Cryoimmobilized enzyme for biosensor construction

A new immobilization material and an immobilization method for a glucose sensor with HEFc (hydroxyethylferrocene) as mediator is described. In the course of three months, the enzyme electrode shows almost no deterioration in its response characteristics. The response time is less than 30 s. The electrode has a wide linear range up to 10 mmol l(-1) with good repeatability. The kinetic parameters have also been calculated.

Cyro-hydrogel for the construction of a tyrosinase-based biosensor

A new immobilization method for construction of a tyrosinase based biosensor is described. A simple physical freezing technique was adopted for preparation. The immobilized enzyme yields specific activities that are more than 22% of the soluble enzyme. The enzyme electrode can be stored in dry state for more than three months without any loss of activity. The biosensor was applied to the determination of several phenols and o-diphenols. The lowest detect limit is 0.02 mu mol/1 and the linear range was 1.0 X 10(-7)-1.0 X 10(-4) mol/1 for catechol. The kinetic parameters have also been calculated.

CONSTRUCTION OF A TYROSINASE-BASED BIOSENSOR IN PURE ORGANIC-PHASE

A novel immobilization method for construction of a tyrosinase-based biosensor applied in pure organic phase is described. This method gives the enzyme a hydrated shell which allows the enzyme to maintain its biocatalytic activity in a pure organic solvent The enzyme electrode was used to determine several phenols and o-diphenols in pure chloroform and chlorobenzene. The biosensor can be stored in dry state for more than 3 months without any loss of the activity. The kinetic parameters have also been calculated and are presented herein.

SENSITIVE AMPEROMETRIC DETECTION OF GLUCOSE BY REVERSED PHASE LIQUID-CHROMATOGRAPHY AT A PRUSSIAN BLUE CHEMICALLY MODIFIED ELECTRODE OF NOVEL CONSTRUCTION

A new liquid chromatography electrochemical (LCEC) scheme for glucose sensing has been developed on the basis of a Prussian Blue chemically modified electrode (CME) of novel construction and characterized in terms of various experimental parameters by the flow injection analysis (FIA) technique. Unique hydrodynamic voltammograms were obtained for the first time at the CME in the flow-through amperometric detection of glucose, and subsequently both anodic and cathodic peaks could be expected on monitoring the operating potential in the modest positive or negative region. The unique pH dependence on the CME response towards glucose makes it perfectly compatible with conventional reversed phase liquid chromatography systems. On the basis of these features, practical application in glucose LCEC detection has been effectively performed; a linear response range over three orders of magnitude and a detection limit of subpicomole level were readily obtained. The capability of the established LCEC mode in the direct sensing of urinary glucose has been demonstrated.

The construction of a cDNA library enriched for immune genes and the analysis of 7535 ESTs from Chinese mitten crab Eriocheir sinensis

Chinese mitten crab Eriocheir sinensis is one of the most important aquaculture crustacean species in China. A cDNA library was constructed from hemocytes of E. sinensis challenged with the mixture of Listonella anguillarum and Staphylococcus aureus, and randomly sequenced to collect genomic information and identify genes involved in immune defense response. Single-pass 5' sequencing of 10368 clones yielded 7535 high quality ESTs (Expressed Sequence Tags) and these ESTs were assembled into 2943 unigenes. BLAST analysis revealed that 1706 unigenes (58.0% of the total) or 4593 ESTs (61.0% of the total) were novel genes that had no significant matches to any protein sequences in the public databases. The rest 1237 unigenes; (42.0% of the total) were closely matched to the known genes or sequences deposited in public databases, which could be classed into 20 or 23 classifications according to "molecular function" or "biological process" respectively based on the Gene Ontology (GO). And 221 unigenes (7.5% of all 2943 unigenes, 17.9% of matched unigenes) or 969 ESTs (12.9% of all 7535 ESTs, 32.9% of matched ESTs) were identified to be immune genes. The relative higher proportion of immune-related genes in the present cDNA library than that in the normal library of E. sinensis and other crustaceans libraries, and the differences and changes in percentage and quantity of some key immune-related genes especially the immune inducible genes between two E. sinensis cDNA libraries may derive from the bacteria challenge to the Chinese mitten crab. The results provided a well-characterized EST resource for the genomics community, gene discovery especially for the identification of host-defense genes and pathways in crabs as well as other crustaceans. (C) 2009 Elsevier Ltd. All rights reserved.

Construction and characterization of a fosmid library for pathogenic bacterium Vibrio anguillarum

Vibrio anguillarum is a common bacterial pathogen in fish. However, little is known about its pathogenic mechanism, in part, because the entire genome has not been completely sequenced. We constructed a fosmid library for V. anguillarum containing 960 clones with an average insert size of 37.7 kb and 8.6-fold genome coverage. We characterized the library by end-sequencing 50 randomly selected clones. This generated 93 sequences with a total length of 57 485 by covering 1.4% of the whole genome. Of these sequences, 58 (62.4%) were homologous to known genes, 30 (32.3%) were genes with hypothetical functions, and the remaining 5 (5.3%) were unknown genes. We demonstrated the utility of this library by PCR screening of 10 genes. This resulted in an average of 6.2 fosmid clones per screening. This fosmid library offers a new tool for gene screening and cloning of V. anguillarum, and for comparative genomic studies among Vibrio species.

Construction and evaluation of DNA vaccines encoding Edwardsiella tarda antigens

Edwardsiella tarda is an opportunistic pathogen that can infect humans, animal, and fish. Two E. tarda antigens, Eta6 and FliC, which are homologues to an ecotin precursor and the FliC flagellin, respectively, were identified by in vivo-induced antigen technology from a pathogenic E. tarda strain isolated from diseased fish. When used as a subunit vaccine, purified recombinant Eta6 was moderately protective against lethal challenge of E. tarda in a Japanese flounder model, whereas purified recombinant FliC showed no apparent immunciprotectivity. Similarly, DNA vaccines based on eta6 and fliC in the form of plasmids pEta6 and pFliC induced, respectively, moderate and marginal protection against E. tarda infection. To improve the vaccine efficacy of eta6, a chimeric DNA vaccine, pCE6, was constructed, which encodes Eta6 fused in-frame to FliC. pCE6 was found to induce significantly higher level of protection than pEta6. Likewise, another chimeric DNA vaccine, pCE18, which expresses FliC fused to a previously identified E. tarda antigen Et18, elicited significantly stronger protective immunity than the DNA vaccine based on et18 alone. Fish immunized with pEta6 and pCE6 produced specific serum antibodies and exhibited significantly enhanced expression of the genes encoding elements that are involved in both innate and adaptive immune responses. Furthermore, the induction magnitudes of most of these genes were significantly higher in pCE6-vaccinated fish than in pEta6-vaccinated fish. (C) 2009 Elsevier Ltd. All rights reserved.

Construction of an attenuated Pseudomonas fluorescens strain and evaluation of its potential as a cross-protective vaccine

Ferric uptake regulator (Fur) is a global transcription regulator that is ubiquitous to Gram-negative bacteria and regulates diverse biological processes, including iron uptake, cellular metabolism, stress response, and production of virulence determinants. As a result, for many pathogenic bacteria, Fur plays a crucial role in the course of infection and disease development. In this study, the fur gene was cloned from a pathogenic Pseudomonas fluorescens strain, TSS, isolated from diseased Japanese flounder cultured in a local farm. TSS Fur can partially complement the defective phenotype of an Escherichia coli fur mutant. A TSS fur null mutant, TFM, was constructed. Compared to TSS, TFM exhibits reduced growth ability, aberrant production of outer membrane proteins, decreased resistance against host serum bactericidal activity, impaired ability to disseminate in host blood and tissues, and drastic attenuation in overall bacterial virulence in a Japanese flounder infection model. When used as a live vaccine administered via the injection, immersion, and oral routes, TFM affords high levels of protection upon Japanese flounder against not only P.fluorescens infection but also Aeromonas hydrophila infection. Furthermore, a plasmid, pJAQ, was constructed, which expresses the coding element of the Vibrio harveyi antigen AgaV-DegQ. TFM harboring pJAQ can secret AgaV-DegQ into the extracellular milieu. Vaccination of Japanese flounder with live TFM/pJAQ elicited strong immunoprotection against both V. harveyi and A. hydrophila infections. (C) 2009 Elsevier Ltd. All rights reserved.

Construction of AFLP-based genetic linkage map for Zhikong scallop, Chlamys farreri Jones et Preston and mapping of sex-linked markers

Zhikong scallop (Chlamys farreri) is an economically important aquaculture species in China; however, frequent mass mortality seriously affects the development of its industry. Genetic linkage map is useful for genetic improvement and selective breeding of C. farreri. Linkage maps were constructed using an intraspecific F-1 cross and amplified fragment length polymorphism (AFLP) markers. Thirty-two selected AFLP primer combinations produced 545 AFLP markers that were polymorphic in either of the parents and segregated in the progeny. Of these segregating markers, 166 were mapped to 19 linkage groups of the female framework map, covering a total of 1503.9 cM, with an average marker spacing of 10.2 cM; and 197 markers were assigned to 20 linkage groups of the male map, covering a total of 1630.7 cM, with 9.2 cM per marker. A sex-linked marker was mapped on the female map with zero recombination and a LOD of 27.3. The genetic length of C farreri genome was estimated as 1889.0 cM for the female and 1995.9 cM for the male. The coverage of the framework map was calculated as 79.6% for the female and 81.7% for the male. When the triplets and doublets were considered, the observed length of the map was calculated as 1610.2 cM with coverage of 85.2% for the female, and 1880.5 cM with coverage of 94.2% for the male. The genetic maps presented here will serve as a basis for the construction of a high-resolution genetic map and mapping of economically important genes. (C) 2004 Published by Elsevier B.V.

Construction and characterization of a bacterial artificial chromosome library of marine macroalga Porphyra yezoensis (Rhodophyta)

A large-DNA-fragment library is necessary for research into the Porphyra genome. In this study, a bacterial artificial chromosome (BAC) library of Porphyra yezoensis was constructed and characterized. The library contains 54,144 BAC clones with an average insert size of about 65 kb and fewer than 0.7% of clones without large inserts. Therefore, its capacity is more than 6.6 P. yezoensis genome equivalents, and the probability of recovering any nuclear DNA sequence from the library is higher than 99%. The library shows good fidelity and stability. A putative trehalose-6-phosphate synthase (TPS) gene was successfully screened out from the library. The above results show that the library is useful for gene cloning and genomic research in P. yezoensis.

Construction of shuttle, expression vector of human tumor necrosis factor alpha (hTNF-alpha) gene and its expression in a cyanobacterium, Anabaena sp. PCC 7120

The construction of the shuttle, expression vector of human tumor necrosis factor alpha (hTNF-alpha) gene and its expression in a cyanobacterium Anabaena sp. PCC 7120 was reported. The 700-bp hTNF cDNA fragments have been recovered from plasmid pRL-rhTNF, then inserted downstream of the promoter PpsbA in the plasmid pRL439. The resultant intermediary plasmid pRL-TC has further been combined with the shuttle vector pDC-8 to get the shuttle, expression vector pDC-TNF. The expression of the rhTNF gene in Escherichia coil has been analyzed by SDS-PAGE and thin-layer scanning, and the results show that the expressed TNF protein with these two vectors is 16.9 percent (pRL-TC) and 15.0 percent (pDC-TNF) of the total proteins in the cells, respectively, while the expression level of TNF gene in plasmid pRL-rhTNF is only 11.8 percent. Combined with the participation of the conjugal and helper plasmids, pDC-TNF has been introduced into Anabaena sg PCC 7120 by triparental conjugative transfer, and the stable transgenic strains have been obtained. The existence of the introduced plasmid pDC-TNF in recombinant cyanobacterial cells has been demonstrated by the results of the agarose electrophoresis with the extracted plasmid samples and Southern blotting with alpha-(32)p labeled hTNF cDNA probes, while the expression of the hTNF gene in Anabaena sp. PCC 7120 has been confirmed by the results of Western blotting with extracted protein samples and human TNF-alpha monoclonal antibodies. The cytotoxicity assays using the mouse cancer cell line L929 proved the cytotoxicity of the TNF in the crude extracts from the transgenic cyanobacterium Anabaena sp. PCC 7120.

Construction and characterization of a live, attenuated esrB mutant of Edwardsiella tarda and its potential as a vaccine against the haemorrhagic septicaemia in turbot, Scophthamus maximus (L.)

The esrB gene of Edwardsiella tarda, which encodes a regulator protein of the type III secretion system, was mutated by the unmarked deletion method and reintroduced by allelic exchange into the chromosome of E. tarda LSE40 by means of the suicide vector pRE 112. The LSE40 esrB mutant was highly attenuated when inoculated intraperitoneally into turbot Scophthamus maximus L., showing a 50% lethal dose of 10(8.1) cfu/fish. The esrB mutants were not recoverable from the internal organs at 14 days post-inoculation. Vaccination with a single dose of 10(5)-10(7) cfu/fish of the esrB mutant elicited significant protection against the wildtype strain of E. tarda LSE40 (relative percentage survival > 50%). The protection correlated well with the antibody titres in the serum of vaccinated fish. (c) 2006 Elsevier Ltd. All rights reserved.

Construction and characterization of two bacterial artificial chromosome libraries of Zhikong scallop, Chlamys farreri Jones et Preston, and identification of BAC clones containing the genes involved in its innate immune system

Large-insert bacterial artificial chromosome (BAC) libraries are necessary for advanced genetics and genomics research. To facilitate gene cloning and characterization, genome analysis, and physical mapping of scallop, two BAC libraries were constructed from nuclear DNA of Zhikong scallop, Chlamys farreri Jones et Preston. The libraries were constructed in the BamHI and MboI sites of the vector pECBAC1, respectively. The BamHI library consists of 73,728 clones, and approximately 99% of the clones contain scallop nuclear DNA inserts with an average size of 110 kb, covering 8.0x haploid genome equivalents. Similarly, the MboI library consists of 7680 clones, with an average insert of 145 kb and no insert-empty clones, thus providing a genome coverage of 1.1x. The combined libraries collectively contain a total of 81,408 BAC clones arrayed in 212 384-well microtiter plates, representing 9.1x haploid genome equivalents and having a probability of greater than 99% of discovering at least one positive clone with a single-copy sequence. High-density clone filters prepared from a subset of the two libraries were screened with nine pairs of Overgos designed from the cDNA or DNA sequences of six genes involved in the innate immune system of mollusks. Positive clones were identified for every gene, with an average of 5.3 BAC clones per gene probe. These results suggest that the two scallop BAC libraries provide useful tools for gene cloning, genome physical mapping, and large-scale sequencing in the species.