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Purification of a New Antibacterial Protein from Housefly(Musca domestica)Larvae

Hui QU ; Youjin HAO ; Yingjun JING ; Desen LI ; Rongqian DU

China Biotechnology.2006;0(09):-.

Insects exhibit a particular resistance to infections.The activation of the innate immune response of insects is involved in the recognition of the infectious nonself and subsequent activation of cellular and humoral reactions.In humoral reactions,insect antibacterial peptides and lysozyme are very important in resistance to infections.Housefly(Musca domestica) is one of the most important kinds of insects and it has strong ability to adapt to the adverse circumstances.It is of momentous theoretical and practical significance to research the immunity system of housefly.The methods of inducement of housefly larvae were firstly studied.Then an antibacterial protein,whose molecular weight is 28kDa,was purified from housefly larvae,induced by 30% H2O2,through saltout,Sephadex G25 column,Sephadex G75 column and CMSepharose Fast Flow column.This antibacterial peptide had activities against most of Gram positive and Gram negative bacteria.

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Construction and Determination of an Episomal Secreting Expression Vector:pYES2/CT/?-factor for Saccharomyces cerevisiae

Yadong HU ; Dongsheng YAO

China Biotechnology.2006;0(12):-.

pPIC9 was used as a template and ?-factor gene of Saccharomyces cerevisiae was amplified by PCR.The gene was cloned in the intracellular expression vector pYES2/CT for Saccharomyces cerevisiae and the secreting expression vector named pYES2/CT/?-factor(pYC?)was constructed.The gene of mannase(man)from recombinant vector of pKLAC1-man(pKLman) was cut by restriction enzymes and linked with pYC?.This recombinant vector pYC?-man was used to determine the secretory ability and stability of pYC?.The excellent secretory ability of pYC? was proved by two experiments.One showed that INVSc1/pYC?-man clones formed the clear rings around the clones on the medium contained trypan blue,while INVSc1/pYC? clones had no rings.Further analysis of mannase activity of extracellular supernatant and intracellular extracts showed that both extracellular and intracellular mannase activities of INVSc1/pYC? were not detected,while INVSc1/pYC?-man had evident extracellular mannase activities and no intracellular mannase activities.The stability of pYC? was also very good proved by continuous cultivation for about 150h.

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The Global Development of the Biopharmaceutical Industry

Shumei WEN

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

The number of new medical products based on chemistry launches at the lowest level for many years. The pharmaceutical industry puts emphasis on biotechnology-derived products. Both genomics and protemics are contributing to understanding and determining more targets which involved in human disease.This means that there will be more opportunities for biopharmaceutical breakthroughs,eventually,more and better biomedicines will be approved.Several trends in development for biopharmaceuticals were analyzed:(1) Biological medicine and Chinese traditional medicine have developed rapidly between 1998~2005, the number of the papers about biological medicine increases dramaticly. (2)The emphasis is on biological medicine in pharmaceutical industry and the biotech market is concentrating in United States and multinational. (3)The proportion of biotech products is becoming larger gradually. More concern is focused on biotech products.(4)In order to reduce the cost of R&D and increase the number of new drugs, more and more M&A occur between the biotech companies.(5)The governments all over the world attach importance to biotech products.(6)Tissue engineering, cell therapy and gene therapy have a good future, full of opportunities and challenges.

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Studies on the Effects of Ultrasound on Pepsin,Trypsin and Catalase

Kang WANG

China Biotechnology.2006;0(05):-.

Three kinds of enzymes,pepsin,trypsin and catalase,were selected to research the effects and mechanisms of ultrasound on protein,taking advantage of the change of their biological activity under sonication as the index.The results indicate that the three kinds of enzymes display different modes and degrees of activity change under sonication.In addition,the effects of ultrasound on the enzymes vary with their concentration,which means that the destruction of ultrasound to enzyme could be reduced by adjusting their concentration.Mannitol,a strong free radical scavenger,and Tween-80,a kind of non-ionic surfactant,could protect the activity of the enzymes during sonication.That implies that free radicals,which are produced through the thermal dissociation of water induced by ultrasound cavitation,and the cavitation bubbles,which could disturb the hydrophobic environment around the enzyme molecules,play important roles during the destruction of sonication to enzymes.However,for different enzyme,different factor play the main role.

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Peanut Cotyledon Bioreactor-a Potential New Force in Peanut Industry

Haiyan YAN

China Biotechnology.2006;0(09):-.

The products from bioreactor of peanut cotyledon are organ-specific, enrichment in content, orally taken directly, etc. These properties made it a better bioreactor than others. Current research background and future perspective in this area were introduced. The techniques needed on peanut bioreactor was summarized. The key of peanut bioactor is construction of transformation vector. The promoters of major storage proteins are best choice for construction of transformation vectors. The promoter of glycinin genes from soybean can also be used in construction of transformation vector.

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Ferment Engineering and Validation Ideas

Mingli LIU

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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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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