Xia Y.,Shanghai Academy of Agricultural science |
Guo J.,Shanghai Academy of Agricultural science |
Ling H.,Qingdao Vland Biotech Group Co. |
Miao D.,Shanghai Academy of Agricultural science
Research Journal of Biotechnology | Year: 2017
The porcine interferon alpha (PoIFN-α) exerts broadspectrum antiviral effects on various viruses. To date, most of recombinant PoIFN-α (rPoIFN-α) prepared from Escherichia coli were deposited as insoluble, inactive inclusion bodies. In the present study, we produced soluble and biologically active recombinant PoIFN-α8 (rPoIFN-α8). This was gained by codon optimization, cold induced expression vector and optimization of expression conditions. An E. coli codons optimized and synthetic mature PoIFN-α8 was cloned into the cold-shock expression vectors of pCold II and then transferred into Escherichia coli BL21 (DE3) to induce by IPTG. By optimizing inducer concentrations and induction duration, rPoIFN-α8 was expressed primarily as a soluble protein. Purification of the recombinant TEE-His-tagged proteins was achieved in a single step by Ni2+- charged column chromatography. The final yield of soluble rPoIFN-α8 was about 100mg from 1L flask culture. The purified rPoIFN-α8 was confirmed by western blot. The antiviral activity of rPoIFN-α8 against VSV on PK-15 cells was up to 1.0×109 IU/mg of protein. Our study provides a feasible and convenient approach to produce large quantities of soluble and biologically active rPoIFN-α8 for both research and industrial purpose.
Zhang C.,Chinese Academy of Agricultural Sciences |
Zhang C.,Qingdao Vland Biotech Group Co. |
Zhang L.,Chinese Academy of Agricultural Sciences |
Cao S.,Shandong New Hope Liuhe Group Co. |
And 6 more authors.
Food Chemistry | Year: 2016
A reliable LC-MS/MS method with high sensitivity was developed and validated for the determination of dipyrone (DIP) metabolites in goat muscle, fat, liver, and kidney samples. Analytes were extracted using acetonitrile mixed with ammonia solution. After dehydration and evaporation to dryness, extracts were purified using an Oasis MAX cartridge. Chromatographic separation was performed on a hydrophilic interaction liquid chromatography column. The analytes were then detected using triple-quadrupole mass spectrometry in positive electrospray ionization and multiple reaction monitoring mode. Calibration plots were constructed using matrix-matched standards and showed good linearity. Limits of quantification for 4-methylaminoantipyrine (MAA), 4-formylaminoantipyrine (FAA), and 4-acetylaminoantipyrone (AAA) ranged from 0.4 μg kg-1 to 6 μg kg-1, while those for 4-aminoantipyrone (AA) ranged from 10 μg kg-1 to 125 μg kg-1 in all tissues. The developed method was successfully applied in the determination of DIP metabolite residues in actual goat tissues. © 2015 Published by Elsevier Ltd.
Yao G.,Shandong University |
Li Z.,Shandong University |
Li Z.,Qingdao Vland Biotech Group Co. |
Wu R.,Shandong University |
And 3 more authors.
Fungal Genetics and Biology | Year: 2016
Filamentous fungi can initiate vegetative growth on complex plant polysaccharides in nature through secreting a large amount of lignocellulose-degrading enzymes. These fungi develop a large amount of asexual spores to disperse and survive under harsh conditions, such as carbon and nitrogen depletion. Numerous studies report the presence of a cross-talk between asexual development and extracellular enzyme production, especially at the regulation level. This study identified and characterized a C2H2-type transcription factor called PoFlbC, which is an Aspergillus FlbC ortholog, in cellulolytic fungus Penicillium oxalicum. Results showed that the native level of PoFlbC was crucial for the normal growth and asexual development of P. oxalicum. Importantly, deletion of the PoflbC gene substantially reduced cellulase and hemicellulase productions. Comparative transcriptome analysis by RNA sequencing revealed a global downregulation of genes encoding cellulases, hemicellulases, and other proteins with functions in lignocellulose degradation. A similar defect was also observed in the OEPoflbC strain, suggesting that the production of cellulolytic enzymes was maintained by native expression of the PoflbC. In this study, an essential activator for both fungal asexual development and cellulase production was established in P. oxalicum. © 2015 Elsevier Inc.
Zhang W.,Qingdao Agricultural University |
Wang B.,Qingdao Agricultural University |
Mao K.,Qingdao Vland Biotech Group Co. |
Ge W.,Qingdao Agricultural University |
And 2 more authors.
Journal of the Chinese Cereals and Oils Association | Year: 2014
This experiment was conducted to study the effects of different choline level added into feed on immune function and liver histology of geese. One hundred and eighty one-day-old "Qingnonghui" geese were selected and randomly divided into 6 groups with 3 replicates in each group and 10 geese in each replicate. Geese in the six groups were fed the corn-soybean basal diets supplemented with 0, 600, 1200, 1800, 2400 and 3000 mg/kg choline, respectively. The experiment lasted for 15 weeks. The results showed as follows: 1) The supplementation of choline significantly increased immune organ index of geese aged 1 to 4 weeks and 1 to 15 weeks (P<0.05 or P<0.01), but there was no significant difference in antibody titre of Bird Flu disease(P<0.05). 2) in no choline group, swelling of hepatocyte was observed and the cytoplasm was full of fat cavitation, and the cell nuclei are squeezed to one side. The supplementation of 1200~3000 mg/kg choline in diets can decrease fat deposition of hepatocyte effectively. In conclusion, the supplementation of choline in diets for "Qingnonghui" geese is feasible, and the optimum supplemental level is 1200 mg/kg.
Zhang W.,Shandong University |
Kou Y.,Shandong University |
Xu J.,Shandong University |
Cao Y.,Shandong University |
And 7 more authors.
Journal of Biological Chemistry | Year: 2013
Proper perception of the extracellular insoluble cellulose is key to initiating the rapid synthesis of cellulases by cellulolytic Trichoderma reesei. Uptake of soluble oligosaccharides derived from cellulose hydrolysis represents a potential point of control in the induced cascade. In this study, we identified a major facilitator superfamily sugar transporter Stp1 capable of transporting cellobiose by reconstructing a cellobiose assimilation system in Saccharomyces cerevisiae. The absence of Stp1 in T. reesei resulted in differential cellulolytic response to Avicel versus cellobiose. Transcriptional profiling revealed a different expression profile in the Δstp1 strain from that of wild-type strain in response to Avicel and demonstrated that Stp1 somehow repressed induction of the bulk of major cellulase and hemicellulose genes.Twoother putative major facilitator superfamily sugar transporters were, however, up-regulated in the profiling. Deletion of one of them identified Crt1 that was required for growth and enzymatic activity on cellulose or lactose, but was not required for growth or hemicellulase activity on xylan. The essential role of Crt1 in cellulase induction did not seem to rely on its transporting activity because the overall uptake of cellobiose or sophorose by T. reesei was not compromised in the absence of Crt1. Phylogenetic analysis revealed that orthologs of Crt1 exist in the genomes of many filamentous ascomycete fungi capable of degrading cellulose. These data thus shed new light on the mechanism by which T. reesei senses and transmits the cellulose signal and offers potential strategies for strain improvement. © 2013 by The American Society for Biochemistry and Molecular Biology, Inc.
Yao G.,Shandong University |
Li Z.,Shandong University |
Li Z.,Qingdao Vland Biotech Group Co. |
Gao L.,Shandong University |
And 4 more authors.
Biotechnology for Biofuels | Year: 2015
Background: In cellulolytic fungi, induction and repression mechanisms synchronously regulate the synthesis of cellulolytic enzymes for accurate responses to carbon sources in the environment. Many proteins, particularly transcription regulatory factors involved in these processes, were identified and genetically engineered in Penicillium oxalicum and other cellulolytic fungi. Despite such great efforts, its effect of modifying a single target to improve the production of cellulase is highly limited. Results: In this study, we developed a systematic strategy for the genetic engineering of P. oxalicum to enhance cellulase yields, by enhancing induction (by blocking intracellular inducer hydrolysis and increasing the activator level) and relieving the repression. We obtained a trigenic recombinant strain named 'RE-10' by deleting bgl2 and creA, along with over-expressing the gene clrB. The cellulolytic ability of RE-10 was significantly improved; the filter paper activity and extracellular protein concentration increased by up to over 20- and 10-fold, respectively, higher than those of the wild-type (WT) strain 114-2 both on pure cellulose and complex wheat bran media. Most strikingly, the cellulolytic ability of RE-10 was comparable with that of the industrial P. oxalicum strain JU-A10-T obtained by random mutagenesis. Comparative proteomics analysis provided further insights into the differential secretomes between RE-10 and WT strains. In particular, the enzymes and accessory proteins involved in lignocellulose degradation were elevated specifically and dramatically in the recombinant, thereby confirming the importance of them in biomass deconstruction and implying a possible co-regulatory mechanism. Conclusions: We established a novel route to substantially improve cellulolytic enzyme production up to the industrial level in P. oxalicum by combinational manipulation of three key genes to amplify the induction along with derepression, representing a milestone in strain engineering of filamentous fungi. Given the conservation in the mode of cellulose expression regulation among filamentous fungi, this strategy could be compatible with other cellulase-producing fungi. © 2015 Yao et al.; licensee BioMed Central.