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Ferment Engineering and Validation Ideas

China Biotechnology.2006;0(01):-.

Fermentation is a technology,validation is a management.Technology and management could not be seperated trom each other.The basic theory of the validation was introduced,the idea of the validation was fused into the ferment technology.The idea of the validation of the ferment system was set forth the whole process of the validation of the ferment system from personal qualification,operation procotol,validation implement,and sum-up reports.It was proved that implementing validation and constructing validation system throughout ferment processing is indispensable for completing a successful fermentation.the validated fermentation system is just a dependable system,the validated ferment technique is a stable technique.

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Formula Western Drug Maybe One of Final Strategic Aims of Modernization of Traditional Chinese Medicine

Zhouzhang ZHI

China Biotechnology.2006;0(04):-.

Systems Biology including Systems Biomedicine is emerging internationally and providing opportunities for revolutions of Traditional Chinese Medicine (TCM) and conventional western drugs (WD), while the latter has been taking more advantages because WD has much stronger research basis at molecular levels than TCM. Thorough understanding of molecular network including gene network would automatically induces design of WD into formula western drugs equipped with TCM philosophy, but such molecular formula drug is eventually one of the final aims of modernization of TCM. So TCM is facing big threats it never met. This situation requires a nationwide and international strategy with intelligence and resolution.

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Activity Study on Antibacterial Protein Isolated From Musca domestica During Larvae-pupae Metamorphosis

Xiaohong CAO ; Zhongli YAN ; Chunling WANG

China Biotechnology.2006;26(1):33-37.

Objective: To research the activity of antibacterial proteins isolated from Musca domestica during larvae-pupae metamorphosis. Methods: The 5-day-old larvae of Musca domestica were injured with a needle. 24h later, the larvae which had changed into pupae were selected. The antibacterial protein was isolated by a four-step protocol including grinding, heating, CM-sepharose F. F. cationexchange chromatography and another CM-sepharose F. F. chromatography. Antibacterial activities of samples were tested by an ultrasensitive radial diffusion assay using Staphylococcus aureus as the indicator organism. The molecular weight of the isolated protein was determined by SDS-PAGE.Results: The molecular weight of this isolated protein was 43kDa, and its antibacterial activity was strong. Conclusions: A kind of protein with strong antibacterial activity can be produced in the Musca domestica during larvae-pupae metamorphosis.

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Increasing the Production of IgG in Batch Tissue-culture with IgG-free Ascites

Ruoyu ZHOU ; Jing YE ; Hong XIE

China Biotechnology.2005;25(8):87-92.

An effective method has been developed for laboratory scale production of IgG. Hybridomas were cultured in serum-free media with 2% IgG-free ascites. Cell density of up to 3.55 × 10 6cells/ml and antibody concentration of 135μ g/ml after purification were abtained, which is four time more than total production of that of IgG concentration in serum-free media. This in vitro method allows great improvement in antibodies production in batch tissue culture. The method reported here is easy to handle and is economical and universally adaptable.

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Construction of a cDNA Library and Cloning of an Arabinosidase cDNA from Armillariella tabescens

Dongsheng YAO ; Hui HUANG ; Long ZHAO ; Chunfang XIE ; Daling LIU

China Biotechnology.2005;25(6):65-70.

The expression cDNA library of A. tabescens was constructed by SMART technique, which useλTriplEx2 as a vector. The titer and the percentage of the constructed library were about 1.0 × 106pfu/mland 98.3% respectively, and the titer and the capacity of the amplified library were about 3.1 × 108pfu/mland 4.2 × 1010. The library was used to provide expressed sequence tags (ESTs). 147 Expressed SequenceTaqs (ESTs) were gained from 176 clones, which were selected randomly and sequenced at the 5'end. Thesequences were submitted to the EMBL database. Blasting the sequences in the GenBank, 43 of them werefound that they have significant similarity with data in GenBank. EST AJ620046 was has significantsimilarity with the arabinosidase of Bacteroides thetaiotaomicron. Using SMART-RACE a full-length cDNA ofAJ620046 was successfully obtained. In order to initially characterize the biochemical properties ofAJ620046, the ORF of AJ620046 named AF was cloned and expressed in Pichia Pastoris yeast.Recombinant pHIL-S1-AF constructed by inserting AF into pHIL-S1 was transformed into Pichia PastorisGS115. Preliminary experiments indicated that AJ620046 was expressed as a 32 kDa protein in recombinantyeast.

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Expression and Purification of Basic Fibroblast Growth Factor Mutant with Reduced Mitogenic Activity

Xiaoping WU ; Xiaokun LI ; Zhijian SU ; Qing ZHENG ; Sixian WU ; Hua XU ; Hongyan QU

China Biotechnology.2005;25(2):49-52.

In order to decrease the potential side-effects of human basic fibroblast growth factor (hbFGF) caused by its broadspectrum mitogenic activity, a single residue of hbFGF, the residue serine 108, was replaced with neutral alanine residue to construct a mutant of hbFGF (mhbFGF) with reduced mitogenic activity. The mutant was overexpressed in Escherichia coli BL21(DE3) by IPTG induction. The expression level of mhbFGF was about 30% of the total cellular protein. The expressed mhbFGF was purified by ionic exchange and heparin affinity chromatography from the supernatant of bacteria lysate. Measured by MTT method, the effect of mhbFGF on Balb/c 3T3 cell proliferation was much lower than that of wild-type hbFGF. The purified recombinant mhbFGF was prepared and sufficient for the following pharmacological and safety studies.

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Anticancer Agents from Endophytic Fungi

Min QIU ; Ruisheng XIE ; Yu SHI ; Haimin CHEN

China Biotechnology.2009;29(1):93-104.

Various cancers have seriously threatened human' s health. Screening newer and more effective anticancer agents from natural sources to cure these diseases is the focus in research. As novel sources of potential medicine, a number of metabolites isolated from endophytic fungi have been proved to have anticancer bioactivity. Usually, these endophytic fungi have special biochemical pathway and they can accumulate anticancer agents in cultures such as taxane, alkaloids, cytochalasins, podophyllotoxin, brefeldin A and so forth. The research advance on anticancer agents purified from endophytic fungi is expatiated systemically. In addition, the strategy of screening anticancer agents as well as the prospect on this area is introduced briefly.

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Agrobacterium-mediated Transformation of an Economically Important Potato Cultivar Using Internodal Stem Explants

Li TANG ; Hui TANG ; Suying WANG ; Xiaoli YANG ; Haengsoon LEE ; Sangsoo KWAK

China Biotechnology.2007;27(7):80-87.

Potato cultivar Atlantic is widely grown for potato chips in the world. However, this economically important potato cultivar exhibits very poor yields and traits under severe environmental stress. To develop an efficient plant transformation system that could be used to produce large scale transgenic potato plants with enhanced tolerance to environmental stress and therefore would be beneficial for potato processing industry, Agrobacterium-mediated transformation of internodal stem explants using both superoxide dismutase (SOD) and ascorbate peroxidase (APX) genes under the control of an oxidative stress-inducible SWPA2 promoter was performed. Comparing to leaf explants, stem internodal explants were less liable to damage during manipulation, more amenable to in vitro conditions. The addition of silver thiosulfate to the selection medium considerably promoted the shoot induction from explant-derived callus. Seven to nine shoots per stem explant were obtained. By combining the best treatments, this system yielded shoot induction frequency of 94.2% and transformation frequency of 80% of internodal stem explants. Stable integration of the transgenes was confirmed by PCR and Southern blot analyses. In conclusion, short duration (7～8 weeks), high efficiency and easy process make this system well suited for wider commercial applications of transgenic Atlantic potato plants.

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Computational Identification of microRNAs and Their Targets

Zhiyun GUO ; Canquan MAO ; Lili XIONG

China Biotechnology.2008;28(10):118-123.

The discovery of microRNAs (miRNAs) has introduced a new paradigm into gene regulatory systems. Since inception, computational methods have been an invaluable tool complementing experimental approaches, and many discoveries have been obtained through combination of experimental and computational approaches. The knowledge that has been accumulated about the principles of miRNAs and target recognition were reviewed. The currently available computational methodologies and software for prediction of miRNA and their target genes also have been discussed.

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Detection and Partial Characterization of γ-glutamyltranspeptidase from Cordyceps sinensis Mycelia

Jianglan YUAN ; Xu KANG ; Zheng HU ; Guolin ZOU

China Biotechnology.2008;28(10):100-105.

γ-glutamyltranspeptidase was detected from the cultured mycelia of Cordyceps sinensis (CSGT). Km and Vmax of CSGT was 2.54×10-4 mol/L and 0.1808 mol/L·min respectively when L-glutamic acid 5-(4-nitroanilide) (GpNA) and glycyglycine was used as its substrate. CSGT was stable from pH 8.0 to 11.0 and at or below 20℃. It was optimally active at pH 9.0～10.0 and 30℃. A series of reducing reagents could activate CSGT, and metal cations such as Zn2+, Cu2+, Hg2+ , Mn2+ inhibited strongly activity of the enzyme, but K+, Ca2+, Mg2+ and Na+ at high concentrations had no effect on its activity, indicating that its active center could contain -SH.

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