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Table of Content

    25 August 2016, Volume 32 Issue 8
    Orignal Article
    Research Progress on Genetic Engineering for Long-chain Polyunsaturated Fatty Acids EPA and DHA
    LI Wen-zong,WANG Lei
    2016, 32(8):  1-7.  doi:10.13560/j.cnki.biotech.bull.1985.2016.08.001
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    Long-chain polyunsaturated fatty acids(LCPUFAs)play an important role in maintaining human health. The demand of edible EPA and DHA has significantly increased in recent years. However,the fish oil resource is obviously reduced due to the environmental pollution and the decline of the fishery resources,thus can’t meet the demands of people. The researchers have successively separated multiple genes that involved in LCPUFA synthesis and clarified the multiple LCPUFA synthetic pathways. LCPUFAs,especially DHA and EPA,have been successfully produced in monocot and dicot plants through genetic engineering. We summarized the latest research progress of LCPUFAs bio-synthesis and genetic engineering in higher plants,and analyzed the issues of LCPUFA synthesis,then proposed the possible solutions,and had an prospect of LCPUFA by genetic engineering.

    Research Progress on the Role of Transcription Factor HIF-1α and Its Signal Pathway in the Pathogenesis
    YANG Meng-si, ZHOU Na, WANG Zhi-gang,HAO Hui-fang
    2016, 32(8):  8-13.  doi:10.13560/j.cnki.biotech.bull.1985.2016.08.002
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    Hypoxia inducible factor-1α(HIF-1α)is a transcription factor under hypoxia condition,which widely exists in mammals and human body. It is a key factor responding to hypoxic stress. HIF-1α is a subunit of hypoxia inducible factor-1(HIF-1)and considered as the master transcriptional regulator of cellular and developmental response to hypoxia,and regulates the activity of HIF-1. During hypoxia,HIF-1α translocates from the cytoplasm to the nucleus,where it dimerizes with HIF-1β and the transcriptionally active HIF-1 complex is formed;the activated HIF complex then associates with HREs in the regulatory regions of target genes to induce gene expression. Forming varied signal pathways with multiple proteins in up- and down- streams,HIF-1α mediates hypoxic signals,then regulates a series of hypoxic compensatory response of cell,which plays a crucial role in body growth,physiological and pathological processes,thus it is a focus of biomedical research. We reviewed the role of transcription factors HIF-1α and its signaling pathway in the occurrence of disease , and introduced to the relationship among HIF-1α and growth, development, inflammation and tumor , then carryed out the prospect, in order to better be used in biomedical.

    Indole-3-acetic Acid-mediated Cross-kingdom Signalling Involved in Plant-bacteria Interactions
    YANG Yang, GAO Ke-xiang, WU Yan, LIU Xiao-guang
    2016, 32(8):  14-21.  doi:10.13560/j.cnki.biotech.bull.1985.2016.08.003
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    As the most common and naturally-occurring phytohormone of the auxin class,indole-3-acetic acid(IAA)involves in regulating many aspects of plant growth and development. The studies revealed that in nature,not only plant may synthesize the IAA,but also a variety of microorganisms including both phytopathogens and plant growth-promoting bacteria possess the ability of producing the auxin phytohormone inducing plant diseases or promoting plant growth. Interestingly,apart from being the secondary bacterial metabolite interfering the hormone homeostasis of host plants,IAA can also be a signaling molecule modulating gene expression and physiology in bacteria,consequently regulating the plant-bacteria interaction through integration into the complex regulatory network in bacteria. This review provides insights into the recent research progresses on IAA biosynthesis pathways and its regulations in bacteria,IAA-mediated control of bacterial gene expression,physiology and behavior,as well as the interaction with host plants varying from pathogenesis to phytostimulation. The review also highlights that IAA can not only modulate the plant growth,development and defense,but also act as cross-kingdom signal molecules playing a critical role in the regulation of the plant-microbe interactions. The review aims to develop novel strategies for the improvement of the plant-growth promotion and tolerance to both biotic and abiotic stresses by further studying and better understanding of the IAA-mediated cross-kingdom signalling mechanisms and genetically manipulating the bacterial IAA signal pathways.
    Current Progress on Ribosome Display
    GUO Yuan
    2016, 32(8):  22-27.  doi:10.13560/j.cnki.biotech.bull.1985.2016.08.004
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    Ribosome display is a cell-free system for the selection of proteins and peptides from large libraries. It uses the principle of coupling the translated proteins(phenotypes)and their corresponding mRNA(genotypes)to ribosome and the stable protein-ribosome-mRNA complexes are formed. Then,the functional proteins and the corresponding encoding mRNAs are obtained by the simultaneous affinity separation of ligands,and they are converted and amplified as DNA for further expressions of related proteins,as well as selection of antibody and protein libraries,in vitro modification of proteins. It may display very large libraries while it is not limited by bacterial transformation. It is also suitable for screening the toxic,proteolytically sensitive,and unstable proteins,and allows the incorporation of modifying amino acids at defined loci. In this paper,research progress on ribosome display systems and their applications in the selection and evolution of proteins are reviewed.
    Research Progress on the Morphology of Spirulina
    WANG Fu-shuang,DONG Shi-rui, WANG Su-ying
    2016, 32(8):  28-33.  doi:10.13560/j.cnki.biotech.bull.1985.2016.08.005
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    The changes in the morphology of Spirulina indicate the decline of the species,which causes the production reduced and the collection of them not easy. At the same time,the easy variations on morphology of Spirulina has increased the difficulty of classifying and identifying it. Therefore,it is important to study the formation mechanism of Spirulina morphology from the aspect of the application prospect of it. This paper reviewed the latest progress on the formation of the Spirulina morphology,summarized the effects of light,temperature,pH,etc. on the morphology of Spirulina,preliminarily analyzed the formation mechanism of Spirulina,and prospected the application of novel proteomics and genomics technologies in studying the formation mechanism of Spirulina in different conditions.
    The Current Status and Future Perspectives of Production of Biopharmaceuticals in Escherichia coli
    CAI Dong-mei, GONG Guo-li
    2016, 32(8):  34-40.  doi:10.13560/j.cnki.biotech.bull.1985.2016.08.007
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    Escherichia coli is the most preferred microorganism to express heterologous protein for therapeutic use,as around 30% of the approved therapeutic proteins are currently being produced using it as a host. Due to its rapid growth,high yield,cost-effectiveness,and easy scale-up process,E. coli is chosen as an expression host in biotech industry for large-scale production of proteins. However,codon preference in E. coli and absences of post-translational modifications,such as glycosylation,phosphorylation and proteolytic processing limit its use for the production of slightly complex recombinant biopharmaceuticals. On purpose of helping E. coli to produce more complex and also glycosylated proteins for therapeutic use,this review summarizes the several novel technological advancements meeting the requirements of biotech industry,mainly focusing on the process of E. coli glycosylating heterologous proteins and expressing complex proteins including full-length glycosylated antibodies via those advancements. Further potential problems and prospects of futures researches are also discussed.
    Research Progress on the Technology of Pig Somatic Cell Nuclear Transfer
    ZHANG Hong-yan, XIN Ji-ge
    2016, 32(8):  41-46.  doi:10.13560/j.cnki.biotech.bull.1985.2016.08.008
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    The technology of pig somatic cell nuclear transfer has been developed for more than ten years,with gradual improvement of the technology,the cloned piglets and genetically modified piglets were generated in many laboratories. Recently,several effective gene targeting and regulating tools have developed quickly,which provides technology basis for the establishment of genetic modified pig models and promotes the transition of the related researches from basic to application. In this paper,its development process and the optimization of key procedures were introduced. The technical advantage,its applications and significances in agriculture and biomedicine were summarized. Meanwhile existing issues and applying prospects were discussed.
    Research Advances on Rapid Detection Methods for Aflatoxin Based on Biological Binders
    ZENG Kun, DU Dao-lin, XUE Yong-lai
    2016, 32(8):  47-55.  doi:10.13560/j.cnki.biotech.bull.1985.2016.08.009
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    Aflatoxins are secondary metabolites secreted by Aspergillus flavus and Aspergillus parasiticus,easily pollute the grains and their processed products,and mainly exist in moldy grain,legumes,nuts and associated food products. Aflatoxins are extremely toxic and carcinogenic,especially aflatoxin B1 that has been found to be the strongest natural carcinogen and severely threat to human health. Current detection methods of aflatoxins in the grains are mainly instrumental analysis by combining the high performance liquid chromatography and liquid mass spectrometry,however,instrumental analysis presents the shortcomings such as instruments cost is very high,analysis process is very complex and needs long-time-trained professionals. In recent years,integrating the rapid identification of aflatoxin based on biological binders(antibody,recombinant antibody,and aptamer)and different signal reporters,a series of analysis methods for aflatoxin were established,and they are fast,flexible,and easy to operate,therefore widely applied in food safety. Here we review the rapid detection methods mainly from 2 aspects of specific biological binders for aflatoxins and signal reporters,compare and evaluate them,also prospect the development trend of rapid methods for aflatoxin.
    Application of Microbial Immobilized Technology in River Pollution Control and Its Research Progress
    YU Lu-ji, LI Ting-mei, LIU Pan-long, FAN Zheng
    2016, 32(8):  56-61.  doi:10.13560/j.cnki.biotech.bull.1985.2016.08.010
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    Microorganisms play important roles in the removal of pollutants,however,river’s conditions limit the permanent and effective purification function;microbial immobilized technology may improve the situation as it enhances the effect of microorganism removing pollutants. This paper analyzed the governance constraints while microbes directly were used in contaminated river,meanwhile expounded the effective mechanism and improvement methods of the microbial immobilized technology. Further,the paper discussed the current issues in the application of the technology and prospected the future research trends,aiming at providing theoretical and technical references for immobilization technology in river pollution control.
    Regulation Techniques for Biosynthesis of Spiramycin in Molecular and Genetic Level Along with Ferment
    ZHENG Yu-qing
    2016, 32(8):  62-68.  doi:10.13560/j.cnki.biotech.bull.1985.2016.08.006
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    Spiramycin is a 16-membered ring macrolide antibiotics,and plays an important role in clinical practice. It is produced by S. ambofaciens. Spiramycin is composed of three parts:forosamine,mycaminose and mycarose. This article describes briefly the physicochemical properties and pharmacological effects of the spiramycin,and recent advances on the biosynthetic pathway and the fermentation process. According to biosynthetic pathway of spiramycin,the techniques for increasing its yield,i.e.,adding precursor or beneficial substances to precursor,and direct overexpression of some important genes,were briefly summarized from 3 aspects of the molecular and genetic level,as well as fermentation process,respectively
    Establishment of Precisely Quantitative Method of Genetically Modified Rice LL62 Based on Digital PCR
    REN Yi-fei, GAO Qin, DENG Ting-ting, LI Xiang, HUANG Wen-sheng, CHEN Shun-sheng, CHEN Ying
    2016, 32(8):  69-76.  doi:10.13560/j.cnki.biotech.bull.1985.2016.08.011
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    In order to promote the smooth implementation of labeling policy for genetically modified(GM)products,we study an precise quantitative detection method for the GM rice LL62 that has not been approved by the Ministry of Agriculture in China,aiming at guarantee the detection and supervision of GM agricultural products in export and import. A digital PCR(dPCR)detection system were designed based on the inserted exogenous fragment of LL62 and 3’ flanking sequence of DNA of rice genome,and a precisely quantitative method was established. The results showed that the designed probes presented high efficiency,the developed dPCR method was highly specific for GM rice LL62 detection. The method was highly repeated,and the relative standard deviation(RSD)values of droplets’ number ranged from 0.60%-11.11%. Results of the quantification of three blind samples showed that the bias between the true value and the measured value was 0.12,0.09 and 0.10,the RSD was 0.09%-10.31%,indicating that the accuracy was high. In conclusion,the established droplet dPCR method for GM rice LL62 detection is simple and convenient with high specificity,solid repeatability and high accuracy,and is suitable for precisely and effectively quantitative analysis of ingredients of GM rice or peeled rice LL62 in agricultural products and foods.
    Analysis on Fe2+ Stress Resistance of Transgenic Japonica Kongyu 131 with NtFer1
    TANG Xin-hua, JIANG Ting-bo, GAO Hong-xiu, WANG Jing-guo, ZOU De-tang
    2016, 32(8):  77-83.  doi:10.13560/j.cnki.biotech.bull.1985.2016.08.012
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    In order to realize the gene’s function and improve the resistance ability of japonica,NtFer1 was transformed into japonica Kongyu 131 by Agrobacterium-mediated method. The results by resistance screening,PCR,Southern blotting,Northern blotting,and RT-PCR demonstrated that the gene was integrated into Kongyu 131’s genome,stably and genetically expressed in offspring. T2 generation was cultured in iron deficiency and overload conditions respectively. Under iron deficiency conditions,SOD,POD activity,relative chlorophyll content,total iron content in leaves and roots of transgenic lines were all significantly higher than the control,while the MDA content was lower than the control. Under iron overload conditions,SOD activity,total iron content in leaves and brown rice,and roots growth of transgenic lines were significantly higher than the control,while MDA content and rice blast index were significantly lower than the control. The results revealed that NtFer1 could improve transgenic plant’s ability of oxidative stress and resistance to rice blast,enhance Fe2+ storage ability and stress resistance in iron deficiency and overload conditions.
    Functional Analysis of Soybean GmAP1
    SONG La-la, ZHANG Xiao-mei, CHEN Fu-lu, FU Yong-Fu, ZHAO De-gang
    2016, 32(8):  84-89.  doi:10.13560/j.cnki.biotech.bull.1985.2016.08.013
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    The full length GmAP1 gene,a homologous gene of AP1,was cloned from the soybean cultivar Tianlong 1 based on the putative sequence of soybean genome in Glycine max Wm82.a2.v1database. GmAP1 was 711 bp in length and encoded a protein of 236 amino acids. The structure analysis of protein sequence showed that GmAP1 protein characterized as MADs-box gene family. To analyze the function of GmAP1,the expression patterns of GmAP1 in different floral organs were analyzed by RT-PCR,and GmAP1 was overexpressed in Arabidopsis. The results showed that GmAP1differently expressed in different floral organs with the highest level in sepals,following by the petals. Transgenic Arabidopsis plants displayed phenotypes of early flowering,dwarf,and increasing number of sepals,petals,and stamens. The result indicated that GmAP1 was one of AP1 homologous genes in soybean with conserved function and played an important role in flowering and floral organ development morphogenesis.
    Cloning and Expression Pattern Analysis of NBS-LRR Like-gene in Peanut
    FENG Yan-fang, GENG Li-li, HAN Rong, ZHANG Jie
    2016, 32(8):  90-95.  doi:10.13560/j.cnki.biotech.bull.1985.2016.08.014
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    Based on peanut’s transcriptome and genome data,a full length sequence of NBS-LRR like-gene was cloned. This gene,which showed 79% similarity with disease resistance protein(KHN40407.1)of Glycine soja,was designated as AhNDrp gene. The length of cDNA of AhNDrp gene was 2 832 bp,and contained no intron. AhNDrp gene harbored 5 typical conserved domains with resistance-related protein,and Domain V was repeat sequence with rich leucine. Real-time quantitative PCR analysis showed that AhNDrp gene was expressed exclusively in root,and the expression was significantly increased after aflatoxin treatment,indicating that this gene was involved in the resistance response.
    mRNA Expression and Bioinformatics Analysis of Cysteine Synthase in Allium sativum
    GUO Tian-lu, ZHANG Huan-huan, DU Jian-zhong, HAO Yao-shan, WANG Yi-xue, SUN Yi
    2016, 32(8):  96-102.  doi:10.13560/j.cnki.biotech.bull.1985.2016.08.015
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    The cysteine synthesized by cysteine synthase is an important source of amino acids containing sulfur in plants. The amino acid with sulfur in garlic is abundant,thus we studied the properties and biological functions of garlic cysteine synthase in order to clarify its role in the metabolism of garlic. Three cysteine synthase genes of garlic,AsGCS2,AsGCS3 and AsGCS4were found from NCBI database,and then bioinformatics analysis and RT-PCR were conducted. The lengths of their open reading frames were 1 152 bp,1 296 bp,and 1 236 bp,respectively. The theoretical relative molecular weights of their encoded enzymes were 40.6 kD,34.1 kD,and 36.1 kD,respectively. By subcellular localization,AsGCS2 was localized in the chloroplasts,while AsGCS3 and AsGCS4 were localized in the cytoplasm. The results of the amino acid sequence alignment revealed that the similarity of AsGCS2 with AsGCS3 was 70%,and that with AsGCS4 was 59%,while it was 68% between AsGCS3 and AsGCS4. Phylogenetic analysis showed that AsGCS2 belonged to Bsas2,AsGCS3 belonged to Bsas1,but AsGCS4 was different from all known cysteine synthases,possibly belonging to Bsas6. RT-PCR expression analysis indicated that the expression levels of CSase varied in various tissues. The expression level of AsGCS2 was the highest in the leaves,the highest expression of AsGCS3 was in the root,and AsGCS4 expressed highly in both leaves and roots. In conclusion,3 cysteine synthase gene AsGCS2,AsGCS3,and AsGCS4 belong to the different subfamilies of Bsas,their tissue expression characteristics were not the same,and they play a role in different tissues of garlic in cysteine biosynthesis.
    The Effect of DNA Methyltransferase Inhibitor 5-Aza-CdR on AID Gene-modified Bovine Fetal Fibroblasts
    AO Xu-dong ,SA Ru-la, WANG Jie, WANG Hui-min ,YU Hai-quan
    2016, 32(8):  103-112.  doi:10.13560/j.cnki.biotech.bull.1985.2016.08.016
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    In order to improve the efficiency of somatic cell nuclear transfer,the researchers used drugs which can alter DNA methylation or histone modifications to benefit somatic cell nuclear transfer. Among them,5-aza-2'-deoxycytidine(5-Aza-CdR)blocked DNA methylation by inhibiting methyl group transfer to adenine or cytosine,and ultimately reduced genomic methylation. In this study,the AID-overexpressing and AID-knocked-down cells were used to study the effects of 5-Aza-CdR(1,2,3,4,and 5 µmol/L)on cell morphology,cell cycle,related gene expression and the changes of methylation status in the gene promoter region. Additionally,the differences between genomic DNA demethylation and site-specific DNA demethylation was analyzed,the methods and its action mechanism of increasing the reprogramming efficiency of somatic cells were discussed. Real-time PCR,BSP(Bisulfite Sequencing PCR),Western blotting,and flow cytometry were employed to analyze the effects of 5-Aza-CdR on the AID-transgenic and AID-knocked-down cells. The results showed that 5-Aza-CdR presented dose-dependent effect on AID-transgenic cells,while the 5-Aza-CdR concentration was 4 µmol/L,obvious toxicity was observed as a lot of cells were dead(P<0.05). When treated by 1-3 µmol/L,the cell’s morphology changed,and proliferation of cells was inhibited. Karyotype analysis showed that 3 µmol/L treatments resulted in the emergence of a small proportion of aneuploidy cells,indicating that high concentrations of 5-Aza-CdR increased the rate of aneuploidy. The number of cells which expressed red fluorescent protein(DsRed)significantly increased after 1 µmol/L 5-Aza-CdR treatment,compared to the control group,the expression of AID and SOX2 were also increased,and the methylation levels in SOX2 promoter region were somehow decreased. After treated by 5-Aza-CdR,the expression of the OCT4 and SOX2genes increased in AID-knocked-down cells,but the expression of the NANOG did not change. The above results revealed that 5-Aza-CdR treatment affected the cell morphology,cell cycle and reporter gene expression in bovine transgenic cells. In conclusion,while treated by 5-Aza-CdR,the genomic demethylation and loci-specific demethylation of AID act synergistically in somatic cell reprogramming.
    Cloning and Specific Expression Analysis of Rat NKx6.1 Promoter
    WANG Da-wei ,WANG Bei-lei, YAO Yuan, CHEN Hao ,ZHANG Xin, GUO Gang, ZHANG Rui
    2016, 32(8):  113-116.  doi:10.13560/j.cnki.biotech.bull.1985.2016.08.017
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    This work aims to construct the reporter vector of rat NKx6.1 promoter and verify the activity of transcription factor T3R in the regulation of NKx6.1 promoter. We cloned a 2.4 kb 5' upstream promoter segment of NKx6.1 from the brain tissue of rat by PCR and predicted the binding sites of potential transcription factor T3R in the segment via bioinformatics method. Three promoter-deficient segments with different lengths were obtained by promoter deletion analysis and then cloned into the expression plasmids of luciferase reporter gene(pGL3-Basic),and corresponding reporter vectors were constructed. The reporter vectors and T3R were co-transfected into rat astroytes,then the activities of the gene’s luciferase were determined. Above results demonstrated that we successfully constructed the reporter vector of NKx6.1 promoter,and the results of dual luciferase assay showed that T3R regulated significantly NKx6.1 promoter,and the region of -1 887 bp-1 507 bp presented the highest activities,i.e.,contained the key cis-regulatory element. In conclusion,we cloned and screened the core promoter region and revealed the transcriptional regulation mechanism of thyroid hormones on NKx6.1 in brain tissue of rat.
    Construction of Lentiviral Vector for CGI99 RNAi and Its Effects on Antlerogenic Periosteum Cell Proliferation in Sika Deer
    LU Xiao ,SUN Hong-mei, CHU Wen-hui, ZHANG Wei, LI Chun-yi
    2016, 32(8):  117-121.  doi:10.13560/j.cnki.biotech.bull.1985.2016.08.018
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    CGI99 of antlerogenic periosteum(AP)cells from Chinese sika deer was interfered using the lentivirus-mediated gene silencing system,and a preliminary study on the gene function on proliferation of AP cells was proceeded. One sequence shRNA targeting CGI99 of sika deer was designed,then reassembled into the lentiviral plasmids pLVTHM. Together with the plasmids pSPAX2 and pMD2.G,recombinant lentivirus was acquired by their co-transfection into HEK 293t cells,and then infected into AP cells. Detecting the down-regulating expression level of CGI99 mRNA in infected AP cells was conducted by RT-PCR,and the effect of silenced CGI99 on AP cell proliferation was assayed by MTT. The results showed that the expression level of CGI99 mRNA in cells that infected with recombinant lentivirus was obviously decreased,the interferential efficiency reached 70.8%;and the curves by MTT assay tended to be consistent. Therefore,we successfully constructed RNAi vector for CGI99 and interfered the expression of CGI99 in AP cells,and preliminarily confirmed that the CGI99 presented insignificant effect on the AP cells’ proliferation.
    Isolation and Biological Characterization of Duck Adipose-derived Mesenchymal Stem Cells
    LIU Xue-ting, YUAN Hong-yi, ZHANG Ming-hai, GUAN Wei-jun
    2016, 32(8):  122-128.  doi:10.13560/j.cnki.biotech.bull.1985.2016.08.019
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    This study aims to establish in vitro culture system for the poultry adipose-derived stem cells(ADSCs),to study the biological characteristics and the multi-differentiation potential. Enzyme digestion method was applied to separate Beijing duck 18-day-old embryos ADSCs and to draw the growth curve. ADSCs were identified by RT-PCR,immunofluorescence and flow cytometric analysis. Our results suggested that the ADSCs had solid proliferative activity and were positive for CD29,CD44 and CD105,the rate of CD29+,CD44+,and CD105+ ADSCs was 95.39%,94.53%,98.19%,respectively. ADSCs differentiated into adipocytes and osteoblasts by induced differentiation in vitro. In summary,under appropriate experimental circumstances,Beijing duck embryo ADSCs have a strong self-renewing ability and multi-differentiation potential in vitro,and it can be an ideal type of seed cell for cellular transplantation therapy.
    Prediction for Secretome from Magnaporthe oryzae at Genome Scale and Its Enrichment Analysis
    CAO Ji-dong, LIU Jun, LI Sui-yan
    2016, 32(8):  129-138.  doi:10.13560/j.cnki.biotech.bull.1985.2016.08.020
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    Secreted proteins play an important role during pathogenic process of Magnaporthe oryzae. However,many of those secreted proteins are actually effect proteins that interfere the resistance of host plants and inhibit the immune responses of host plants. Therefore,the prediction of secreted proteins by M. oryzae and its functional analysis are necessary and hot topics in the interaction of plant and microbe molecules. The software SignalP,TMHMM,and SecretomeP were applied to complete the prediction of the secretome by M. oryzae. The analyses of classical secreted proteins(CSPs)containing signal peptide by GO function enrichment,KEGG pathway,and statistics of domains were performed,further the CSPs involved in the degradation of plant derived compounds were predicted. Total 789 CSPs were found in M. oryzae genome and the amino acid lengths of CSPs were mainly concentrated between 100 to 500 aa exclusively. GO function analysis of CSPs indicated that they were enriched in the secreting pathways and in the interactions with host. Interestingly,the results of KEGG metabolism and domain analysis of CSPs suggested that some of them contributed to sugar metabolism. Around 156 CSPs were recruited in the degradation of cell walls of plants. Besides,many non-classical leaderless secreted proteins were discovered in the M. oryzae secretome. In summary,by designing the informatics procedure,we predicted the secretome of M. oryzae,CSPs were able to degrade plant derived compounds such as cell walls,and some were involved in sugar metabolism. In addition,M. oryzae. harbored many non-classical leaderless secreted proteins.
    Prokaryotic Expression of MCP Gene from Largemouth Bass Ulcerative Syndrome Virus and the Immune Effect Analysis of Recombinant Protein
    MA Dong-mei, DENG Guo-cheng, BAI Jun-jie, JIANG Xiao-yan, CAO Ting-ting, CAI Lei
    2016, 32(8):  139-144.  doi:10.13560/j.cnki.biotech.bull.1985.2016.08.021
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    For preventing the outbreak of viral ulcerative syndrome in largemouth bass(Micropterus salmoides)in Guangdong province,the ORF of major capsid protein(MCP)gene,as target antigen protein gene,from largemouth bass ulcerative syndrome virus(LBUSV)was inserted in vector pBV220,then transformed into Escherichia coli DH5α,and the recombinant engineering bacterium for expressing MCP was constructed. Following 42℃ temperature inducement and SDS-PAGE analysis,the recombinant bacterium was detected to produce a special expression protein with molecular weight about 51 kD,and the proportion of recombinant MCP protein was nearly 30% of total bacterial protein. After washed,purified,dissolved,renatured and dialyzed,the purity of the recombinant protein reached over 90%. The purified protein was mixed and emulsified with incomplete Freund's adjuvant,then was injected in largemouth bass as vaccine. The immunized fish were challenged with LBUSV,and the highest relative percentage of survival reached 67.7% after 20 days. The results indicate that the MCP of LBUSV has the immunogenicity and can be selected as candidate protein for recombinant vaccine.
    Cloning,Expression and Bacterial Binding of Peptidoglycan Recognition Proteins-SA Gene from Musca domestica
    LUO Man, WANG Yu, HU Ya, XIU Jiang-fan, WANG Tao, PENG Jian, SHANG Xiao-li, WU Jian-wei
    2016, 32(8):  145-151.  doi:10.13560/j.cnki.biotech.bull.1985.2016.08.022
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    This work is to clone and express the Musca domestica peptidoglycan recognition proteins-SA(PGRP-SA)gene and research the microbial binding activity of it. The PGRP-SA gene was isolated from M. domestica larvae cDNA library,then primers were designed using cDNA plasmid as the template,and the complete encoding sequence of PGRP-SA was acquired by PCR. The bioinformatics methods were employed to predict and analyze the gene and its encoded proteins. The recombinant plasmid pET-28a(+)-PGRP-SA was expressed in Escherichia coli for induced expression and protein purification. RT-PCR was used to test the varied transcription levels of PGRP-SA gene in different tissues. The microbial binding activity of PGRP-SA protein was studied by the microorganism binding assay. The results indicated that the ORF full-length of PGRP-SA gene was 615 bp,encoding 204 amino acids. The molecular weight was 22.8 kD and pI 9.11 and had the conserved PGRP domain. After the recombinant plasmid pET-28a(+)-PGRP-SA was successfully constructed,it was expressed in E. coli by IPTG. SDS-PAGE and Western blot analysis showed that the functional protein purified by Ni2+ affinity chromatography was in consistent as the predicted in size. PGRP-SA was expressed in three instars hemolymph,fat body,foregut,midgut,trachea,malpighian tube except hindgut,the highest in the hemolymph,indicating that the expression of PGRP-SA was tissue-specific. Recombinant PGRP-SA protein bound to Staphylococcus aureus and E. coli,but not Monilia albica. Conclusively,the M. domestica PGRP-SA protein was successfully expressed and purified,and also it was confirmed that PGRP-SA protein from M. domestica bound to S. aureus and E. coli.
    Expression of Keratinase Gene Derived from Stenotrophomonas maltophilia in Pichia pastoris
    LI Guang-lei, ZHANG Juan, FANG Zhen, LONG Ming-xing, DU Guo-cheng, CHEN Jian
    2016, 32(8):  152-160.  doi:10.13560/j.cnki.biotech.bull.1985.2016.08.023
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    kerD,a keratinase gene derived from Stenotrophomonas maltophilia BBE11-1,was optimized by Pichia codon,and then the recombinant vector pPIC9k-kerD was constructed;the recombinant vector was integrated into the Pichia SMD1168 genome,and Mut+ recons were screened;recons resisting G418 were induced by methanol,and the best recombinant strain with the highest enzyme production was screened. The recombinant enzyme was purified and detected by SDS-PAGE,while part of the enzymatic properties was investigated. We found that the optimal reaction pH of recombinant enzyme was 10,and the optimal reaction temperature was 60℃. In order to further increase the yield of recombinant enzyme,multi-carbon source feeding strategy,using methanol mixed with sorbitol and mannitol as carbon source,was used to optimize the formation of producing keratinase by the recombinant strain. The results showed that when mixing ratio of methanol to mannitol was 20∶0.5,fermenting 168 h,production of recombinant keratinase reached 2 048 U/mL,which was improved 87.2% from single methanol carbon.
    Cloning,Expression,and Characterization of cotA from Alkalophilic Bacillus clausii
    LIU You-xun, YAN Ming-yang, GENG Yuan-yuan, HUANG Juan
    2016, 32(8):  161-168.  doi:10.13560/j.cnki.biotech.bull.1985.2016.08.024
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    In order to obtain a multi-copper oxidase with unusual features,a cotA gene from alkalophilic Bacillus clausii KSM-K16 was cloned and expressed in Escherichia coli. According to the CotA sequence from B. clausii KSM-K16 and the codon preference in E. coli,the gene of full-length sequence was designed,synthesized,and cloned into the expression vector of pET28a. Then the pET28a-cotAwas transformed to E. coli BL21(DE3)that was induced to express recombinant protein,which was subsequently purified and biochemically characterized. The purified recombinant CotA was about 62 kD,showed the blue green and had the characteristic absorption peak of Cu2+ at 609 nm. Also the recombinant CotA oxidized the substrates such as ABTS,SGZ and bilirubin. When SGZ as substrate,the optimal temperature and pH was 90℃ and 7.5,respectively. CotA maintained more than 70% of its original activity after incubating at 80℃ for 2 h. Furthermore,it was quite stable over pH ranging 4.0-11.0,more than 90% of its original activity after incubating at 37℃ for 1 h still remained. The results showed that the CotA from alkalophilic B. clausii was a typical multi-copper oxidase with the activities of laccase and bilirubin oxidase,and presented strong acidic-,alkali-,and thermo-stability.
    Screening and Identification of Maize Growth-promoting Rhizobacteria and Its Promoting Effects on Maize
    WAN Bing-bing, LIU Ye, WU Yue, ZHANG Dong-yan, WANG Guo-wen, JIANG Ying
    2016, 32(8):  169-176.  doi:10.13560/j.cnki.biotech.bull.1985.2016.08.025
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    Five maize plant growth-promoting rhizobacteria(PGPR)strains designated as YM1,YM2,YM3,YM4,and YM5 respectively were isolated and purified from the sandy soil. Based on their nitrogenase activity,ability of dissolving phosphorus and potassium,and indole acetic acid(IAA)synthetic quantity,the active microbial inoculum was screened for producing efficient microbial fertilizer. The results of shake flask culture indicated that strain YM4 was observed to have a stronger ability of dissolving phosphorus and potassium,and higher nitrogenase activity accompanied with the strongest synthetic quantity of IAA than other strains. Its nitrogenase activity reached 15.53 nmol C2H4/(h·mL),the transformation amounts of tricalcium phosphate and potassium silicate were 128.90 mg/L,17.40 mg/L respectively,and the synthetic quantity of IAA was 37.18 mg/L. YM4 was identified as Bacillus pumilus based on morphological observation,physiological and biochemical characteristics test and the conserved sequence analysis of 16S rDNA. Compared to the control treatment,pot experiment results suggested that the concentrations of IAA,available nitrogen,phosphorous,and potassium in the potting soil inoculated with YM4 significantly increased by 136.36%,42.1%,67.41%,and 14.29%,respectively;moreover,the morphologies of plant roots changed significantly,i.e.,the root length,surface area,root volumes and root tips of maize increased by 172.24%,141.73%,112.14%,and 104.53%,respectively;additionally,the average wet weight,the height of maize,SPAD value increased by 130.07%,150.65%,and 151.56% and the total N,P,K content of maize significantly increased by 120.99%,166.33% and 138.21% respectively. We may conclude that YM4 possess the abilities of efficiently fixing nitrogen,solubilizing phosphorus and potassium,and synthetizing IAA,thus has great application potential in the development and utilization of agricultural production.
    Screening and Identification of Mixed Culture,and Its Bioleaching Capacity
    NIE Yi-lei ,CHEN Hong ,LUO Li-jin, JIA Wei ,CHEN Xing-wei
    2016, 32(8):  177-183.  doi:10.13560/j.cnki.biotech.bull.1985.2016.08.026
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    It is aimed to screen the highly efficient leaching bacteria from acid mining water of low-grade copper sulfide mine,and also preliminarily investigate the bioleaching capacities of the bacteria. Firstly,a mixed culture FIM-201304 was obtained by 9K enrichment from acid mining water of a low-grade copper sulfide mine in Fujian province. Then,one dominated bacterium D-1 was isolated from the FIM-201304 using 9K solid culture medium. Sequencing technology by high-throughput Illumina Miseq was used to analyze the community structure of FIM-201304. D-1 was identified by phenotypic characteristics and 16S rRNA gene sequencing analysis. Flask experiment was utilized to compare the effects of leaching low-grade copper- and nickel-sulfide between the mixed culture FIM-201304 and a single bacterium D-1. Results from high-throughput sequencing analysis showed that the mixed culture FIM-201304 contained the dominated bacteria of Acidithiobacillus ferrooxidans(Abundance accounted for 85.02%),Pseudomonas sp.(Abundance accounted for 10.07%),Acidiphilium acidophilum(Abundance accounted for 1.30%)and Sphingomonas sp.(Abundance accounted for 1.21%). According to phenotypic characteristics and 16S rRNA gene sequence analysis,strain D-1 was identified as Acidithiobacillus ferrooxidans.The results after 20 days of bioleaching with flasks showed that leaching ratio of copper and nickel by FIM-201304 was 65% and 56% respectively,while the leaching ratio by D-1 was 41% and 36% under the same conditions,i.e.,the bioleaching ratio of copper and nickel by mixed culture increased 24% and 20% compared to that by single bacterium,respectively. Conclusively,the mixed culture resulted in the better leaching effect as heterotrophic bacteria promoted the metal-leaching capacity of autotrophic bacteria from the ore.
    Screening of Marine Crude Oil-degrading Bacteria and Construction of Microbial Consortium
    WU Bing-qi, LIU Shu-jie, CHEN Fu-ming, ZHOU Chu-ying
    2016, 32(8):  184-193.  doi:10.13560/j.cnki.biotech.bull.1985.2016.08.027
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    For the purpose of controlling marine oil contamination by biological treatment technology,using crude oil acting as sole carbon source and enrichment and spread plate method,high-performance oil-grading bacteria were isolated from five sampling points in the sea near Shenzhen,and bacterial consortium was constructed by mixing and orthogonal experiments. Physiological and biochemical experiments and 16S rRNA gene sequence analysis were used to identify the strains. Single-factor experiment was employed to optimize the conditions of oil biodegradation by the consortium,and gas chromatography and mass spectrum(GC-MS)were utilized to analyze its biodegradation characteristics. The results showed that 22 strains of high-performance oil-degrading bacteria were isolated,and the degrading rates varied from 34.5% to 52.2%. The degrading rate by microbial consortium SQ1 composed of S1-30,S1-38,and S2-13 strains reached 68.3%. These three strains were identified as Corynebacterium sp.,Dietzia sp. and Labrenzia sp. SQ1 was able to degrade the oil by 73.5% in 11 days under optimized conditions,referring to 30℃,pH7.6,oil concentration 20 g/L. The GC-MS results showed that consortium SQ1 was able to degrade the total alkane by 91.7%,and the more refractory C21-C35 by nearly 100%. The study shows that consortium SQ1 has great application potential of bioremediation for marine oil contamination.
    Development of Wettable Powder of Trichoderma reesei FS10-C and Its Plant Growth-promoting Effects
    LUO Yang, TENG Ying, LUO Xu-qiang, LI Zhen-gao
    2016, 32(8):  194-199.  doi:10.13560/j.cnki.biotech.bull.1985.2016.08.028
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    Optimal formula of the wettable powder of Trichoderma reesei FS10-C,with functions of plant growth-promoting and heavy-metal-resistant effect,was defined by screening carriers,wetting agents,dispersing agents,and protecting agents,and a pot experiment was conducted to determine its effects on the growth of Sedum plumbizincicola. The results showed that the optimal formula of the wettable powder was confirmed as follows:10% Trichoderma reesei FS10-C conidia,74.6% kaolin,5% dodecyl-benzene sulfonic acid sodium,10% sodium lignosulphonate,and 0.4% vitamin C. The suspension rate of the formula was 72.48%,which matched the national formulation standard. Results of the pot experiment demonstrated that 500 times dilution of Trichoderma reesei FS10-C conidia WP presented a favorable growth-promoting effect on S. plumbizincicola,under which the aboveground fresh weight and dry weight of the plant increased by 14.70% and 23.14% respectivelycompared with the controls.
    Antagonistic Efficacy and Growth-Promoting Effect of Bacillus methylotrophicus Isolated from Dendrobium huoshanense
    WU Li-qin ,GU Hai-ke ,WANG Qing, SHANG Hong-zhong, LIU Gui-jun ,BAO Fang
    2016, 32(8):  200-206.  doi:10.13560/j.cnki.biotech.bull.1985.2016.08.029
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    Isolating endophytic bacteria form Dendrobium huoshanense aimed to obtain endophytic bacteria that have broad-spectrum antimicrobial activity and plant growth-promoting trait. Plate confrontation method was applied to screen the endophytic bacteria with antimicrobial activities against the black spot disease of D. huoshanense,of which the antimicrobial spectrum and plant growth-promoting(PGP)traits were determined. Total 22 endophytes were isolated from D. huoshanense,and strain RA presented strong inhibitory activities against Alternaria tenuissima and it was identified as Bacillus methylotrophicus on the basis of 16S rDNA sequence analysis and was selected for further studies. Strain RA was capable of co-producing IAA,siderophore,protease,biosurfactant,and biofilm,and significantly inhibited 8 plant pathogens and had broad-spectrum antimicrobial effects. A significant improvement in corn seedlings growth was observed when seeds of corn were bacterized prior to sowing. In conclusion,results clearly suggest that strain RA is a potential candidate for use as biocontrol agent and in biofertilizer.
    Screening for the Interaction Proteins of SOC1 from Phyllostachys violascens Using Yeast Two-Hybrid System
    PAN Xian-fei ,SHI Quan, LIN Xin-chun, XU Ying-wu ,CAO You-zhi
    2016, 32(8):  207-213.  doi:10.13560/j.cnki.biotech.bull.1985.2016.08.030
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    This work aims to study the target proteins of suppressor of overexpression of constans1(SOC1)in flowering way of Phyllostachys violaceus and to explore the action mechanism of SOC1. The pGBKT7-PvSOC1 vector was constructed as the bait protein,then the cDNA library of P. violascens flowering was established using SMART technology,and the interaction proteins with SOC1 were screened by yeast two-hybrid system as well as analyzed and identified. The results showed that the cDNA library for yeast two-hybrid was constructed successfully,the conversion rate of the library was about 3.0×106 converters per μg pGADT7-Rec,and the capacity of the library was up to 7.5×106 cfu/mL and the insert fragments were distributed from 0.25 kb to 2 kb. The protein interacted with the bait protein PvSOC1 was screened through the yeast two-hybrid technology. The screened target protein was identified as a protein kinase containing plentiful leucine which amino acid sequence was very conservative in different species,and had the highest affinity with rice.
    The Expression of Elastin-like Polypeptide Tag in Periplasmic Space of Escherichia coli
    LI Jing-quan, LIN Heng, DING Ning, ZUO Xiao-xue, SHI Jin-ping, XU Yong-ping, LENG Chun-ling, YANG Chun-guang
    2016, 32(8):  214-220.  doi:10.13560/j.cnki.biotech.bull.1985.2016.08.031
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    This work aims to investigate the expression of elastin-like polypeptide[I]40(ELP[I]40)in the periplasmic space of Escherichia coli. The expression vectors of pIG6LH/ELP[I]40and pIG6LH/ELP[I]40+Trx were constructed and transfected into E. coli BLR(DE3)and induced to express by IPTG. Recombinant ELP fusion protein were purified by inverse transition cycling(ITC). Inverse temperature transition(Tt)of ELP[I]40 and ELP[I]40+Trx protein were measured. The effects of the concentrations of ELP[I]40 and ELP[I]40+Trx proteins and NaCl on phase-transition temperature were investigated. ELP[I]40 and ELP[I]40+Trx protein were purified by 3 round ITC. When the protein concentration of ELP[I]40and ELP[I]40+Trx were 10 μmol/L,25 μmol/L,50 μmol/L,75 μmol/L,and 100 μmol/L,the Tt were 31.5℃,29℃,27℃,26℃,25℃ and 31.8℃,29.5℃,27.5℃,26℃,25.5℃ respectively. When the final concentrations of ELP[I]40 and ELP[I]40+Trx were 25 mmol/L respectively and the NaCl concentration was 0.25 mol/L,0.5 mol/L,0.75 mol/L,1.00 mol/L,and 1.25 mol/L respectively,the Tt of ELP[I]40 decreased from 29℃ to 24.5℃,22℃,19℃,15℃,and 11.5℃ respectively and the Tt of ELP[I]40+Trx from 29.5℃ to 25℃,23℃,20.2℃,15.5℃,and 11.8℃ respectively. The ELP[I]40 in the periplasmic space of E. coli possessed the same physicochemical properties as the intracellular one,and the ELP could be used as an isolation and purification tag of expressed proteins in E. coli’s periplasmic space.
    Isolation and Identification of a Bacterium Disturbing Molecular Cloning,and Analysis of DNA-Degrading Activity of Its Fermentation Broth
    DIAO Wen-tao, WANG Xue-yan, CHEN Xiao-fei, FENG Fei, NING Meng, ZHOU Fu-zhong
    2016, 32(8):  221-225.  doi:10.13560/j.cnki.biotech.bull.1985.2016.08.032
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    This work aims to analyze the reason that resulted in the failure of cloning and to check if there are some microorganisms strongly degrading DNA in the environment of electrophoresis chamber. The colony morphology,gram staining and the 16S rDNA sequence were utilized to identify the strain degrading DNA,and agarose gel electrophoresis was for analyzing the DNA-degrading activity. As results,a strain was isolated from DNA electrophoresis chamber,and identified as gram-negative bacterium. Then it was cultured in YPD medium,and the capacity of the fermentation broth degrading DNA was detected using plasmid pUC19 as substrate. The strain quickly and sufficiently degraded DNA at the optimal temperature 45℃,thus this strain was designated DD(DNA degrading). The sequence alignment of the DD by the 16S rDNA showed that this DD had 100% identity with the that of Serrtia marcescens. Conclusively,the bacterial strain DD isolated from DNA electrophoresis chamber is a Serrtia marcescens strain possessing significant DNA-degrading activity based on the results of colony morphology,gram staining,and 16S rDNA sequence.
    Metabolic Engineering for Modifying Corynebacterium glutamicum to Produce More Pyruvate
    YU Ying, XU Mei-xue ,LIU Jin-lei, FAN Rong ,FENG Hui-yong, LI Tian-ming
    2016, 32(8):  226-232.  doi:10.13560/j.cnki.biotech.bull.1985.2016.08.033
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    In order to increase the accumulation of pyruvate while decrease the production of by-products during the metabolic process of Corynebacterium glutamicum,the homologous recombination was employed to knock out the key genes of pyruvate:quinone oxidoreductase (pqo),pyruvate dehydrogenase(pdh)and L-lactate dehydrogenase(lldh)in the tributary metabolic pathways,thus the gene deletion mutation strains were constructed. Using compound medium with 4.5% glucose as carbon sources,the concentration of pyruvate with mutation strain at shake flask fermentation for 48 hours reached 14.6 g/L,while that with the wild type only was 0.45 g/L. The conversion rate of sugar to acid with the mutation strain was 33.18% and 32.5 times higher than the wild type.
    Effects of Metal Ions on Antibody Production and Charge Heterogeneity in CHO Cell Cultures
    ZHANG Xin-tao ,TANG Hong-ping ,ZHAO Liang ,FAN Li, LIU Xu-ping, MIAO Shi-wei, TAN Wen-song
    2016, 32(8):  233-241.  doi:10.13560/j.cnki.biotech.bull.1985.2016.08.034
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    The objective of this work is to get insights into the role of metal ions in the production of the monoclonal antibody(mAb)during Chinese hamster ovary(CHO)cell cultures. Cell growth,antibody production and antibody charge distribution at different concentrations of copper and zinc ions during cell cultures were comprehensively investigated. It was found that 120 nmol/L copper ion and 50 µmol/L zinc ion were the optimal concentrations for cell growth and antibody production. Excessively low or high levels of copper and zinc ions brought negative impacts on cell growth and viability maintenance. Additionally,copper ion and zinc ion,as the inhibitor and cofactor of basic carboxypeptidase respectively,showed considerable effects on antibody charge distribution. The medium containing 50 nmol/L copper ion and 50 μmol/L zinc ion was the most conducive to the reduction of charge variants and the increase of main species. Both excessively low or high concentrations of copper and zinc ions led to the increase of the antibody charge variants and the reduction of main species. The concentrations and ratios of copper and zinc ions altered the enzymatic digestion at antibody C-terminal lysine,subsequently affected the content of the basic variant. Additionally,the working space of copper and zinc ions for process control of antibody charge variants was preliminarily discussed. In conclusion,the metal ions play important roles in cell growth,mAb production and mAb charge distribution during CHO cell cultures.
    Preparation of Hierarchical Microsphere BiOBr Catalyst and Its Photocatalytic Disinfection Performance Under Visible Light
    WANG Ya, LIN Li, LI Bei-bei, HUANG Man-hong, CHEN Liang
    2016, 32(8):  242-248.  doi:10.13560/j.cnki.biotech.bull.1985.2016.08.035
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    The BiOBr,three-dimensional hierarchical microsphere,was successfully fabricated by one-pot solvent-thermal method,and was characterized by scanning electron microscopy(SEM),X-ray diffraction(XRD)and UV-vis. Performance of photocatalytic disinfection of Escherichia coli using BiOBr under visible light irradiation was studied,also the disinfection mechanism was explored. The results showed that the BiOBr was microsphere with diameter of about 2 µm,and had solid visible light response. Sterilization experiments showed that the BiOBr presented the enhanced photocatalytic inactivation of gram-negative bacterium E. coli under visible light irradiation(λ> 420 nm). The disinfection rate reached over 70% with the 0.5 mg/mL photocatalytic materials for 12 h visible light irradiation. Reactive capture experiments demonstrated that 3D hierarchical microspheres BiOBr destroyed bacterial cell membranes and walls via hole oxidation,therefore achieved the purpose of killing bacteria under visible light. ?
    Content
    2016, 32(8):  300-300. 
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    Copyright
    2016, 32(8):  400-400. 
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