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Zhu X.,Shanghai JiaoTong University | Chen L.,Shanghai Normal University | Shen P.,Development Center for Science and Technology | Jia J.,Shanghai Key Laboratory of Agricultural Genetics and Breeding | And 2 more authors.
Journal of Agricultural and Food Chemistry

Protein-based detection methods, enzyme-linked immunosorbent assay (ELISA) and lateral flow strip, have been widely used for rapid, spot, and sensitive detection of genetically modified organisms (GMOs). Herein, one novel quantum dot-based fluorescence-linked immunosorbent assay (QD-FLISA) was developed employing quantum dots (QDs) as the fluorescent marker for the detection of the Cry1Ab protein in MON810 maize. The end-point fluorescent detection system was carried out using QDs conjugated with goat anti-rabbit secondary antibody. The newly developed Cry1Ab QD-FLISA assay was highly specific to the Cry1Ab protein and had no cross-reactivity with other target proteins, such as Cry2Ab, Cry1F, and Cry3Bb. The quantified linearity was achieved in the value range of 0.05-5% (w/w). The limits of detection (LOD) and quantification (LOQ) of the QD-FLISA were 2.956 and 9.854 pg/mL, respectively, which were more sensitive than the conventional sandwich ELISA method. All of the results indicated that QD-FLISA was a highly specific and sensitive method for the monitoring of Cry1Ab in GMOs. © 2011 American Chemical Society. Source

Yu R.,Shanghai Key Laboratory of Agricultural Genetics and Breeding
Wei sheng wu xue bao = Acta microbiologica Sinica

A new method used to heterologously express [FeFe]-hydrogenase in Escherichia coli was investigated in our present study. By homologous recombination, three assistant genes (hydE, hydF and hydG) for hydrogenase were integrated into the chromosome of E. coli BW 25113-10, in which all hydrogenase genes were inactivated. A hydrogenase structural gene hydA from Clostridium butyricum was used to test the hydrogenase maturation ability of the recombined E. coli. BW 25113-13. The corrected integration of the three assistant genes was confirmed by PCR, and RT-PCR results indicated that the three accessory genes were transcripted in the recombinant. The active expression of hydA indicated that the constitutively expressed accessory proteins could assist the maturation of the [FeFe]-hydrogenases. A simplified [FeFe]-hydrogenase expression recombinant E. coli BW25113-13 was constructed. It would lay foundations for the functional screening of [FeFe]-hydrogenases and the construction of novel hydrogen producing pathways in E. coli. Source

Shi L.,Nanjing Agricultural University | Fang X.,Nanjing Agricultural University | Li M.,Nanjing Agricultural University | Mu D.,Nanjing Agricultural University | And 3 more authors.
World Journal of Microbiology and Biotechnology

In this study, we report the development of a simple and efficient system for genetic transformation of the medicinal fungus Ganoderma lucidum. Various parameters were optimized to obtain successful Agrobacterium tumefaciens-mediated transformation. Co-cultivation of bacteria and protoplast at a ratio of 1,000:1 at 25°C in medium containing 0. 2 mM acetosyringone was found to be the optimum condition for high efficiency transformation. Four plasmids, each carrying a different promoter driving the expression of an antibiotic resistance marker, were tested. The construct carrying the Ganoderma lucidum glyceraldehyde-3-phosphate dehydrogenase (GPD) promoter showed good transformation efficiency, whereas constructs with the GPD promoter from ascomycetes were ineffective. Our analysis showed that over 70% of the transformants tested remained mitotically stable even after five successive rounds of subculturing. We were able to detect the expression of EGFP and GUS reporter genes in the Ganoderma lucidum transformants by fluorescence imaging and histochemical staining assays respectively. Our results demonstrate a new transgenic approach that will facilitate Ganoderma lucidum research. © 2011 Springer Science+Business Media B.V. Source

Jin H.,Jiangnan University | Liu G.,Jiangnan University | Ye X.,Shanghai Key Laboratory of Agricultural Genetics and Breeding | Duan Z.,Jiangnan University | And 2 more authors.
Biochemical Engineering Journal

Porcine interferon-α (pIFN-α) production by recombinant Pichia pastoris with standard induction strategy at 30°C often suffers problems such as low antiviral activity, long cells adaptation period, etc. To solve these problems, a combinational induction strategy by simultaneously controlling induction temperature at 20°C and dissolved oxygen concentration (DO) over 50% was proposed and the relevant fermentation runs were conducted in a 5l bioreactor. With this control strategy, pIFN-α antiviral activity could be continuously enhanced and eventually reached a level of 3.62×107IU/ml, which was about 16-fold of the maximum obtained when induction was done at 20°C but without DO control, and more than 100-fold of the maximum obtained with the standard induction strategy at 30°C. The novel control strategy could enhance alcohol oxidase (AOX) activity and relieve oxygen supply limitation in oxidative phosphorylation reaction to accelerate ATP regeneration simultaneously. As a result, the metabolic flux towards pIFN-α synthesis was enhanced and the adaptation period was shortened, enabling the entire system to be operated in a most efficient way. © 2010 Elsevier B.V. Source

Gao M.-J.,Jiangnan University | Li Z.,Shanghai Key Laboratory of Agricultural Genetics and Breeding | Yu R.-S.,Shanghai Key Laboratory of Agricultural Genetics and Breeding | Wu J.-R.,Jiangnan University | And 4 more authors.
Bioprocess and Biosystems Engineering

The production of porcine interferon-α (pIFN-α) by Pichia pastoris was largely enhanced when adopting sorbitol/methanol co-feeding induction strategy at 30°C in a 10-L fermentor. Analysis of energy regeneration pattern and carbon metabolism revealed that major energy metabolism energizing pIFN-α synthesis shifted from formaldehyde dissimilatory energy metabolism pathway to TCA cycle under the methanol/sorbitol co-feeding induction strategy. The sorbitol/methanol co-feeding induction strategy weakened formaldehyde dissimilatory pathway and repressed the accumulation of toxic metabolite-formaldehyde, reduced theoretical oxygen consumption rate and oxygen supply requirement, and increased energy/methanol utilization efficiency so that more methanol could be effectively used for pIFN-α synthesis. As a result, pIFN-α antiviral activity reached a highest level of 1.8 × 107 IU/mL which was about 10- to 200-folds of those obtained under pure methanol induction at 20 and 30°C, respectively. © 2012 Springer-Verlag. Source

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