A Modified Quantum Dot-Based Dot Blot Assay for Rapid Detection of Fish Pathogen Vibrio anguillarum

( Yang Zhang ),( Jingfan Xiao ),( Qiyao Wang ),( Yuanxing Zhang ) 한국미생물 · 생명공학회 2016 Journal of microbiology and biotechnology Vol.26 No.8

Vibrio anguillarum, a devastating pathogen causing vibriosis among marine fish, is prevailing in worldwide fishery industries and accounts for grievous economic losses. Therefore, a rapid on-site detection and diagnostic technique for this pathogen is in urgent need. In this study, two mouse monoclonal antibodies (MAbs) against V. anguillarum, 6B3-C5 and 8G3-B5, were generated by using hybridoma technology and their isotypes were characterized. MAb 6B3-C5 was chosen as the detector antibody and conjugated with quantum dots. Based on MAb 6B3- C5 labeled with quantum dots, a modified dot blot assay was developed for the on-site determination of V. anguillarum. It was found that the method had no cross-reactivity with other than V. anguillarum bacteria. The detection limit (LOD) for V. anguillarum was 1 × 10³ CFU/ml in cultured bacterial suspension samples, which was a 100-fold higher sensitivity than the reported colloidal gold immunochromatographic test strip. When V. anguillarum was mixed with turbot tissue homogenates, the LOD was 1 × 10³ CFU/ml, suggesting that tissue homogenates did not influence the detection capabilities. Preenrichment with the tissue homogenates for 12 h could raise the LOD up to 1 × 10² CFU/ml, confirming the reliability of the method.

High brightness and precise adjustment of multicolor-tunable luminescence of Lu2GeO5:Tb3+, Eu3+ phosphors for white LEDs

Shaoan Zhang,Yuanxing Li,Wenfeng Li,Zhenzhang Li,Minfan Qiu,Zhongfei Mu,Yang Li,Yihua Hu 한국물리학회 2019 Current Applied Physics Vol.19 No.9

High brightness and precise adjustment of luminescence colour of phosphors are two main targets in the research of phosphor-converted white LEDs. However, few feasible strategy can be employed to achieve the multicolortunable luminescence under the premise of maintaining high quantum efficiency. Here, we demonstrate a highefficiency energy-transfer process from Tb3+ to Eu3+ ions with a higher luminescent quantum efficiency (64.5% and 53.4%, respectively), and green-red multicolor emission in Lu2GeO5 host via varying the doping content of Tb3+ and Eu3+ ions. Besides, Lu2GeO5:Tb3+, Lu2GeO5: Eu3+ and Lu2GeO5: Tb3+, Eu3+ all exhibit weak thermal quenching which ensures the stable use of white LED device in the high temperature environment. This paper provides a novel multicolor-tunable phosphor with high brightness, efficient energy transfer and weak thermal quenching, which presents a potential application for UV-converted white LEDs.

Expression and Purification of Soluble Recombinant Human Endostatin in Escherichia coli

Cuihong Du,Xiaoping Yi,Yuanxing Zhang 한국생물공학회 2010 Biotechnology and Bioprocess Engineering Vol.15 No.2

Endostatin, a 20 kDa C-terminal fragment of collagen XVIII, is a specific inhibitor of endothelial cell proliferation and angiogenesis. In the present study, we produced soluble and biologically active recombinant human endostatin (rhEndostatin) in Escherichia coli by expressing via fusion with solubility-promoting peptides and optimizing the expression conditions. The rhEndostatin was expressed via fusion with glutathione S-transferase (GST) and NusA protein, respectively. It revealed that NusA protein enhanced the production of soluble rhEndostatin; but GST didn’t. By optimizing the expression conditions, the production of soluble NusA-rhEndostatin fusion protein was about 50% of total cellular proteins and about 90% of the products appeared in the cellular supernatant fraction. The soluble NusA-rhEndostatin fusion protein was purified by one-step hydrophobic interaction chromatography and NusA was removed by thrombin. Then rhEndostatin was purified by affinity chromatography and gel filtration chromatography. As a result, a simple and economical purification procedure for rhEndostatin isolation was obtained. The biological activity of the rhEndostatin was demonstrated in vitro using a human vascular endothelial cells (HuVECs) proliferation assay. Our study provides a feasible and convenient approach to produce soluble and biologically active rhEndostatin.

Transcriptome analysis of Δmig1Δmig2 mutant reveals their roles in methanol catabolism, peroxisome biogenesis and autophagy in methylotrophic yeast Pichia pastoris

Lei Shi,Xiaolong Wang,Jinjia Wang,Ping Zhang,Fei Qi,Menghao Cai,Yuanxing Zhang,Xiangshan Zhou 한국유전학회 2018 Genes & Genomics Vol.40 No.4

Two catabolite repressor genes (MIG1 and MIG2) were previously identified in Pichia pastoris, and the derepression of alcohol oxidase (AOX) expression was realized in Δmig1 or Δmig1Δmig2 mutants grown in glycerol, but not in glucose. In this study, genome-wide RNA-seq analysis of Δmig1Δmig2 and the wild-type strain grown in glycerol revealed that the expression of numerous genes was greatly altered. Nearly 7% (357 genes) of approximately 5276 genes annotated in P. pastoris were significantly upregulated, with at least a two-fold differential expression in Δmig1Δmig2; the genes were mainly related to cell metabolism. Approximately 23% (1197 genes) were significantly downregulated; these were mainly correlated with the physiological characteristics of the cell. The methanol catabolism and peroxisome biogenesis pathways were remarkably enhanced, and the genes AOX1 and AOX2 were upregulated higher than 30-fold, which was consistent with the experimental results of AOX expression. The Mig proteins had a slight effect on autophagy when cells were grown in glycerol. The expression analysis of transcription factors showed that deletion of MIG1 and MIG2 significantly upregulated the binding of an essential transcription activator, Mit1p, with the AOX1 promoter, which suggested that Mig proteins might regulate the AOX1 promoter through the regulation of Mit1p. This work provides a reference for the further exploration of the methanol induction and catabolite repression mechanisms of AOX expression in methylotrophic yeasts.

Biosynthesis and uptake of glycine betaine as cold-stress response to low temperature in fish pathogen Vibrio anguillarum

Yue Ma,Qiyao Wang,Xiating Gao,Yuanxing Zhang 한국미생물학회 2017 The journal of microbiology Vol.55 No.1

Fish pathogen Vibrio anguillarum, a mesophile bacterium, is usually found in estuarine and marine coastal ecosystems worldwide that pose a constant stress to local organism by its fluctuation in salinity as well as notable temperature change. Though V. anguillarum is able to proliferate while maintain its pathogenicity under low temperature (5–18°C), so far, coldadaption molecular mechanism of the bacteria is unknown. In this study, V. anguillarum was found possessing a putative glycine betaine synthesis system, which is encoded by betABI and synthesizes glycine betaine from its precursor choline. Furthermore, significant up-regulation of the bet gene at the transcriptional level was noted in log phase in response to cold-stress. Moreover, the accumulation of betaine glycine was only found appearing at low growth temperatures, suggesting that response regulation of both synthesis system and transporter system are cold-dependent. Furthermore, in-frame deletion mutation in the two putative ABC transporters and three putative BCCT family transporters associated with glycine betaine uptake could not block cellular accumulation of betaine glycine in V. anguillarum under coldstress, suggesting the redundant feature in V. anguillarum betaine transporter system. These findings confirmed that glycine betaine serves as an effective cold stress protectant and highlighted an underappreciated facet of the acclimatization of V. anguillarum to cold environments.

An Effective Method for Deproteinization of Bioactive Polysaccharides Extracted from Lingzhi (Ganoderma atrum)

Yi Chen,Mingyong Xie,Wenjuan Li,Hui Zhang,Shaoping Nie,Yuanxing Wang,Chang Li 한국식품과학회 2012 Food Science and Biotechnology Vol.21 No.1

Deproteinization procedure is a fundamental step for analyzing polysaccharide from natural plants. In this study, in the course of refining bioactive polysaccharides from lingzhi (Ganoderma atrum), an effective deproteinization method using lead acetate solution was established by comparing with other available methods. The percentages of deproteinization, polysaccharide loss, and its antioxidant activities loss were used as the index to evaluate and optimize the precipitation experimental conditions. The results showed that the modified method, precipitation with the addition of 0.4-0.52%(w/v) lead acetate, was superior to the others, as evidenced by the highest deproteinization efficiency (88%), as well as the lowest polysaccharide loss (17%). And notably its antioxidant activity also remained good (loss 15%). It provides a simple prefractionation step for the analysis of polysaccharide from natural plants. Polysaccharide isolated by this method is in the native state. Our method may offer a rapid method for removing protein from plant polysaccharides in large scale.

Selection of Stable Reference Genes for Real-Time Quantitative PCR Analysis in Edwardsiella tarda

( Zhongyang Sun ),( Jia Deng ),( Haizhen Wu ),( Qiyao Wang ),( Yuanxing Zhang ) 한국미생물 · 생명공학회 2017 Journal of microbiology and biotechnology Vol.27 No.1

Edwardsiella tarda is a gram-negative pathogenic bacterium in aquaculture that can cause hemorrhagic septicemia in fish. Many secreted proteins have already been identified as virulent factors of E. tarda. Moreover, since virulent phenotypes are based on the expression regulation of virulent genes, understanding the expression profile of virulent genes is important. A quantitative RT-PCR is one of the preferred methods for determining different gene expressions. However, this requires the selection of a stable reference gene in E. tarda, which has not yet been systematically studied. Accordingly, this study evaluated nine candidate reference genes (recA, uup, rpoB, rho, topA, gyrA, groEL, rpoD, and 16S rRNA) using the Excel-based programs BestKeeper, GeNorm, and NormFinder under different culture conditions. The results showed that 16S rRNA was more stable than the other genes at different culture growth phases. However, at the same culture time, topA was identified as the reference gene under the conditions of different strains, different culture media, and infection, whereas gyrA was identified under the condition of different temperatures. Thus, in experiments, the expression of gapA and fbaA in E. tarda was analyzed by RT-qPCR using 16S rRNA, recA, and uup as the reference genes. The results showed that 16S rRNA was the most suitable reference gene in this analysis, and that using unsuitable reference genes resulted in inaccurate results.

De novo transcriptome sequencing of marine-derived Aspergillus glaucus and comparative analysis of metabolic and developmental variations in response to salt stress

Shaomei Liu,Jiaxin Li,Yuan Wu,Yanna Ren,Qi Liu,Qiyao Wang,Xiangshan Zhou,Menghao Cai,Yuanxing Zhang 한국유전학회 2017 Genes & Genomics Vol.39 No.3

Aspergillus glaucus HB1-19 is a typical marinederived fungus preferring the dependence on sea water for its growth, asexual development and polyketides biosynthesis. Therein, salt stress greatly functions even in superior to light illumination, which is also a critical regulation signal for fungi. Here, comparative RNA-seq analysis of this strain was performed under conditions of saltstress ? dark (group A), non salt-stress ? dark (group B), salt-stress ? light (group C). The RNA-seq generated a total of 19,024 unigenes with an average length of 1415 bp. Differentially expressed genes were very similar between group A and group C but greatly differed between group A and group B, proving that salt stress functioned superior to light illumination globally. Salt stress highly enhanced primary metabolism and activated Ras and MAPK signaling pathways. There seems no direct interaction between asexual development and polyketides biosynthesis. Salt stress inhibited terpenoids biosynthesis but showed little influences on polyketide pathway as well as other secondary metabolism pathways. These findings provide a better understanding of marine fungi adapting to marine environment. Also, it indicates that the so-called ‘salt stress-induced’ may truly be a ‘metal ions-induced’ for biosynthesis of secondary metabolites in marine fungi.