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Purification of a New Antibacterial Protein from Housefly(Musca domestica)Larvae

Hui QU ; Youjin HAO ; Yingjun JING ; Desen LI ; Rongqian DU

China Biotechnology.2006;0(09):-.

Insects exhibit a particular resistance to infections.The activation of the innate immune response of insects is involved in the recognition of the infectious nonself and subsequent activation of cellular and humoral reactions.In humoral reactions,insect antibacterial peptides and lysozyme are very important in resistance to infections.Housefly(Musca domestica) is one of the most important kinds of insects and it has strong ability to adapt to the adverse circumstances.It is of momentous theoretical and practical significance to research the immunity system of housefly.The methods of inducement of housefly larvae were firstly studied.Then an antibacterial protein,whose molecular weight is 28kDa,was purified from housefly larvae,induced by 30% H2O2,through saltout,Sephadex G25 column,Sephadex G75 column and CMSepharose Fast Flow column.This antibacterial peptide had activities against most of Gram positive and Gram negative bacteria.

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Construction and Determination of an Episomal Secreting Expression Vector:pYES2/CT/?-factor for Saccharomyces cerevisiae

Yadong HU ; Dongsheng YAO

China Biotechnology.2006;0(12):-.

pPIC9 was used as a template and ?-factor gene of Saccharomyces cerevisiae was amplified by PCR.The gene was cloned in the intracellular expression vector pYES2/CT for Saccharomyces cerevisiae and the secreting expression vector named pYES2/CT/?-factor(pYC?)was constructed.The gene of mannase(man)from recombinant vector of pKLAC1-man(pKLman) was cut by restriction enzymes and linked with pYC?.This recombinant vector pYC?-man was used to determine the secretory ability and stability of pYC?.The excellent secretory ability of pYC? was proved by two experiments.One showed that INVSc1/pYC?-man clones formed the clear rings around the clones on the medium contained trypan blue,while INVSc1/pYC? clones had no rings.Further analysis of mannase activity of extracellular supernatant and intracellular extracts showed that both extracellular and intracellular mannase activities of INVSc1/pYC? were not detected,while INVSc1/pYC?-man had evident extracellular mannase activities and no intracellular mannase activities.The stability of pYC? was also very good proved by continuous cultivation for about 150h.

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Computational Identification of microRNAs and Their Targets

Zhiyun GUO ; Canquan MAO ; Lili XIONG

China Biotechnology.2008;28(10):118-123.

The discovery of microRNAs (miRNAs) has introduced a new paradigm into gene regulatory systems. Since inception, computational methods have been an invaluable tool complementing experimental approaches, and many discoveries have been obtained through combination of experimental and computational approaches. The knowledge that has been accumulated about the principles of miRNAs and target recognition were reviewed. The currently available computational methodologies and software for prediction of miRNA and their target genes also have been discussed.

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Detection and Partial Characterization of γ-glutamyltranspeptidase from Cordyceps sinensis Mycelia

Jianglan YUAN ; Xu KANG ; Zheng HU ; Guolin ZOU

China Biotechnology.2008;28(10):100-105.

γ-glutamyltranspeptidase was detected from the cultured mycelia of Cordyceps sinensis (CSGT). Km and Vmax of CSGT was 2.54×10-4 mol/L and 0.1808 mol/L·min respectively when L-glutamic acid 5-(4-nitroanilide) (GpNA) and glycyglycine was used as its substrate. CSGT was stable from pH 8.0 to 11.0 and at or below 20℃. It was optimally active at pH 9.0～10.0 and 30℃. A series of reducing reagents could activate CSGT, and metal cations such as Zn2+, Cu2+, Hg2+ , Mn2+ inhibited strongly activity of the enzyme, but K+, Ca2+, Mg2+ and Na+ at high concentrations had no effect on its activity, indicating that its active center could contain -SH.

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Purification and Characterization of One Alkaline Protease from Actinomucor elegans AS3.2778

Jinquan PAN ; Xiaochun LUO ; Mingquan XIE

China Biotechnology.2008;28(9):111-118.

One alkaline protease from Actinomucor elegans AS3.2778 was purified protein. The enzyme was purified using ammonium sulfate precipitation, ion exchange chromatography, hydrophobic chromatography and size exclusion chromatography method, and its properties were also investigated. The molecular weight of this enzyme is 32 kDa with SDS-PAGE method, optimum temperature is 60℃, optimum pH is 8.5 to 10.5, it is stable in the pH range of 6.0 to 9.0 at < 40℃ temperature, and being completely inhibited by the serine protease inhibitor, PMSF, indicated that it belongs to the serine protease family. Specificity test indicated this protease has extensive selectivity to peptide bones, especially to peptide bones composed of Leucine residue.

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Molecular Cloning and Expression of Cardiotoxin Ⅲ from Naja naja atra in E.coli and Yeast Pichia pastoris

Xingyong CHEN ; Ping LU ; Jing LIU ; Kangsen XU

China Biotechnology.2008;28(8):23-30.

Chinese cobra (Naja naja atra) cardiotoxins are three-fingered family with 60～62 amino acids bind by four disulfide bonds. CardiotoxinⅢ (CTXⅢ) is one of the major toxic component which can cause hemolysis and cytotoxicity. However, there is no report on the fusion expression of CTXⅢ in soluble form so far. The cloning, expression and purification of recombinant CTX Ⅲ (rCTXⅢ) from Naja naja atra in E. coli and in yeast Pichia pastoris were reported here. CTXⅢ gene, fused with enterokinase in E.coli His-patch Thioredoxin expression system, were expressed in soluble form and released by osmotic-shock treatment. CTX Ⅲ gene was also cloned and expressed in the methylotropic yeast Pichia pastoris pPIC9K expression vector in the first time. The yield of the secretion level was 9.5 mg/L. Using straightforward one-step chromatography procedure, the rCTXⅢ, with three additional amino acids (GYT) at the N-terminal site, was purified to a purity of more than 90% and recovery yield of 65%. The purified rCTX Ⅲ was further characterized by cytotoxic assay with IC50 4.66μg/ml. An effective expression and purification system for recombinant CTXs in P. pastoris was developed, this system will permit us the ready isolation of active cardiotoxins. This protocol can also be easily used for the production of the toxin in a larger scale with low cost.

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Expression, Purification and Characterization of [Gly14]-Humanin, a Novel Neuroprotective Peptide

Baofeng YU ; Jun XIE ; Xianjiu CHEN ; Yuehong ZHANG ; Huizhen WANG ; Niuliang CHENG ; Bo NIU

China Biotechnology.2008;28(4):21-26.

Humanin (HN, its analogue [Gly14]-Humanin, HNG) was originally identified as an endogenous peptide that protects neuronal cells from apoptosis induced by various types of Alzheimer's disease-related insults. But the relative low content of this peptide in its natural sources limits its further characterization. An expression vector pET32a/HNG was corstructed and transformed it into E. coli BL21 trxB (DE3). HNG was expressed as a fusion protein in the soluble fraction and was purified by nickel affinity chromatography. Subsequently, the purified fusion protein was cleaved by enterokinase and was further purified by reverse-phase HPLC. A 23 mg recombinant HNG (rHNG) from 1 L bacterial culture was purified. The molecular weight of rHNG determined by ESI-MS was 2876.5 Da which was the expected size for correctly processed peptide. The N-terminal amino acid sequence of rHNG determined by Edman degradation method is identical to the theoretical sequence. Neuroprotective bioassay studies of rHNG exhibited its potential neuroprotective effect comparable to that of the natural HNG peptide.

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The Assembly of a Novel Enzyme Biosensor for Aflatoxin B1 Detection

Daling LIU ; Yi SHEN ; Jing ZHANG ; Dongsheng YAO

China Biotechnology.2008;28(3):44-52.

A novel biosensor for aflatoxin B1 detecting has been reported. The biosensor electrode for AFB1 detecting was assembled by immobilized aflatoxin-oxidoreductase using open-ended multi-walled carbon nanotubes as matrix. Its linear range was between 0.16μM and 3.2μM. And if the specific anti-aflatoxin B1 antibody and aflatoxin oxidoreductase were both immobilized on the electrode with Multi-Walled carbon nanotubes, the detection limit of the modified electrode could be 16 nM with a 10 times improved sensitivity. The aflatoxin enzyme biosensor assembled this way strode one step forward its practical application.

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Improving Potato Plants Oxidative Stress and Salt Tolerance by Gene Transfer Both of Cu/Zn Superoxide Dismutase and Ascorbate Peroxidase

Li TANG ; Hui TANG ; Sangsoo KWAK ; Haengsoon LEE ; Suying WANG ; Xiaoli YANG

China Biotechnology.2008;28(3):25-31.

In plants, oxidative stress is one of the major causes of damage as a result of various environmental stresses and it is primarily due to the excessive accumulation of reactive oxygen species. To develop transgenic potato plants with enhanced tolerance to environmental stress, transgenic potato plants (Solanum tuberosum L. cv. Atlantic) expressing the Cu/ZnSOD and APX genes in chloroplasts were generated under the control of the oxidative stress-inducible promoter. To investigate oxidative stress tolerance, transgenic plants were evaluated at the level of leaf discs and plantlets after methyl viologen (MV) and salt treatment. Leaf discs from transgenic potato plants showed 13% less membrane damage compared to non-transgenic (NT) plants suffering 10 μmol/L MV treatment of 48 h, and showed 1.6-fold higher chlorophyll contents than those of NT plants at 1.0mol/L NaCl treatment (31% vs. 19%). In addition, transgenic potato plants maintained higher rooting rates (75%) during 100mmol/L NaCl treatment than those (12%) from NT plants. Moreover, the tolerance to salt stress in transgenic plants was consistent to increased transcript levels and higher activities of SOD and APX compared to NT plants. These results suggest that expression of Cu/ZnSOD and APX in chloroplasts could be used in plants to enhance the tolerance to environmental stresses.

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Photolithotrophic Cultivation of Laminaria japonica Gametophyte Cells in Stirred Tank Photobioreactors: Studies in Different Pulse Feeding Modes

Siye CHEN ; Hanshi QI

China Biotechnology.2008;28(1):36-43.

Photoautotrophic gametophyte cells of the brown macroalgae Laminaria japonica were cultivated in 500ml stirred tank photobioreactors under seven pulse feeding modes and one batch mode.It is the first time for the study of effects of the feeding time points and feeding quantity on macroalgal cell growth and nutrient consumption.Results showed that, with inoculum density of 50mg DCW/L, in modified APSW artificial seawater medium at 13℃, light intensity of 60μE/m2.s, light cycle of 16/8h L/D, aeration rate of 50ml/min, and agitation speed of 100r/min, feeding the culture with small nutrient quantity was beneficial for the synchronization between nitrate and phosphate absorption, and further for biomass production.Feeding when ambient nutrient was abundant or depleted was quite weak for large amount of biomass accumulation, which might be due to the slowing nutrient absorption, nutrient storage, or the divergence absorption between nitrate and phosphate.Feeding nutrient frequently with small quantity from mid-exponential growth of macroalgal cells, that is maintaining medium nutrient concentration between 1/3 and 1/2 of its initial concentration, was the most effective way for biomass production, with biomass increased by 12.270 times of for 51 days' cultivation.

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