25 September 2013, Volume 33 Issue 9

    

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  Rescue of the Minigenome of Human Respiratory Syncytial Virus Based on T7 Promoter Expression System

  YUAN Rui, FU Yuan-hui, HE Jin-sheng, JIAO Yue-ying, JIANG Gui-yun, ZHANG Mei, PENG Xiang-lei, KAN Xue-tong

  China Biotechnology. 2013, 33(9): 1-9.

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  Objective:To establish a T7 promoter based reverse genetics system competent for the rescue of human respiratory syncytial virus. Methods:We construct four helper plasmids of px8δT-PT1-N, px8δT-PT1-P, px8δT-PT1-M2-1 and px8δT-PT1-L encoding RSV nucleocapsid proteins, respectively, and one minigenome plasmid of pSC11-E containing open reading frame of the enhanced green flurorecent protein and cis-acting elements including RSV leader region/promoter, gene start, gene end and trailer region/antigenomic promoter. All these plasmids are under the control of T7 promoter and identified by restriction endonucleoase analysis and Western blot. The pSC11-E is rescued by RSV and the above helper plasmids in BSR T7/5 cells, respectively. Then, the fluorescence expression is observed over time with fluorescence microscopy. Results:We successfully constructed a reverse genetics system based 5 plasmids undert the control of T7 promoter and finished the rescue operation to the minigenome of RSV. Conclusion:This system can be further applied to investigate the function of RSV genome by deletion and mutation of its genes.
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  Combined Enzymatic Digestion Method and Explants Culture Method Used on Primary Culture and Biological Characteristic Identification of Pulmonary Artery Smooth Muscle Cells of mice

  XIN Yi, GONG Da, XI Xin, SHI Hong-tao, LIU Sa, XU Xiu-fang, LI Na, HUANG Yi-min

  China Biotechnology. 2013, 33(9): 10-16.

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  Objective:To investigate a technical method to isolate, cultivate and identify the mice pulmonary arterial smooth muscle cells(PASMCs). Methods:Isolation of pulmonary arterial smooth muscle cells (PASMCs) has been performed using Trypsin-typeⅠ collagen and papain digestion method and tissue explants culture method. The cell growth of PASMCs was observed under invert microscope. Cell viability rate of the different cells passage was evaluated by trypan blue staining. The PASMCs proliferation profile was analyzed by curve of growth, MTT assay and DNA cell cycle analysis respectively,and were identified by immunofluorescence as well. Result:After one day culture in the enzyme digested method, the adherent cells were observed to grow quickly with a typical "peak-valley" after 4 days under invert microscope. The cells can be passaged 7-8d after the rapid growth. But in the explants culture method, the cells started to amplify to grow gradually from the center to the periphery. Cells began to grow rapidly after 7d culture and single compact arrangement then fused 80% after 10 day. The cell viabilities from both isolation methods were more than 96% tested by trypan blue staining. There was over 90% positive expression in intracellular a-smooth muscle actin (a-SMA) in immunofluorescence staining.The shape of the second passage cell growth curves were similar in both methods ; There were also no significant differences in cell cycle and MTT assay in both methods. In cell cycle over 85% of cells were in G0/G1 phase. Conclusion:The PASMCs can be effectively isolated both by enzyme digestion method and explant methods which would provide us ideal seeding cells for pulmonary vascular diseases.
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  Tat-PTDs Mediated Metallothionein High-yield Expression in Escherichia coli and Its Heavy Metal Resistance Studying

  LI Hua-ling, WANG Kai, QING Yan, LIU Dan-dan, CHEN Wen-fei

  China Biotechnology. 2013, 33(9): 17-23.

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  Objective:To explore the feature of Tat-PTDs for metallothionein (MT) high-yield expression in E. coli and its heavy metal resistance in vitro. Methods:the MT-1, 2 encoding genes in Balb/c mice liver tissue was amplified using polymerase chain reaction (PCR) initially, and subcloned into the prokaryotic expression plasmid pET28b-Tat and pET28b followed direct sequencing separately. The validated plasmids were transformed into E. coli BL21 (DE3) cells and induced using 1 mmol/L IPTG, and the protein samples at 0 h, 2 h, 4 h and 6 h were prepared and identified by SDS-PAGE and Western blot. Subsequently, the bacterial cells at 4 h induced were diluted using LB medium and study its resistance of 5 mm copper. Results:This study demonstrated that Tat-PTDs could obviously increase the expression of MT-1, 2 in E. coli and Tat fusion protein mainly expressed in the bacterial supernatant. The Tat fusion protein expression level was 5~8 folds compared to its control according to the results of Western blot and histogram analysis. Furthermore, the over-expressed Tat-MT-1, 2 bacterial cells had an obvious resistance to 5mm copper compared with its control (P<0.01). Conclusion:the data showed that protein transduction based on Tat-PTDs can promote genes of mouse metallothionein high-yield and soluble expression in prokaryotic system. The Tat fusion protein expression level was significantly improved compared to non-Tat fusion protein and its resistance to copper was also increased.
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  Expression and Fusion Protein TAT-NLS-Nkx6.2 in E.coli and Its Purification and Biological Analysis

  SUN Shao-fei, WANG Bei-lei, YUAN Ting, ZHANG Bing, GUO Gang, ZHANG Ru

  China Biotechnology. 2013, 33(9): 24-30.

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  Objective:To obtain a fusion protein TN-Nkx6.2, which Nkx6.2 links with TAT and NLS in a recombinant prokaryotic system and to analysis its bioactivity. Methods:In this research, we engineer human immunodeficiency virus HIV-1 trans-activating factor TAT and nuclear location sequence (NLS) into transcription factor Nkx6.2 protein ligate to synthetic gene TAT-NLS-Nkx6.2, recombinant plasmid pET-28a- TN-Nkx6.2 and transform into E.coli BL21 (DE3) for expression by IPTG induction.Then by SDS-PAGE and Western Blot to to identified fusion protein,which purified by histidine Ni²⁺ affinity chromatography.Results:TN-Nkx6.2 was with above 90% purity and good solubility was obtained in vitro,which can combine with brain cells in mice and internalize into the cytoplasm and nucleus proved by fluorescent immunohistochemical.Conclusion:Obtain the fusion protein of TN-Nkx6.2 has biological activity, and can cross the blood-brain barrier into the mice brain cells in nucleus cytoplasm, which lay the material foundation for further explortion of the molecular mechanism that Nkx6.2 hox genes in thyroid reduced model role.
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  Subcellular Localization and Functional Studying of SCBP60g in Arabidopsis

  WAN Yong-qing, LI Rui-li, ZOU Bo, WAN Dong-li, WANG Rui-gang, LI Guo-jing

  China Biotechnology. 2013, 33(9): 31-37.

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  The GFP fused SCBP60g constructs and SCBP60g complementation constructs were made by Gateway cloning technology, and the transgenic plants were obtained. Subcellular localization SCBP60g was observed by confocal laser-scanning microscope, and the function of plant defense against pathogens was examined by in planta bacterial growth assay. In addition, SCBP60g overexpression vector was constructed and the sensitivity to ABA of the transgenic plants was also examined. The results showed:SCBP60g protein localized in the nuclei; SCBP60g failed to restore the sensitivity of cbp60g-1 mutant to Pseudomonas syringae; overexpression of SCBP60g did not affect the response of Arabidopsis to ABA. These results indicated that SCBP60g was not involved in Arabidopsis thaliana response to pathogens and ABA.
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  Prokaryotic Expression of CM of T-protein from E.coli and Its Crystal Optimization Guided by NMR

  RUI Bin, XIE Chang-lin, JIANG Na, WEN Han

  China Biotechnology. 2013, 33(9): 38-44.

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  More and more researches have shown that the pathogenicities of many bacteria are related to their chorismate mutases in the shikimate pathway. CMs represent a hot selective target for the development of antibiotics and fungicides. By PCR the recombinant CM_T was expressed in competent cells E.coli BL21-Gold(DE3) successfully. The purity could reach electrophoresis purity after purification by Ni-NTA affinity chromatography, GE superdex75 gel filtration column and GE MonoQ cation-exchange chromatography. The result of mass spectra matched with the original sequence of CM_T basically. And then CM_T crystal were found in the condition of 30% PEG 4000,100mmol/L NaAc pH4.6,200mmol/L NH₄Ac. The application of ¹H-¹⁵N HSQC NMR provided guidance for optimization of CM_T crystal, and finally we successfully obtained the high diffraction CM_T crystal. This work lays the foundation for the structure of CM_T.
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  Study on Washing Method of Recombinant sTNFR1IBs

  LUO Li, Qin, Jiao-rong, WANG Ming-rong

  China Biotechnology. 2013, 33(9): 45-52.

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  Objective:Overexpression of recombinant protein in prokaryotic cell often results in the formation of aggregates called inclusion bodies(IBs). IBs need to be washed repeatedly before denaturing to remove impurities with one washing buffer, but it often result in low yield, long time, or low purity, which directly affect the yield and quality of the recombinant protein. By optimizing the washing condition of sTNFR1 IBs, its industrial production can be guided. Methods:First, screening one better way from the 25 repeat-washing buffers which composed of two factors——sodium deoxycholate and urea, and five levels in each factor, and the yield of target protein is as the evaluation index. Second, according to the ANOVA results of repeat-washing, a new step-washing method is proposed, which is different from the repeat-washing method. In addition, the above two different washing methods be compared by enlarging sample amount and different batches of fermentation. Results:ANOVA results of repeat-washing method showed that sodium deoxycholate or urea alone is superior to both, and W3(1mol/L Urea wash 3 times) obtain the highest yield of target protein. In step-washing method, FW6(2% sodium deoxycholate is used first and 2% sodium deoxycholate+2mol/L Urea is used in the second step) can effectively improve the purity of target protein and save time. The same batch of fermentation (about 20% target protein) of different scale sample (3g and 50g) washed by W3 and FW6, purity of target protein was increased to 26% and 31% respectively, and the later target protein yield is higher than the former. The results indicated that step-washing method is better than repeat-washing method. For the different level of fermentation sample (about 10% and 60% target protein), step-washing is better than repeat-washing and step-washing method can obviously improve the target protein purity for the low level of fermentation sample. Conclusion:A step-washing method which is more effective than the repeat-washing method is proposed by the optimization of IBs washing conditions, which improves the purity of target protein, saves the washing time, but also provides a new idea for IBs washing.
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  An Efficient Fungal Protein Extraction Method

  HU Bin-bin, LIN Lian-bing, WEI Yun-lin, JI Xiu-ling, ZHANG Qi

  China Biotechnology. 2013, 33(9): 53-58.

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  Protein extraction is the first step of proteomics research. Its quality directly affects the protein expression analysis studies and the accuracy of the results. In order to extract total cellular protein from Mortierella isabellina strain M6-22 for further proteomics and some other related research, five protein extraction methods were tried. Protein concentration and SDS-PAGE analysis of protein abundancy were used to evaluate the effects of these methods, and one of them was selected and modified, by which, a total protein concentration of 2.99 mg/ml was obtained. The modified method had advantage of simpler procedure than the original one and could obtain higher protein abundancy. The modified method was further used to extract the total protein of five other fungi, and the results showed that similar extraction effect could be obtained, indicating that this method can be potentially used as an efficient total protein extraction method for fungi.
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  Optimization of Electroporation Condition for Buffalo Fetal Fibroblast Cells

  LUO Chan, GONG Yun, REN Yan-ping, YANG Su-fang, RUAN Qiu-yan, GUAN Xiao-mei, JIANG Jian-rong, SHI De-shun

  China Biotechnology. 2013, 33(9): 59-65.

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  A significant aspect of applying somatic cell nuclear transfer (SCNT) technology to generate transgenic animal is the in vitro method utilized to obtain viable donor nuclei for cloning procedures. Therefore, the present study was undertaken to explore the optimal electroporation parameters for transfection of buffalo fetal fibroblasts (BFFs) and determine the ability of these electroporation parameters to produce stably transfected fibroblast colonies. First, the BFFs was obtained by tissue mass inoculation method and were confirmed of possessed a normal diploid karyotype. And then, the transfection efficiency of different voltage (150V, 200V, 250V, and 300V), different pulse length (5ms, 10ms and 15ms) and different cells passage were compared. Results show that one 10ms pulse of 300V can introduced DNA into the cells more effectively, and the passage of cells also has remarkable influence on transfection efficiency. Finally, the utilization of these conditions can produced numerous transgenic fibroblast colonies following G418 selection. This study provides a suitable electroporation condition for introducing DNA into BFFs and improves the efficiency of transgenic SCNT technology.
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  Optimization of Genetic Transformation System of Sugarcane Callus Mediated by Agrobacterium

  QIN Cui-xian, CHEN Zhong-liang, GUI Yi-yun, WANG Miao, ZHOU Jian-hui, LIAO Qing, LI Yang-rui, HUANG Dong-liang,

  China Biotechnology. 2013, 33(9): 66-72.

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  To establish high-efficiency sugarcane transformation system, the optimal concentration of antibiotic (Km) was screened for subculture and differentiation of sugarcane calli, as well as seedling rooting, respectively. The factors that influenced sugarcane transformation, such as pre-culture time, infection time and co-culture time were also studied. The results showed that the optimal concentrations of antibiotic (Km) for subculture, differentiation and seedling rooting were 300 mg/L, 40 mg/L and 20 mg/L, respectively. The optimal pre-culture time, infection time and co-culture time were 4d, 30min and 4d, respectively. PCR detection showed that 61.6% of resistance plants were transformed plants. This work lays a solid foundation for further study on gene function and transgenic research of sugarcane.
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  The Optimization of Medium for Coenzyme Q₁₀ Fermentation by Artificial Neural Network associated with Genetic Algorithms

  ZHOU Yong, ZHENG Yi, SONG Li-dan

  China Biotechnology. 2013, 33(9): 73-78.

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  In order to improve the yield of Coenzyme Q₁₀ produced by Rhodobacter sphaeroides F3-40. First, the reasonable concentration ranges of 4 important medium components were determined by single-factor experiments. On the basis of the above results, uniform design method was adopted to optimize their combination. Square stepwise regression analyses, artificial neural network associated with genetic algorithms optimization (ANN-GA) were used to optimize their concentrations respectively. From results, the ANN-GA showed better optimization effect. In the end, the yield of Coenzyme Q₁₀ by ANN-GA reached 245mg/L, increased 10.86%,16.11% and 63.33% more than that of square stepwise regression analysis (221mg/L), single-factor analysis (211 mg/L) and the primary (150mg/L) respectively.
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  Protein Acetylation in Prokaryotes

  BI Jing, ZHANG Xue-lian

  China Biotechnology. 2013, 33(9): 79-84.

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  In addition to histones, recent evidences suggest many non-histone proteins are subject to lysine acetylation. Furthermore, lysine acetylation status has been shown to influence several fundamental cellular pathways, including cellular differentiation and metabolism. Protein lysine acetylation and its regulatory enzymes have thus emerged as a frontier for research in mammalian cells. Recent studies support that protein acetylation occurs prevalently in prokaryotes and broadly impacts prokaryotes physiology. An emerging theme from these studies is that metabolic enzymes involved in central metabolic pathways and intermediary metabolisms are subjected to acetylation. To explore more rapidly the impact of protein acetylation in bacteria, we will summarize the current examples of protein acetylation in prokaryotes, discuss the emerging link between acetylation and central metabolism and understand the importance of protein acetylation in bacteria.
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  Current Situation and Prospects of Therapeutic Antibody

  GE Liang-peng, DING Ning, LAN Guo-cheng, ZOU Xian-gang, LIU Zuo-hua

  China Biotechnology. 2013, 33(9): 85-93.

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  The paper reviewed the current situation and prospects of human therapeutic antibodies in the aspect of the progress of fully humanized antibody, the clinical research situation and future development of therapeutic antibody. Firstly,we compare the difference between fully humanized antibody and humanized antibody, and introduce the progress,advantage and disadvantage, and develop direction of fully humanized antibody produced by Phage Display technology and transgenic animal technology in detail. Then we also discuss several key technical problems of therapeutic antibodies in clinic research, including the current clinic research situation, therapeutic antibodies mechanism,the choice of target antigen, and challenge in antibodies production and application etc. Finally, we outlined the future trends for therapeutic antibody development in small therapeutic antibody, immunoconjugates, bispecific antibodies, Intracellular antibodies, Fc-modified antibody, antibody glycosylation and polyclonal antibody.
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  The Progress of Hybrid Peptides on Design and Biological Activity

  WU Ru-juan, ZHANG Ri-jun

  China Biotechnology. 2013, 33(9): 94-100.

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  With the over-use of antibiotic in feed, antimicrobial peptides (AMPs) attract more and more attention as new medicine because of small molecular, broad antimicrobial spectrum, high activity, heat tolerance, drug resistance and safe. With the deep research in structure, function and antimicrobial mechanism of AMPs, people try to design new hybrid antimicrobial peptides which possess higher antimicrobial activity and broader spectrum. The paper summarizes the progress of hybrid AMPs from the following aspects:design, biology activity and the future focus.
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  Research Progress of DNA Methylation on Plant Regulation

  MA Lang-lang, JIANG Zhou, HUANG Xiao-bo, SHEN Ya-ou, PAN Guang-tang

  China Biotechnology. 2013, 33(9): 101-110.

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  DNA methylation, the process that methyl transfers from one of SAM to cytosine by the catalysis of DNA methyl enzyme and generating 5-methyl cytosine, is an important modification in eukaryotic cell genome and plays an essential role in the process of plant growth and development. The article reviewed the characteristics, maintenance mechanism of methylation regulation, as well as epigenetic phenomenon, biological significance, research methods, application in plant genetics and breeding, and prospects for future research, directing the extensive application of DNA methylation on crop breeding.
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  Research Progress in Different Substrates Used by Oleaginous Yeast for Lipid Production

  XU Ji-fei, ZHANG Yan-fen, ZHAO Gui-qi, ZHAO Ji

  China Biotechnology. 2013, 33(9): 111-118.

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  The oleaginous yeast is a kind of oleaginous microorganisms which generate and accumulate liposome such as triacylglycerol, and have significant implications for the increasingly serious energy and environmental security issues. The search for the cheap, freely available and with high conversion efficiency fermentation substrate will promote the industrialization process of the microbial oil production. In this review, the utilization of oleaginous yeast on a variety of substrates, including mixed sugars, cellulosic biomass, substances produced during fermentation, food industry wastewater and other organic matter, is introduced. Additionally, characteristics of lipid accumulation and oil production are summarized and compared; the current utilizing questions and future direction of development of all kinds of substrates oleaginous yeast can utilize are indicated also.
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  The Types, Safety and Efficacity of the Transplanted Cells

  WANG Dian-liang

  China Biotechnology. 2013, 33(9): 119-125.

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  The cells used for cell transplantation therapy mainly consist of hepatocyte, islet cells, dendritic cells, cytokine-induced killer cells, lymphokine activated killer cells, embryonic stem cells, hemopoietic stem cells, and mesenchymal stem cells, etc. Every cell kind has different biological characteristics. Before clinic application, according to the related documents of State Food and Drug Administration, the specific cell agent is studied on its biological characteristics, pharmacology, and toxicology, etc. Animal experiments and clinical tests are carried on to assure its indications and contraindications for assessment of safety and efficacity. After a license has been obtained from State Food and Drug Administration, the related clinical researches and applications can be carried on.