live attenuated V. anguillarum and showed the availability of zebrafish model in the evaluation of the vaccine candidate.
Fish & Shellfish Immunology 04/2012; 33(1):36-41. · 3.32 Impact Factor
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ABSTRACT: The aim of this study was to reveal functional redundancy and variation of the two catalases KatB and KatG in Edwardsiella tarda.
Genome sequencing of fish pathogen Edw. tarda EIB202 reveals that it contains two genes putatively encoding catalases, katB (ETAE_1368) and katG (ETAE_0889). Under free-living conditions, single disruption in katB or katG resulted in no growth impairment, whereas double mutation of the two genes led to moderate decrease in growth, indicating that these two catalases were together essential for the physiological fitness by dissipating the endogenous H(2) O(2) . katG mutant exhibited much more elevated sensitivity to exogenous H(2) O(2) than katB mutant did, indicating that KatG was quasi-essential in detoxifying external reactive oxygen species (ROS) in Edw. tarda EIB202. Further comparative analysis indicated that katB or katG disruption showed different effects on the virulence-related processes of Edw. tarda such as haemolysin production, bile and serum resistance, as well as the internalization within fish epithelial cells. Moreover, both of the katB and katG mutants exhibited incapacity to replicate in murine macrophage J774 cell model, although the deficiency was seen much severe for ΔkatB/katG mutant. With regard to in vivo virulence, katB and katG mutants displayed delayed lethality and increased LD(50) values for zebrafish.
KatB and KatG in Edw. tarda serve for the physiological fitness, and pathogenesis related the bacterial survival in macrophage and in vivo of fish.
Counteracting ROS for systemic infection, Edw. tarda catalase KatG and KatB merits as potential targets for attenuated live vaccine construction.
Letters in Applied Microbiology 02/2012; 54(5):425-32. · 1.62 Impact Factor
