25 November 2013, Volume 33 Issue 11

    

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  N-acetyl-L-cysteine Protect BMCSs Against Apoptosis Induced by H₂O₂ Via Regulaing the Activity of PI3K-Akt Signaling Pathways

  XIE Rong-hui, YIN Chang-chang, YIN Ming, CHEN Wei-cai, WU Ya-hua, HE Ding-wen, LIN Si-wen, GENG Shu-guo

  China Biotechnology. 2013, 33(11): 1-7.

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  Objective: To study whether the N-acetyl cysteine (NAC) has direct cytoprotective effects on bone marrow mesenchymal stem cells (BMSCs) against apoptosis induced by H₂O₂ and and if the PI3K-Akt has partticipated in the mechanism of anti-apoptosis.Methods: BMSCs were extracted from SD rats and cultured by whole bone marrow adherence method. cultured cells were randomly divided into normal control group, H₂O₂ treatment group, the NAC group, NAC + H₂O₂ group and LY294002 intervention group. Then the cellular morphology was observed under inverted phase contrast microscope; Cell survival rate of BMSCs was determined by MTT assay in the different concentration of H₂O₂ and NAC; The apoptosis rate of BMSCs was detected by flow cytometry;the expressions of p-Akt and Akt were analyzed by Western blot.Results The apoptosis of BMSCs could be induced by H₂O₂,its cell survival rate occurred coincident with H₂O₂ concentrations,and its morphology appears typical apoptotic characteristic.Compared with H₂O₂ group,the rate of apoptosis between normal control group and NAC+H₂O₂ group were lower;but in LY294002 intervention group,its rate of apoptosis was higher and has no significant difference as compared with that of H₂O₂ group;Compared with normal group, NAC could increase the expression levels of p-Akt and be related to time of exposure; Compared: with H₂O₂ group,the expression of p-Akt increased,there was no significant difference between H₂O₂ group and LY294002 intervention group.Conclusion: N-acetyl-L-cysteine can protect the BMCSs against apoptosis induced by H₂O₂ via the activation of PI3K-Akt signaling pathways.
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  Expression, Purification and Enzymatic Characterization of Arabidopsis L-Cysteine Desulfhydrase

  YUAN Hui-hong, LIANG Ya-li, SHEN Jie-jie, ZHANG Li-ping, LIU Zhi-qiang, PEI Yan-xi

  China Biotechnology. 2013, 33(11): 8-13.

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  Objective: The recombinant Arabidopsis thaliana L-Cysteine Desulfhydrase (AtLCD) that catalyze the generation of endogenous H₂S was expressed in recombinant E. coli BL21 (DE3)/pET28a-LCD strains. We characterized a AtLCD from E. coli and optimized its the condition of induction and purification. Methods: AtLCD were purified from E. coli BL21 (DE3)/pET28a-LCD to analyze its enzymatic properties after it was induced by isopropyl-b-D-thiogalactopyranoside (IPTG). Results: The optimized conditions were: the AtLCD protein was induced by 0.1 mmo/L IPTG at 30℃ for 3 h, and purified through Ni–AKTA column, and the purified AtLCD of optimized imidazole was 500 mmol/L. The results shows that the Optimal pH value and temperature of the AtLCD were 9.5 and 37℃, respectively. Under the optimum conditions, Km value and Vmax of the AtLCD for hydrolysis of L-Cysteine was 1.572 mmol/L and 1.52 nmol/ (mg·min). Effects of metal ions on the activity of recombination AtLCD showed that Mg²⁺, Fe³⁺ and EDTA inhibited the enzyme activity lightly, while Ba²⁺ , Ca²⁺ and Co²⁺ enhanced the enzyme activity, Co²⁺ enhanced it obviously. Moreover, the recombination AtLCD activity was inhibited varying degrees by SDS and hydroxylamine. Conclusion: Enzymatic properties of AtLCD was obtained and optimized its the condition of induction and purification. It will provide theoretical basis for AtLCD and response to adversity stress mechanism of an important gasotransmitter H₂S in plants.
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  BMP9-induced Osteogenic Differentiation is Partially Inhibited by miR-30a in Mesenchymal Stem Cell Line C3H10 T1/2

  LI Bao-lin, BAI Hui-Li, ZHANG Ru-Yi, YAN Shu-Juan, HE-Fang, YANG Dang-Dang, LIU-Chen, SHI Qiong

  China Biotechnology. 2013, 33(11): 14-20.

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  Objective:To study the role of miR-30a in bone morphogenetic protein 9 (BMP9) induced osteogenic differentiation of mesenchymal stem cell line C3H10T1/2.Method:C3H10T1/2 cells were treated with Adenovirus GFP and BMP9,the early osteogenic marker Alkaline phosphatase(ALP) activity was detected by ALP quantitative assay and staining assay,the later osteogenic marker calcium precipitation was detected by Alizarin Red S staining,OPN was another later marker,which was detected by PCR and Western blot.The expression of miR-30a in C3H10T1/2 cells quantified by Real-time PCR.Effects of miR-30a on BMP9-induced osteogenic differentiation of C3H10T1/2 cells was detected by ALP,OPN and calcium precipitation.Runx2 was detected in order to study its mechanism. Results: BMP9 was not only increased the activity of ALP on days 5,7,the calcium precipitation on 14 day was also increased compared with Ad-GFP group.miR-30a reduced the expression of ALP on BMP9-induced osteogenic differentiation.The expression of OPN and calcium precipitation were also inhibited by miR-30a.Conclusion:BMP9-induced osteogenic differentiation is partially inhibited by miR-30a in mesenchymal stem cell line C3H10 T1/2.
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  Optimization of Culture Medium and Prediction of Antibacterial Activity by Bacillus Amyloliquefaciens Q-426 Fermentation

  ZHOU Guang-qi, MA Peng-bo, LIU Qiao, QUAN Chun-shan, FAN Sheng-di

  China Biotechnology. 2013, 33(11): 21-26.

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  Improve the antibacterial activity produced by Bacillus amyloliquefaciens Q-426 fermentation. To this end, response surface method is employed to optimize culture medium. The optimized medium is (g/L): glucose 3.92, ammonium chloride 0.19, magnesium chloride 3.83, beef extract 5.00, and the optimization raised the diameter of inhibition zone (DIZ) from 24 mm to 29mm.In addition, we present a model based on BP neural network to predict the DIZ according to the medium components. The BP neural network prediction model is trained using the culture medium components as inputs and the DIZ as output. The fitting error of our prediction model is -2.9629%~2.8571% (absolute mean is 1.1979%); the predicting error is -1.1111%~1.1538% (absolute mean is 0.9931%). Therefore, our study shows the feasibility of the prediction model for DIZ using BP neural network.
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  Screening and Indetification of Anti-rabies Virus Single Chain Fragments Antiboy from Human Phage Display Library

  CHEN Ji-jun, MAO Xiao-yan, QIAO Yu-ling, BI Si-ying

  China Biotechnology. 2013, 33(11): 27-31.

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  Objective: Construct a human anti-rabies virus single-chain fragments (scFV) antibody phage antibody library. Screening, expression and indeticification of human single chain(scFv) with in vivo anti-rabies neutralizing activity by phage display. Methods: Based on phage display libraries which originate from the peripheral blood of 21 high titers of healthy donors vaccinated rabies vaccines, purified rbies virus was coated on immune tubes. Three round screens was executed. The postivie rate of monclone phage was detected by ELISA. Sequences of positive clone were analysed by Vbase2 and DNAStar. E.coli BL21(DE3) recombinant expression system were constructed. ScFv was expressed by IPTG induction and purified by Ni affinity chromatography. Determined the vivo neutralizing activity of scFv by RFFIT and detected the coss-binding capacity with 3aG, CVS, CTN and PV rabies virus stain by indrect ELISA. Results: 40 scFv sequences were obtained. 7 scFv-BL21(DE3) recombinant expression system were constructed. 3/7 scFv-BL21(DE3) were soluble expression. 4/7 scFv had in vivo neutralizing activity more than 500IU/mg. All of the scFv had evidented coss-binding capcity with 3aG, PV and CTN starin. Conclusion: ScFv were obtained for anti-rabies virus from phage antibody libirary. RFFIT results show that expressed scFvs have in vivo anti-rabies virus neturilizing activity.
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  The Effect of Carbon and Nitrogen Sources on Laccase Production and Properties from Fomes fomentarius

  SHANG Jie, WU Qiu-xia, LIAN Xiao-long, WANG Qiu-yu

  China Biotechnology. 2013, 33(11): 32-37.

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  The production of laccase by Fomes fomentarius was studied. Cultures conditions involving variations in carbon and nitrogen sources and different C:N ratios were examined at constant temperature and pH, with the aim of increasing yield of laccase. And its enzymatic properties were determined. The best results were obtained when using wheat bran and peptone as carbon and nitrogen sources respectively with a C:N ratio of 10.4. Compared to the initial medium, the highest laccase yield observed is approximately increased by 7.88 times under the optimized conditions. The optimum pH and temperature for its activity is 3.0 and 50℃ with the corresponding K_m and V_max of 0.20 mmol/L and 2.58 mmol/L·min respectively. DTT(0.1mmol/L) and NaN₃(10mmol/L) nearly inhibit all activity of the laccase, as well as the metal ions especially Ba²⁺, Ca²⁺, Co²⁺ and Fe²⁺(10mmol/L). In summary, our results will provide basic information to the production of laccase by Fomes fomentarius and the utilization of environmental engineering in the future.
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  Isolation, Identification and Characterization of a Biofilm Formation Strain

  LI Long-jie, ZHOU Gang, SHI Qing-shan, OUYANG You-sheng, CHEN Yi-ben, HU Wen-feng

  China Biotechnology. 2013, 33(11): 38-43.

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  Objective: Isolate and screen a strain that has the ability to form biofilms from the samples of industrial spoilage with isothiazolinone, meanwhile research its preliminary characterization of biofilm formation. Methods:The streak plate and crystal violet staining methods were used to isolate and screen the biofilm-forming strain respectively. Morphological, physiological characteristics and 16S rDNA sequence analysis were adopted to determine the phylogenetic position of the isolated strain. The biofilms formed on the glass surface were observed under confocal scanning laser microscopy, including exopolysaccharides and live or dead cells. The influence of culture time, pH and different carbon sources on biofilm formation was also researched by crystal violet staining method in the microtiter plates. Results: A biofilm-forming strain was successfully isolated and named as BF-17, which was identified and clarified into Enterobacter cloacae. The highest biofilm biomass was found at the time of 96 h or pH 5.0, respectively. After static cultured for 4 days, biofilms of BF-17 formed on the glass surface exhibited a representative morphological characteristics and structures of the typical biofilms. Meanwhile, the highest biofilm formation was formed in the M9 medium with the supplementation of α-lactose or D-fructose respectively. Whereas, addition of citric acid in M9 medium made the lowest biofilm formation. Conclusion: E. cloacae BF-17 shows a stable capacity of biofilm formation and can be used as a sample for further biofilm study. Moreover, the biofilm formation of BF-17 can be influenced by various external environment factors.
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  The Optimization Research of Fermentation Medium of γ-Polyglutamic Acid(γ-PGA) Produced by Bacillus natto

  ZHANG Wen, ZHANG Shu-qing, MA Xiao-tong, HE Cui-cui

  China Biotechnology. 2013, 33(11): 44-50.

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  Optimization of fermentation medium for Bacillus natto ZW-2 to produce γ-polyglutamic acid(γ-PGA)was studied to increase the yield of γ-polyglutamic with the shaking-flask culture. With Design-Export software, Plackett-Burman Design was used to evaluate the effect of the factors on Bacillus natto ZW-2 to produce γ-PGA based on single-factor optimization experiment. Then steepest ascent experiments was designed to approach the maximum production area of γ-PGA.At last, Box-Behnken experiment was used to get the optimal fermentation medium.The results showed that the optimal composition was sodium glutamate 68.76g/L, NH₄Cl1.16g/L, K₂HPO₄2.16g/L. On this condition the average yield of the verifying experiment was 36.421g/L, which was 1.54 times the original medium. Plackett-Burman design and Box-Behnken Design were simultaneously used in the optimization of the fermentation of Bacillus natto ZW-2 to produce γ-PGA, which was proved to be effective and economical.
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  A Novel Purification Method for Lysosomes by Lmmunoprecipitation

  ZHANG Lei, YANG Yu, WU Chuan-fang

  China Biotechnology. 2013, 33(11): 51-55.

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  A novel purification method for lysosomes by IP lysosomal membrane protein was explored in this paper. LAMP2-Flag was ligated into the eukaryotic expression vector pcDNA3.1, and was transfected into Hela cell line. The lysosomal location of LAMP2-Flag was conformed by Immunofluorescence labeling. Hela cell lines that can stably express LAMP2-Flag were cultured in large numbers. Highly pure lysosomes were isolated from cell extractions using anti-Flag immunoprecipitation under native condition. The purifiedlysosomes contained highlyenriched lysosomal marker proteins and enzymems, and no mitochondrial contamination. Our rResults indicate the efficiency of this method in the isolation of lysosomes of high purity from LAMP2-Flag expressing Hela cell lines.
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  Development of an Aptasensor for Electrochemical Detection of Tetracycline

  CHEN Dan, YAO Dong-sheng, XIE Chun-fang, LIU Da-ling

  China Biotechnology. 2013, 33(11): 56-62.

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  The residual antibiotics in agricultural products became one of the most noticeable problems for animal derived food security, which caused many known and potential harm to public health. One of the most common antibiotics used in fodder animals is tetracyclines. Developing the rapid, simple and sensitive biosensor system for tetracyclines detection is very important in food safety control. In this paper, a tetracycline binding aptamer, whose recognition is confirmed by Isothermal Titration Calorimetry, is used as bio-recognizer. The developed biosensor in tetracycline detection and the electrochemical behavior are investigated. Results: By using isothermal titration calorimetry, the aptamer shows a high affinity to tetracycline, the dissociation equilibrium constant is at Kd=51.8μmol/L. According to the differential pulse voltammetry (DPV) analysis, there is a linear relationship between the log concentration of tetracycline and the charge transfer resistance (ΔIp) in the tetracycline conc. ranges from 5.0 to 5.0×10³μg/L with correlation coefficient of 0.987 6. The detection limit is at 1.0μg/L within a detection time of 15 min. The detection limit lies obviously lower than the National limited residue of tetracycline (6.0×10²μg/L) and also lower than other reported aptasensor for tetracycline.
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  An Improved Method to Measure Bioactivity of the Fusion Protein GGH Based on the Intracellular cAMP Level

  GENG Yan, REN Yi-lin, XU Zheng-hong, DOU Wen-fang

  China Biotechnology. 2013, 33(11): 63-67.

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  Objective: To improve a method to determine bioactivity of the fusion protein GGH based on the intracellular cAMP level. Methods: According to GGH is the analog of GLP-1 which can promote islet β cells to produce second messenger cAMP, detecting intracellular cAMP levels by enzyme-linked immunosorbent assay (ELISA) can be used to characterize GGH biological activity. Results: Exenatide or GGH was used to stimulate rat insulinoma cell line RIN m5f to produce cAMP. The optimal cAMP protective agent and drug diluent were 100nmol/L IBMX and DPBS. We found that freezing and thawing cells in liquid nitrogen 2 times led to maximum and stable production of cAMP in the supernatant measured by ELISA. Conclusion: An in vitro high-throughput screening method for detecting the bioactivities of the fusion protein GGH was successfully modified. Using this method, the biological activity of GGH and GLP-1 analogs can be rapidly identified.
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  Fusion Expression and Purification of Human Beta Defensin-3 in E.coli

  ZHU Jun-ping, LI Gai-rui, DU Qi-ke, GUO Zhong-min, LU Jia-hai

  China Biotechnology. 2013, 33(11): 68-74.

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  Antimicrobial peptides (AMPs) which are produced by most living organisms have shown robust activity against a wide variety of pathogens, including drug-resistant bacteria. They are important components of the innate and adaptive system. Human beta-defensin-3 is an endogenous, cysteine-rich antimicrobial peptide that resistants to high salt and contributes more powerful to host defense against multi-resistant bacteria compared with other beta-defensins. There is a great need for a less expensive and increasing efficiency peptide-production platform.This research aims at expressing human beta-defensin-3 and a novel sequence human beta-defensin-3i based on E. coli codon preference in E.coli BLR(DE3) via pET-EI plasmid vector. HβD-3i were 12 times higher yields than HβD-3. All the peptides, expressed as fusion protein, were isolated from the protein debris by the method called Inverse Transition Cycling(ITC).Fully reduced peptides that were purified exhibited expected antimicrobial activity that towarded to Staphylococus aureus was stronger than Salmonella.It is an attractive way to express human beta-defensin-3 via pET-EI prokaryotic plasmid vector. This approach described here is a low-cost, convenient and potential way for large-scale human beta-defensin-3 production.
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  Development and Application of DPO-PCR Detection method for Enterotoxigenic E.coli

  XU Yi-gang, LI Dan-dan, LIU Zhong-mei, CUI Li-chun, LI Su-long

  China Biotechnology. 2013, 33(11): 75-80.

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  Dual-priming oligonucleotide (DPO) is a novel primer design method with the features of simple design, strong specificity and wider annealing temperature range. In this study, a pair of DPO primers was designed based on enterotoxigenic E.coli (ETEC) LT gene and following optimization of reaction system factors that Taq DNA polymerase, Mg²⁺ and dNTP, a DPO-PCR detection method for ETEC was established and its sensitivity, specificity and annealing temperature insensitivity were analyzed. Results showed that detection sensitivity of the method was 1.24×10² CFU/ml and the target gene could be efficiently amplified by the DPO primers at 45℃~65℃ of annealing temperature range. The DPO-PCR method showed high specificity, in tested strains, 4 ETEC strains were positive results and other strains were negative results, and no nonspecific amplifications were produced. Compared with conventional PCR methods, the DPO-PCR method should not require to repeatedly optimize primer parameters in particular annealing temperature, at the same time, the special structure of DPO primers would enhance detection specificity, which provided a new method for fast and accurate detection of pathogens.
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  The Role of Acid Stress on Improved Performance of Selenium/glutathione-Enriched Candida utilis

  WANG Da-hui, XU Hong-qing, WANG Cheng-fu, WEI Gong-yuan

  China Biotechnology. 2013, 33(11): 81-85.

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  Intracellular organic selenium and glutathione contents are the two most important characteristics of high performance of selenium-enriched yeast. In order to improve the performance of selenium/glutathione-enriched Candida utilis, the effects of diverse pH conditions on cell growth, glutathione biosynthesis and the biotransformation of inorganic selenium to organic selenium during selenium enrichment were investigated. It was indicated that the moderate acid stress was favor of the increased intracellular contents of organic selenium and glutathione, as well as the decreased level of the oxidized compound as malondialdehyde. The maximum levels of the intracellular organic selenium and glutathione were obtained at pH 3.5 as 1.13 mg/g and 12.3 mg/g, respectively. Based on the analysis of residual methionine in the medium, intracellular pH, the ratios of NADH/NAD⁺ and ATP/ADP, the reason of the improved performance of selenium/glutathione-enriched Candida utilis by moderate acid stress were partly interpreted. The results presented will give a feasible approach for further enhanced nutrimental function of Candida utilis.
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  Purify Cellulose of Corn Straw by Acid and Alkali Pretreatment and Research of Reducing Sugar by Enzymatic Hydrolysis

  WU Chong-hui, KOU Wei, SHAO Li-jie, ZHANG Huan, CAO Yan-xin, ZHANG Da-lei

  China Biotechnology. 2013, 33(11): 86-91.

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  In order to improve reducing sugar yield of corn straw, corn straw was pretreated by dilute acid and alkali. The finding by analysis showed that the content of cellulose was increased from 39.15% to 91.34%, and most of hemicellulose and lignin was eliminated, yet the mass loss of cellulose was only 2.01%. X-ray diffraction showed the crystalline degree of cellulose increased by 124.13%; Infrared spectrum analysis found that most of the structure of hemicellulose and lignin was destructed after corn straw was pretreated by acid and alkali; Scanning electron microscope pictures found that cracks and holes increased obviously on the surface of fiber bundle. Further cellulose enzymolysis experiment showed that the yield of reducing sugar of untreated corn straw was only 13.66%, while the yield of corn straw treated by acid and alkali can reach 65.17%,improved 377.09% compared with the materials untreated, while enzymolysis time could be reduced about 24h. The result showed that dilute acid and alkali pretreatment could obviously increase the conversion efficiency of corn straw cellulose.
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  Recent Developments in N-linked Glycoproteins Production in Escherichia coli and Glycoprotein Vaccines

  MA Zhong-rui, HAN Dong-lei, ZHAO Jun-fei, CHEN Meng-lin, CHEN Min

  China Biotechnology. 2013, 33(11): 92-98.

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  In the last decade, the investigations into protein N-linked glycosylation system from Campylobacter jejuni become deeper and its heterologous expression in Escherichia coli through introduction of key enzyme PglB and exogenous carrier proteins continue to progress rapidly. Now several kinds of N-linked glycoprotein could be produced using this Escherichia coli recombinant system. This method provides a new and broad way for producing glycoprotein vaccines. In the meantime, such efforts as enhancement of the expression of PglB and selection of glycosylation sites as well as development in enhancing the immune efficacy of glycoprotein have all significantly improved N-glycosylation efficiency in Escherichia coli, which lead to a good future for large-scale production of glycoprotein vaccines.
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  Progress in Protein Fragment Complementation Assay

  HUANG Xin-yuan, FAN Hong-bo, ZOU Li-ping

  China Biotechnology. 2013, 33(11): 99-105.

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  Protein fragment complementation assay(PCA) is an important method to study in vivo protein-protein interactions(PPIs). In PCA, PPI is coupled to refolding of a reporter protein from two cognate fragments where reconstitution of reporter activity acts as the detector of a protein interaction. PCAs based on different types of reporter proteins have been developed to detect in vivo PPIs as well as their modulation or spatial and temporal changes. To date, successful applications of PCA have been widely reported in many areas such as the study of interactome networks, screening for binding partners and drug discovery, owing to its high sensitivity, high signal-to-background ratio, and high throughput. Different types of PCAs and their application aspects are summarized, together with the limitations and development prospects.
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  Expression and Purification of Disulfide-rich Peptide Toxins

  ZHANG Ke-jun, LI Li, DAI Qiu-yun

  China Biotechnology. 2013, 33(11): 106-111.

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  The disulfide-rich peptide toxins possess low molecular weight, tight disulfide bridges and low folding efficiency in vitro, hence they are difficult to be chemically synthesized efficiently when sequences are more than 40 amino acids. This review briefly summarizes the progress of genetic expression of disulfide-rich peptide toxins (such as conotoxins, scorpion and spider toxins), especially focuses on the technical features of their expressions and purifications in Escherichia coli and yeast.
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  Research Progress in Aquaculture Ghost Vaccines

  JIANG Na, XING Wei, MA Zhi-hong, LI Tie-liang, LUO Lin

  China Biotechnology. 2013, 33(11): 112-117.

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  Bacterial ghost, a novel inactivated non-denatured bacterium, is intact bacterial envelope which is lysised by the lysis gene E of PhiX174. Bacterial ghost has been researched as fish bacteria vaccine just in recent 8 years. It not only has high biological safety, but also can induce humoral and cellular immunity. So bacterial ghost is possible to be used in oral immunization and has a unique advantage in fish vaccine. Much more attention has been paid on ghost being used as a recombinant vaccine or drug delivery system. This paper summarizes the international latest developments in fish bacterial ghost vaccine, discusses their immune response and protective effect in different immunization ways using different strategies.