Yuanxing Zhang

East China University of Science and Technology, Shanghai, Shanghai Shi, China

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Publications (144)345.07 Total impact

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    ABSTRACT: Edwardsiella tarda is a leading fish pathogen haunting worldwide aquaculture industry. In E. tarda, two-component system EsrA-EsrB positively regulates type III and VI secretion systems (T3SS and T6SS) and negatively regulates hemolysin EthA, which has been demonstrated to be essential for the invasion processes in fish. In order to develop a live attenuated vaccine (LAV) with high invasiveness to be practically and economically used as immersion-administered vaccine in aquaculture, here, we generated a random mutation library of esrB sequences by error-prone PCR and introduced them into the E. tarda esrB deletion mutant. The mutant YWZ47 with significantly increased hemolytic activity and low T3SS and T6SS secretion was screened. Phenotypes including extracellular protein profiles, invasion in macrophages, lethality toward fish, and infection kinetics were investigated in the wild-type strain EIB202 and the mutants ΔesrB, ΔT3SS, ΔT6SS, ΔT3SS/ΔT6SS, and YWZ47. Compared to the documented LAV strain ΔesrB, YWZ47 showed higher invasive capability and low in vivo virulence toward fish. Significantly higher relative percent survival (RPS) could be generated in turbot (Scophthalmus maximus) against the challenge of the wild-type EIB202 when inoculated through immersion route, and the RPS was comparable with that of ΔesrB through intraperitoneal (i.p.) injection inoculation. Two mutated points, K167M and H197L, were found by sequence analysis of EsrBYWZ47 variant. These structural modifications underpin the variations in the regulatory functions of the mutant and wild-type EsrB. This study promoted understanding of virulence regulation by EsrB in E. tarda and presented a promising candidate of invasive attenuated vaccine used in aquaculture industries.
    Applied microbiology and biotechnology. 11/2014;
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    ABSTRACT: As important zoonotic organisms causing infections in humans, Edwardsiella bacteria are also notorious leading fish pathogens haunting worldwide aquaculture industries. However, the taxa are now widely recognized to be misclassified, which hurdled the understanding of the epidemiology and development of effective diagnostics and vaccines. Currently the genus Edwardsiella consists of three species Edwardsiella tarda, E. ictaluri, and E. hoshinae. Previous phylogenomic analysis revealed that E. tarda strains display two major highly divergent genomic types (genotypes), EdwGI and EdwGII, and the former represents a genotype of fish-pathogenic isolates and being recently proposed as a novel species E. piscicida, sp. nov. Here multiple phylogenetic analyses and the genome-level comparisons of EdwGI strains disclose that the phylogroup strains from diseased eel formed an obviously distinct cluster that could be equated with a new species status. The phylogenetic evidence for the new species assignment was also supported by corresponding DNA–DNA hybridization estimation values and by phenotypic characteristics. Interestingly, further comparative genomics reveals that these strains have acquired the locus of enterocyte effacement (LEE) genes and as a result these bacteria contain at least 2 sets of distinct T3SS and 3 sets of T6SS gene clusters, respectively. It is therefore proposed that the phylogroup strains from diseased eel should be classified as Edwardisella anguillarum sp. nov., and the type strain is ET080813T (= DSM27202T = CCUG 64215T = CCTCC AB2013118T = MCCC 1K00238T). These findings will contribute to development of species-specific control measures against Edwardsiella bacterium in aquatic animals, while also shedding light on the pathogenesis evolution in Edwardsiella bacterium.
    Systematic and Applied Microbiology 11/2014; · 3.29 Impact Factor
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    ABSTRACT: Edwardsiella tarda, an enteric opportunistic pathogen, is associated with acute to chronic edwardsiellosis in cultured fish, resulting in heavy losses in aquaculture. To date, the pathogenesis of E. tarda has been extensively studied and a great deal of vaccine candidates have been attempted. However, the research on the predisposition of E. tarda infection is poorly reported. In this study, the effects of intestinal inflammation on E. tarda infection were investigated using a zebrafish model that influenced by perturbation of intestinal microbiota. Featured symptoms of edwardsiellosis were observed in intestinal inflammatory zebrafish compared with healthy fish. Higher bacterial numbers were detected in both mucosal tissues (intestine, skin and gills) and lymphoid tissues (liver, spleen and kidney) of inflammatory zebrafish while the bacterial loads in healthy zebrafish appeared to be relatively lower by 10-100 folds. Moreover, significant up-regulation of IL-1β, TNF-α and iNOS was noticed in multiple tissues of zebrafish with intestinal inflammation between 6 and 72 h post infection. However, only moderate elevation was observed in the gills and liver of healthy fish. Furthermore, the expression of genes involved in neutrophil recruitment (mpx, IL-8 and LECT2) and antimicrobial response (β-defensin and hepcidin) showed notable up-regulation in the intestine of inflammatory zebrafish. These results demonstrate that fish with intestinal inflammation is more susceptible to E. tarda and the antimicrobial response during E. tarda infection might inhibit the growth of intestinal microbiota that E. tarda could resist. Our results suggest that maintaining good management to avoid intestinal inflammation is a feasible prevention measure against edwardsiellosis.
    Fish &amp Shellfish Immunology 09/2014; · 2.96 Impact Factor
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    ABSTRACT: Edwardsiella tarda and Vibrio anguillarum are the two main pathogenic bacteria that cause edwardsiellosis and vibriosis in various species of fish raised in aquaculture. In our previous study, the live attenuated vaccines E. tarda WED and V. anguillarum MVAV6203 showed robust relative protection when vaccinated zebrafish or turbot were challenged with virulent E. tarda or V. anguillarum, respectively. Additionally, vaccinated fish processed the two vaccines through different pathways of antigen processing and presentation. Here, the immune protection of a combination vaccination consisting of E. tarda WED and V. anguillarum MVAV6203 was initially evaluated in zebrafish. After challenge with E. tarda and V. anguillarum at 1 month post-vaccination, the vaccinated zebrafish exhibited the relative protective survival of 70% and 90%, respectively. The expression of genes related to antigen recognition, processing and presentation were measured in the liver and spleen of vaccinated zebrafish. Gene expression profiling showed that more than one Toll-like receptor signaling pathway was activated and that both MHC I and II pathways of antigen processing and presentation were evoked. Later, the immune protection of the combination vaccine was evaluated in turbot and it showed similarly effective immune-mediated protection. By ELISA analysis, we found that the specific antibody levels in vaccinated turbot increased compared to those of fish vaccinated by a single vaccine during 2 months post-vaccination. Meanwhile, the expression levels of MHC I and II in the liver, spleen and kidney of vaccinated turbot were both up-regulated, suggesting that the MHC I and II pathways of antigen processing and presentation are activated in vaccinated turbot, similar to vaccinated zebrafish. In summary, a combination vaccine of live attenuated E. tarda WED and V. anguillarum MVAV6203 is effective and could be used widely in the future.
    Vaccine. 09/2014;
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    ABSTRACT: The evpP gene in fish pathogen Edwardsiella tarda, coding the T6SS secretory protein EvpP and carrying an evpA-evpO independent promoter region, was crucial for host cell invasion. The transcription of evpP was positively regulated by either the two-component system EsrA-EsrB or iron concentration, and its overexpression was known to enhance the invasion ability in our previous study. This work demonstrated that the H-NS protein, a pleiotropic regulator of gene expression, was a new transcriptional modulator of evpP gene. The results showed that in vivo the transcriptional level of evpP was down-regulated by H-NS and in vitro this global regulator interacted directly with evpP promoter region. Moreover, DNase I footprinting experiments mapping the interaction regions of H-NS and evpP revealed that this global regulator bound to evpP promoter and neighbouring areas at multiple sites. We provided a new insight into evpP regulation network and demonstrated the repression of H-NS to the transcription of evpP gene.This article is protected by copyright. All rights reserved.
    Letters in Applied Microbiology 08/2014; · 1.63 Impact Factor
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    ABSTRACT: In recent years, increasing diseases especially bacterial diseases have brought a host of losses with the expansive cultivation of turbot (Scophthalmus maximus). In order to do more research about the immune system of turbot for better understanding the mechanism of resisting diseases, the immunoglobulin genes related to secretory and membrane-bound IgM (s-IgM and m-IgM) of turbot were cloned using homology sequences cloning and SMART RACE PCR method. The heavy chain of s-IgM cDNA is 1900 bp in length including a leader region, a variable region, four constant regions (CH1, CH2, CH3 and CH4) and a C-terminal while the cDNA of m-IgM is 1795 bp with the same leader region, variable region, three constant regions (CH1, CH2 and CH3) and two transmembrane regions (TM1 and TM2). The sequence of IgM gene was also obtained and the structure consisted of V-CH1-CH2-CH3-CH4-TM1-TM2 is similar to other fishes. The highest level of s-IgM expression was observed in spleen, followed by kidney, gills, eyes, skin of the healthy turbot whereas the same profile of m-IgM expression is found with low level. And s-IgM takes up dominant proportion of total IgM expression. Also the relative expressions of s-IgM and m-IgM were analyzed in turbot vaccinated with the live attenuated vaccine Vibrio anguillarum. Not only the transcriptions of both s-IgM and m-IgM in liver, spleen and kidney of turbot injected with V. anguillarum MVAV6203 were up-regulated but also the expressions of s-IgM and m-IgM in spleen, kidney, gut, skin and gills of bath-vaccinated turbot were increased. Comparing the ratio changes of relative expression of m-IgM and s-IgM in vaccinated turbot, we found that the proportion of m-IgM were increasing in both administration routes, which probably indicated that the increasing expression of m-IgM strengthen the phagocytic ability of B cells.
    Fish &amp Shellfish Immunology 07/2014; · 2.96 Impact Factor
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    ABSTRACT: Live attenuated vaccine is one of the efficient vaccine candidates in aquaculture, which can be easily delivered to fish via bath-vaccination. An outstanding advantage of bath-vaccination is that vaccine delivery is through the same route as that utilized by many fish pathogens, generating specific mucosal immune responses. In this work, we investigated the mucosal immune responses induced by a live attenuated Vibrio anguillarum vaccine in zebrafish via bath-vaccination. Bacteria proliferated rapidly in 3 hours after vaccination and maintained at a high level until 6 hours in the intestine. Besides, bacteria persisted in the intestine for a longer time whereas decreased rapidly in the skin and gills. Moreover, a significant up-regulation of TLR5 triggering a MyD88-dependent signaling pathway was observed in the intestine, which implied that flagella were the crucial antigenic component of the live attenuated vaccine. And macrophages and neutrophils showed active responses participating in antigen recognition and sampling after vaccination. Furthermore, an inflammation was observed with plenty of lymphocytes in the intestine at 24 h post vaccination but eliminated within 7 days. In conclusion, the live attenuated V. anguillarum vaccine induced notable mucosal immune responses in the intestine which could be used as a mucosal vaccine vector in the future.
    Fish &amp Shellfish Immunology 07/2014; · 2.96 Impact Factor
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    ABSTRACT: Recombinant glycoprotein drugs require proper glycosylation for optimal therapeutic efficacy. Glycoprotein therapeutics are rapidly removed from circulation and have reduced efficacy if they are poorly sialylated. Ricinus communis agglutinin-I (RCA-I) was found highly toxic to wild-type CHO-K1 cells and all the mutants that survived RCA-I treatment contained a dysfunctional N-acetylglucosaminyltransferase I (GnT I) gene. These mutants are named CHO-gmt4 cells. Interestingly, upon restoration of GnT I, the sialylation of a model glycoprotein, erythropoietin, produced in CHO-gmt4 cells was shown to be superior to that produced in wild-type CHO-K1 cells. This addendum summarizes the applicability of this cell line, from transient to stable expression of the recombinant protein, and from a lab scale to an industrial scale perfusion bioreactor. In addition, CHO-gmt4 cells can be used to produce glycoproteins with mannose-terminated N-glycans. Recombinant glucocerebrosidase produced by CHO-gmt4 cells will not require glycan remodeling and may be directly used to treat patients with Gaucher disease. CHO-gmt4 cells can also be used to produce other glycoprotein therapeutics which target cells expressing mannose receptors.
    Bioengineered. 06/2014; 5(4).
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    ABSTRACT: For filamentous fungi, the basic growth unit of hyphae usually makes it sensitive to shear stress which is generated from mechanical force and dynamic fluid in bioreactor, and it severely decreases microbial productions. The conventional strategies against shear-sensitive conundrum in fungal fermentation usually focus on adapting agitation, impeller type and bioreactor configuration, which brings high cost and tough work in industry. This study aims to genetically shape shear resistant morphology of shear-sensitive filamentous fungus Aspergillus glaucus to make it adapt to bioreactor so as to establish an efficient fermentation process.
    Microbial Cell Factories 05/2014; 13(1):73. · 3.31 Impact Factor
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    ABSTRACT: A cyclin-like protein from Aspergillus nidulans, ClgA, was identified. Its cyclin-like subunit shares 28.3% identity to Saccharomyces cerevisiae Clg1. Deletion of clgA slightly influenced fungal growth, but repressed asexual development and made it more sensitive to temperature variations. It also downregulated expression of brlA, abaA and wetA, which are critically responsible for asexual development. Sexual development was impaired in the ΔclgAmutant. Its related genes, veA and nosA, were expressed weakly in the ΔclgAmutant, while nsdD expression showed the opposite behavior. Generally, ClgA functioned differently from other reported cyclins in developmentof A.nidulans.
    Research in Microbiology 04/2014; · 2.89 Impact Factor
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    ABSTRACT: The real-time distribution of anticancer 1403C in fermentation broth of marine fungus Halorosellinia sp. was investigated. It was closely related with pH variations, which was, 1403C in the supernatant decreased while that in the mycelia increased with pH rising. There was only 0.5 % of the total 1403C left in the supernatant when pH reached 7.0. The scanning electron microscope then provided information that compounds precipitated on the mycelia when pH rose. Then, the pH-regulation experiments proved that 1403C mainly secreted extracellular and easily dissolved in acidic condition but precipitated and absorbed on the mycelia with the increase of broth pH. Thereby, a pH-regulation strategy was proposed and applied to accumulate 1403C on the mycelia before draw-off of fermentation broth. It significantly simplified purification process and is critical for 1403C preparation of industrial scale.
    Journal of Industrial Microbiology 04/2014; · 1.80 Impact Factor
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    ABSTRACT: It is an attractive strategy to develop a recombinant bacterial vector vaccine by expressing exogenous protective antigen to induce the immune response, and the main concern is how to enhance the cellular internalization of antigen produced by bacterial vector. Cell-penetrating peptides (CPPs) are short cationic/amphipathic peptides which facilitate cellular uptake of various molecular cargoes and therefore have great potentials in vector vaccine design. In this work, eleven different CPPs were fused to the C-terminus of EGFP respectively, and the resultant EGFP-CPP fusion proteins were expressed and purified to assay their cross-membrane transport in macrophage J774 A.1 cells. Among the tested CPPs, TAT showed an excellent capability to deliver the cargo protein EGFP into cytoplasm. In order to establish an efficient antigen delivery system in Escherichia coli, the EGFP-TAT synthesis circuit was combined with an in vivo inducible lysis circuit PviuA-E in E. coli to form an integrated antigen delivery system, the resultant E. coli was proved to be able to lyse upon the induction of a mimic in vivo signal and thus release intracellular EGFP-TAT intensively, which were assumed to undergo a more efficient intracellular delivery by CPP to evoke protective immune responses. Based on the established antigen delivery system, the protective antigen gene flgD from an invasive intracellular fish pathogen Edwardsiella tarda EIB202, was applied to establish an E. coli recombinant vector vaccine. This E. coli vector vaccine presented superior immune protection (RPS = 63%) under the challenge with E. tarda EIB202, suggesting that the novel antigen delivery system had great potential in bacterial vector vaccine applications.
    Fish &amp Shellfish Immunology 04/2014; · 2.96 Impact Factor
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    ABSTRACT: Edwardsiella tarda the etiological agent for edwardsiellosis, a devastating fish disease prevailing in worldwide aquaculture industries was subjected to a molecular genetic study. To research into the influence when RpoN (σ54) and RpoS (σ38) were deleted simultaneously, the double deletion mutant of RpoN (σ54) and RpoS (σ38), namely rnrs, was constructed. Firstly, RpoN and RpoS are both essential for H2O2, starvation, high osmotic pressure and acid resistance, which have synergistic effect. Secondly, virulence of rnrs reduces significantly compared to E. tarda EIB 202 WT, ΔrpoN mutant and ΔrpoS mutant. Furthermore, transcriptional control of rpoS by rpoN in stationary phase was observed through qRT-PCR, while rpoS had no influence on rpoN in the level of transcription. Meanwhile, regulation of flagellar sigma factor σF (FliA) and other flagella-related genes including flgA, flgK, flgL, motA, and motB by rpoS, and rpoN was found. fliA and other flagella-related genes were controlled positively by rpoN, while negatively by rpoS. At last, two differential expression genes in transcriptional level of rnrs strain were detected by DD-RT-PCR, namely cheY and narK. This study therefore indicated interaction between sigma factors RpoN and RpoS, which modulates stress response, virulence, motility, and provides new insights into the regulatory networks of E. tarda.
    Journal of Basic Microbiology 03/2014; · 1.20 Impact Factor
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    ABSTRACT: Here, we report a simple and ultrasensitive upconversion fluorescent strip sensor based on NaYF4:Yb,Er nanoparticles (NPs) and the lateral flow immunochromatographic assay (LFIA). Carboxyl-modified β-NaYF4:Yb,Er NPs were successfully synthesized by a facile one-pot solvothermal approach, upon further coupling with monoclonal antibody, the resultant UCNPs-antibody conjugates probes were used in LFIA and served as signal vehicles for the fluorescent reporters. V. anguillarum was used as a model analyte to demonstrate the use of this strip sensor. The limit of the detection for the fluorescent strip was determined as 10(2) CFU mL(-1), which is 100 times lower than those displayed by enzyme-linked immunosorbent assays, while the time needed for the detection was only 15 min. Furthermore, no cross-reaction with other eight pathogens was found, indicating the good specificity of the strip. This developed LFIA would offer the potential as a useful tool for the quantification of pathogens analysis in the future.
    Nanoscale 02/2014; · 6.73 Impact Factor
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    ABSTRACT: Sodium butyrate is commonly used in mammalian cell cultures to increase the productivity of recombinant proteins. A Chinese hamster ovary (CHO) cell line producing recombinant human erythropoietin (rhEPO) was cultured in commercial medium. Addition of 0.5 mM butyrate inhibited the over-growth of the cells after the medium was changed from serum-added medium to serum-free medium. At the 6th day, the addition of butyrate lowered the transcriptional level of sialidases I, II, and III compared to that of control groups by 56, 87, and 59 %, respectively. Extracellular sialidase activity was decreased by 53 % by addition of butyrate. The inhibition of cell over-growth and the decrease of extracellular sialidase activity helped to increase the acidic isoform content of rhEPO expressed by this CHO cell strain.
    Biotechnology Letters 02/2014; · 1.85 Impact Factor
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    ABSTRACT: Edwardsiella tarda is a rod-shaped Gram-negative pathogenic bacterium that causes hemorrhagic septicemia in fish. Nucleoid-associated protein HU is a basic DNA-binding protein with structural specificity in regulating genes expression. In wild-type E. tarda EIB202, HU is composed of two subunits HUα (hupA) and HUβ (hupB), and exists in homodimer or heterodimer forms. Different from the wild-type and ΔhupB mutant, ΔhupA mutant was found to be defective in cell growth, H2S production, acid adaptation, and exhibited abnormal cell division resulting in a filamentous phenotype in log phase bacteria. The qRT-PCR result showed that deletion of hupA significantly up-regulated the transcription levels of recA and sulA, which in turn stimulated RecA-dependent pathway to prevent cell division, resulting in filamentous morphology in E. tarda. Furthermore, the elongated ΔhupA cells showed a striking defect in EPC cell invasion, and the adhesion and internalization rates were reduced to 25% and 27% of the wild-type in log phase cultures. Confocal laser scanning microscopy revealed that filamentous bacteria failed to adhere to and could not be internalized into EPC. When some of the bacteria regained the rod-shape morphology in stationary cultures, the ΔhupA mutants showed increased adhesion and internalization rates into EPC. Moreover, ΔhupA mutant exhibited delayed mortalities (for two days) in zebrafish but the LD50 increased 17 folds. Immunohistochemical analysis showed that ΔhupA mutant reduced proliferation abilities in the muscle, liver and intestine of zebrafish. This study indicates that HU protein and strains morphology play essential roles in the virulence network of E. tarda.
    Veterinary Microbiology 01/2014; · 3.13 Impact Factor
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    ABSTRACT: This study aimed to investigate the protective mucosal immunity elicited by live attenuated Vibrio anguillarum in fish. Zebrafish were immunized by bath or injection way, and undertook bath challenge at 28 days post vaccination. The results implied that bath vaccination was the better delivery route for inducing the protective immunity against bath challenge in zebrafish. The expressions of genes related to Th1, Th2 and Th17 cells were measured in the mucosal tissues of vaccinated and challenged zebrafish. Gene expression profiles showed that Th17-like responses were induced in mucosal immune system by vaccination via bath and injection routes while Th1 and Th2-like responses were not remarkable. Compared to injection vaccination, bath vaccination elicited the intense Th17-like immune responses in the gut tissue of zebrafish. Additionally, in gills and skin, Th17-like mucosal immunity elicited by injection vaccination occurred later than that by bath vaccination. Our results proved the immunological importance of gut in bath vaccination and the presence of two-compartmental model for immune response in zebrafish. In conclusion, bath vaccination more efficiently elicited protective Th17-like immunity than injection vaccination in mucosal tissues of vaccinated zebrafish. In turbot, effective immune protection against wild-type V. anguillarum was obtained by bath-vaccinated and the Th17-like responses were found in mucosal and systemic tissues.
    Fish &amp Shellfish Immunology 01/2014; · 2.96 Impact Factor
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    ABSTRACT: It is an attractive strategy to develop a recombinant bacterial vector vaccine by expressing exogenous protective antigen to induce the immune response, and the main concern is how to enhance the cellular internalization of antigen produced by bacterial vector. Cell-penetrating peptides (CPPs) are short cationic/amphipathic peptides which facilitate cellular uptake of various molecular cargoes and therefore have great potentials in vector vaccine design. In this work, eleven different CPPs were fused to the C-terminus of EGFP respectively, and the resultant EGFP-CPP fusion proteins were expressed and purified to assay their cross-membrane transport in macrophage J774 A.1 cells. Among the tested CPPs, TAT showed an excellent capability to deliver the cargo protein EGFP into cytoplasm. In order to establish an efficient antigen delivery system in E. coli, the EGFP-TAT synthesis circuit was combined with an in vivo inducible lysis circuit PviuA-E in E. coli to form an integrated antigen delivery system, the resultant E. coli was proved to be able to lyse upon the induction of a mimic in vivo signal and thus release intracellular EGFP-TAT intensively, which were assumed to undergo a more efficient intracellular delivery by CPP to evoke protective immune responses. Based on the established antigen delivery system, the protective antigen gene flgD from an invasive intracellular fish pathogen Edwardsiella tarda EIB202, was applied to establish an E. coli recombinant vector vaccine. This E. coli vector vaccine presented superior immune protection (RPS = 63%) under the challenge with E. tarda EIB202, suggesting that the novel antigen delivery system had great potential in bacterial vector vaccine applications.
    Fish &amp Shellfish Immunology 01/2014; · 2.96 Impact Factor
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    ABSTRACT: Insulin precursor fusion protein expressed in Pichia pastoris is a single-chain protein with a spacer peptide (EEAEAEAEPK) localized at its N-terminal. Currently, the one-step transpeptidation reaction with low yield and high cost is generally employed to convert the insulin precursor fusion protein into human insulin ester. In this study, a two-step transpeptidation reaction was proposed that separating cleavage step from coupling step so that each reaction was performed under its optimal conditions. Using this method, the total efficiency doubled and the reaction time was shortened compared to the one-step method. In addition, the amount of O-t-butyl-L-threonine t-butyl ester and trypsin dosages were reduced by 50% and 75%, respectively. This two-step transpeptidation strategy was simple and efficient and could be used for the pharmaceutical production of human insulin. This article is protected by copyright. All rights reserved.
    Biotechnology and Applied Biochemistry 12/2013; · 1.35 Impact Factor
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    ABSTRACT: The regulation of protein tyrosine phosphorylation mediated by the protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs) is essential for cellular homeostasis. The co-expression of PTKs with PTPs in Pichia pastoris was used to facilitate the expression of active PTKs by neutralizing its apparent toxicity to cells. In this study, the gene encoding phosphatase PTP1B with/without blue fluorescent protein (BFP) or peroxisomal targeting signal 1 (SKL) was cloned into expression vector pAG32 to produce four vectors followed by Pichia pastoris GS115 transformation. The tyrosine kinases EGFR-2 and PDGFRβ were expressed on vector pPIC3.5K fused with His-tag and green fluorescent protein (GFP) at their N-terminus. The two plasmids were transformed into P. pastoris with/without PTP1B to get 10 strains. The EGFR-2 and PDGFRβ fusion proteins were purified by Ni(2+) affinity Sepharose. In the recombinant P. pastoris, the PTKs co-expressed with PTP1B exhibited higher kinase catalytic activity than those expressing PTKs alone. The highest activities were achieved by targeting the PTKs and PTP1B into peroxisomes. Thus, the EGFR-2 and PDGFRβ fusion proteins expressed in P. pastoris could be used as attractive targets in the drug screening for anti-cancer therapeutics.
    Journal of Microbiology and Biotechnology 11/2013; · 1.40 Impact Factor

Publication Stats

812 Citations
345.07 Total Impact Points

Institutions

  • 2003–2014
    • East China University of Science and Technology
      • School of Pharmacy
      Shanghai, Shanghai Shi, China
  • 2011
    • University of Vienna
      Wien, Vienna, Austria
  • 2010
    • Keck Graduate Institute
      Claremont, California, United States
  • 2006
    • Fudan University
      • Department of Chemistry
      Shanghai, Shanghai Shi, China
    • Nanjing University of Science and Technology
      Nan-ching, Jiangsu Sheng, China