AbstractGermany, the largest economy in Europe, has always been attaching great importance to biotechnology aiming at cultivating bioindustry to be the third largest industry after automobile and manufacture industry in the country. A series of efforts has been made by German government to enhance biotechnology and bioindustry such as promoting basic research and technology commercialization, pioneering in the frontier of life science by maximizing Germany advantages, elevating biotechnology innovation systematically by arrays of technology programs, reinforcing workforce training, exploiting advantages of regional bioclusters in different level, giving priority to industrial biotechnology development, investing greatly to support innovative biotechnology companies.
AbstractThe bioindustry of New Zealand does not rank high world widely in the sense of the scale. However it is the indispensable part of the country's economy. Prestigious fields of New Zealand biotechnology focus on agribiotechnology and biomedicine research, e.g. large animal and plantbased research, the researches on biosafety, environment protection, neuroscience, cardiovascular diseases, tuberculosis and asthma, diabetes, tumor, and etc. The New Zealand government has attached great importance to the development of biotechnology and bioindustry. The officially announced “The Growth and Innovation Framework” in 2002 listed biotechnology as one of the 3 pillars underpinning the future economy of New Zealand and planned to increase the investment in biotechnology and bioindustry as the important measure for the New Zealand's transformation to hightech economy.
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AbstractBiotechnology in Africa lags greatly behind the rest of the world. Most African countries have not developed modern biotechnology until recent years. Biotechnology is being attached greater importance by African countries nowadays through enhanced training efforts, active international cooperation, and reinforcement of biosafety by means of widely involvement in UNEP/GEF biosafety programs. Particularly, the High Level Panel on Modern Biotechnology (ABP) has been established with the help and guidance of African Union and related international organizations to elaborate “African Biotechnology Development Report” which is dedicated to the vision of the African biotechnology development in the future. The understanding on the status and trends of African biotechnology development could have significant and positive effects on Chinese biotechnology and SinoAfrica cooperation.
As a member of tyrosine kinases (TK) family, Epidermal growth factor receptor(EGFR) has an activity of intrinsic protein tyrosine kinase, which plays an essential role in the regulation of signal transduction in the cells. Due to the abnormal expression of EGFR-TK, the certain type cancers may developed and progressed. Based on that, the inhibitors of EGFR-TK could be effective medicines for the treatment of cancer. In this study, the EGFR-TK domain was amplified by RT-PCR with RNA of HUVCEs cells as the template and expressed in E.coli BL21(DE3) using plasmid pET30a as vector. The recombinant protein was purified with the affinity chromatography (Ni-NTA), which was identified to have kinase activity catalyzing the substrate phosphorylated with ATP in the enzymatic reaction. Using the recombinant EGFR-TK as target, the screening model for enzymatic inhibitors was constructed.
To construct and express human SNAP25 gene mediated by recombinant adenovirus vectors．human SNAP25 gene was coloned by RT-PCR and directly cloned into vector Pentr-TOPO to fulfill TOPO-SNAP25 plasmid.The recombinant plasmid TOPO-SNAP25 was identified and confirmed by PCR and sequencing.SNAP25 gene was cloned into the pAD／CMV／V5-DESTTM gateway vector by LR recombination reaction with pAD／CMV／V5-DESTTM gateway vectors and TOPO-SNAP25 plasmid. The ccdB-CmR gene in pAD／CMV／V5-DESTTM was replaced by SNAP25 gene.The recombination vector was digested by Pac I enzyme and transfected into HEK-293A cells by Lipofectamine 2000 to obtain recombinant adenovirus vectors pAD／CMV／V5一DEST-SNAP25, which were detected with mouse anti-human monoclonal SNAP25 antibody in transfected HEK-293A cells. The results indicate that recombinant adenovirus pAD-SNAP25 was packaged in HEK-293A cells. Rat islet β-cells infected with adenovirus pAD-SNAP25, level of insulin secretion were enhanced by expression of exogenous SNAP-25 at high glucose concentration.
Objective: To express the B subunit of Shiga-like Toxin type Ⅱ, and analyze its expression form and receptor-binding activity. Methods: The slt2b gene was obtained from EHEC O157:H7 by PCR, and cloned to the expression vector pET22b(+).The genetically engineered bacteria pET22b(+)-stx2B/BL21 expressed the recombinant StxB after induced with IPTG. The renatured inclusion bodies were purified by ion exchange chromatography. The expression form of rStx2B was investigated by denaturing and native electrophoresis. The receptor-binding activity was confirmed by fluorescence detection and flow cytometer. Result: The constructed genetically engineered bacteria expressed the rStx2B at a high level. The purified protein was obtained after denaturation, renaturation and ion exchange chromatography. According to the denaturing and native electrophoresis, the rStx2B was expressed in a dimmer form, which consists of two monomers cross linked with disulfide bridge. The rStx2B showed good receptor-binding activity by Hela-binding assay. Conclusion: The genetically engineered bacteria were constructed successfully. The receptor-binding activity of rStx2B was independent of the pentamers.
TTI gene coding for Tsetse thrombin inhibitor was modified with E.coli bias codon and expressed in Escherichia coli with high efficiency. Recombinant protein was purified to more than 98% purity. Assay for enzyme activity determination was set up. The result showed that the fusion protein exhibited inhibiting activity for thrombin. Inhibitory rate of purified TTI was 73% when concentration of thrombin and substrate was 10U/ml and 250 mol/L respectively. Inhibition pattern was determined as competitive with ki at 35 mol/L.
Escherichia coli was used to produce acidic fibroblast growth factor(aFGF) of high purity, and its effect on proliferation of 3T3 and improvement of bald rats were studied. The result shows that in vitro aFGF at the level from 1.95 ng/ml to 1000ng/ml can promote the proliferation of balb/c 3T3, which has significant difference when compared with the control group(P<0.05，P<0.01). In vivo aFGF at the level of 2μg/ml and 4μg/ml can improve the bald hair of the rats, which also has significant difference when compared with the control group (P<0.05). Pathology result proves that aFGF can improve the hair numbers of the bald rats, as well as the hyperaemia situation of the veins. We conclude that aFGF has good future in clinical research.
Human immunodeficiency virus (HIV) is the etiologic agent of AIDS.Immunogenic epitopes of HIV resides on virus-encoded envelope glycoproteins. To prepare HIV-1 gp160 protein and used it for clinical diagnosis, three gene fragments of HIV-1 env containing highly immunogenic epitopes were amplified by PCR from plasmid of HIV-1 HXB2. The resulting PCR products were linked and cloned into a prokaryotic expression vector pET28a(+) and the accuracy of the inserted fragments was identified by sequencing. To express the HIV-1 gp160 fusion epitopes in E. coli cells and identify fusion protein, the induced protein was checked with SDS-PAGE and Western Blot (WB). The identified HIV positive serum samples were tested by Western Blot (WB) to analysis immunogenicity of the purified protein. The length of the chimeric fragment derived from HIV-1 gp160 was 969bp, and encoded 37kDa amino acid residues. Procaryotic expression plasmid was constructed successfully which can highly effectively express gp160 fusion protein. Recombinant fusion protein was expressed in Eserichia coli BL21(DE3) as an insoluble protein. Procaryotic expression plasmid which can highly effectively express gp160 fusion protein was constructed successfully. The founding and subjects were provided for the research of diagnosis kit.
The octapeptide Lys-Gly-Asp-Glu-Ser-Leu-Ala, also called beefy meaty peptide (BMP), has previously been demonstrated to be a savory seasoning. In order to produce BMP in pilot scale, recombinant P. pastoris GS115-16B2 secreting the fusion peptide containing 16 tandem repeated BMP was inoculated in a 500L fermentor to carry out a three-step fermentation process, i.e., glycerol batch phase and a glycerol fed-batch phase to achieve high cell densities, followed by a methanol fed-batch induction phase. The cells were grown to desired cell density on glycerol at 30°C and pH 5.0 and then induced on methanol for expression of BMP at 28°C and pH 3.0. Dissolved oxygen (DO) was maintained above 20%-35% saturation by adjusting agitation rate, aeration and fermentor pressure. Samples were analyzed to determine the cell density (OD600), wet cell weight and BMP production. It is found that，after the culture for 118 hours, the cell density(OD600), wet cell weight and the yield of target protein are respectively 184.5, 318.7g/L and 172 mg/L.
Bio-production of 1, 3-propanediol (1,3-PD) has attracted much attention recently, and it exhibits a wide range of potential utilizations, especially as monomer for the synthesis of polypropylene terephtalate (PPT). Bio-conversion from glycerol to 1,3-PD by bacterial species Klebsiella pneumoniae was investigated by fed-batch fermentation and regulated by nutrient substances limited culture. Depending on product formation directly linked to cell growth, evidence was provided that nitrogen could constitute a limiting factor for manipulating cell growth by controlling NH4Cl concentration, it is found that an adequate nitrogen level for glycerol fermentation into 1,3-PD was efficient in converting glycerol into 1,3-PD. Under less nitrogen concentration, biomass yield decreased resulting a less 1,3-PD yield. Under excess nitrogen concentration, an increase of biomass yield and specific growth rate did not help production, this was attributed to much more consumption of glycerol on cell growth and metabolize. It was clearly shown that the volumetric productivity and molar convertion yield reached a high level by controlling nitrogen supply with the NH4Cl fed rate of 0.41 g/(L•h) and the residual concentration below 0.1 g/L. After 25-28 hours, the final concentration, the volumetric productivity, molar convention yield of 1,3-PD in fed-batch fermentation under nutrient limited conditions were 52.03 g/L, 2.04 g/(L•h), 0.66 respectively, increased 28.0 %, 35.1 % and 29.4 % over control. In this way an efficient microbial production 1,3-PD from glycerol was demonstrated by controlling the specific growth rate of cell through limited NH4Cl regulation, and then both the productivity and molar yield would increase effectively.
5-enol-Pyruvylshikimate -3-phosphate synthase (EPSPS) is an enzyme on the pathway toward the synthesis of aromatic amino acids in plants and microorganism, it also is the target of the broad-spectrum herbicide glyphosate。there were two distinct epsps genes: epsps1 and epsps2. and they had a high homologious with others epsps genes. The result of experiment suggest the expression of gene epsps1 maitained invariance in the stress of glyphosate, While expression of gene epsps2 in stress of glyphosate of glyphosate became 1.8 to 2.3 times than expression of non-glyphosate induced. we consider that it is a stress response of plant when it is in the stress of glyphosate。
In this paper, the type of basic media and the contents of plant growth substances were investigated by orthogonal design experiment, and also the effects of different culture conditions on the growth of suspension cells and the accumulation of total flavonoids in Eucommia ulmoides were studied. The results showed that B5 medium supplemented with 0.5mg/L NAA, 0.6mg/L 6-BA and 30g/L sucrose, at initial pH 5.0-5.5, 20g (FW)/L inoculation quantity and 110 rpm of rotation speed was a preferable culture conditions for E. ulmoides suspension cells growth and flavonoids synthesis. The results of metabolic kinetics analysis for E. ulmoides cell suspension culture showed that the logistic and Luedeking-Piret equations can be used for describing the kinetics of cell growth, sucrose consumption and flavonoids production during the process. The maximum specific growth rate ( m), the actual growth yield based on sucrose (YG) and maintenance coefficient (m) were 0.417d-1, 0.619g/g and 0.0206g/(g·d-1) respectively. All these outcomes in this paper could give a basis for establishing the suspension cell culture of E. ulmoides and production of the natural active components in large-scale.
The SMA modified PVDF membrane were prepared by the copolymerization of styrene with maleic anhydride on the surface of PVDF membrane in the supercritical carbon dioxide. Free β-galactosidase was immobilized on the membranes by the formation of amido bonds between the amino groups of the enzyme and the anhydrides on the surface of the membranes. The effects of immobilization on the properties of the immobilized β-galactosidase were studied. The immobilized membrane showed the highest activity when β-galactosidase was immobilized for 6h under the condition of pH 8.2, 4 C, and enzyme/membrane 1:10. The activity of immobilized enzyme and protein binding capacity reached 13.5U and 68.2 g/cm2 membrane. The specific activity of immobilized enzyme could reach 280.0U/mg protein. The relative activity was 89.0%, compared with the free β-galactosidase. The immobilized enzyme had higher optimum temperature（55oC） and pH（7.8） compared with that of free β-galactosidase and showed excellent operational and storage stability.
The fermentation conditions of alkaline lipase producing by Pseudomonas cepacia PCL-3 were optimized. Based on the analysis of single factorial experiments, dextrin was the most suitable carbon source， peptone and urea were the suitable compound nitrogen sources among the examined materials. Three significant factors (urea，inoculum and initial pH) were selected from the eight factors related to lipase production by Plaekett-Burman method, and were further optimized with response surface analysis. And then， steepest ascent procedures were applied to define the optimal response region of the three factors. The obtained optimal conditions were urea 0.15％, inoculum 3.05% and initial pH 8.38, under which conditions, the enzyme activity was improved from 25.37 U/ml to 48.88 U/ml, enhanced 1.93 folds. Starting from the flask conditions, the highest lipase activity of 47.69U/mL was achieved by batch fermentation in a 10 L fermentor after 52 h of the cultivation
Benzoate dioxygenase gene（benABC）cluster was amplified from Pseudomonas sp.B3-1 DNA in vitro, using PCR techniques and directionally cloned into plasmid pLAFRJ. The recombinant plasmid was transformed into E．coli DH5α by electroporation, then mobilized into Pseudomonas sp.B3-1 by triconjugation. Selecting for the medium with Amp and Tc bioantics, the recombinant strain named Pseudomonas sp.B4 were obtained. After optimizing the fermentation conditions of this strain for producing catecho1，the optimized conditions were sodium benzoate 6.0 g/L, polypepton 2.5 g/L, pH6.0, incubated at 32℃and shaken at 200 rpm for 36 hour. Under these conditions，the catechol yield improved about 20% compared with the wild strain B3-1 and reached 0.7 mg/ml.
Nerve growth factor (NGF) was firstly discovered as a member of neurotrophin family, and the research and development of NGF has been lasting more than fifty years since its discovery. To this end, a two-step and high -yield chromatographic method which consists of cation ion-exchange chromatography and reversed-phase chromatography was reported to isolate recombinant human beta nerve growth factor (β -NGF) secreted by constructed Chinese hamster ovary cells (CHO/dhfr-) from the culture media. Through the process of purification, the purity of protein which was determined by SDS-PAGE and RP-HPLC has reached to 95%, and the recovery of β -NGF routed by RP-HPLC could be 70%. Furthermore, the biological activity of final purified protein evaluated by PC12 cells and dorsal root ganglia (DRG) exhibited the same performance as the standard protein of β -NGF bought from Sigma, which indicated that there is no loss of biological activity through the isolation process. This study's conclusion suggested that an economical isolation method of recombinant human β -NGF could be practiced on the industrial process of purification.
Abstract Kosteletzkya virginica oil as raw materials was used to produce biodiesel. The single factor experiment and the orthogonal experiment combination design were adopted to study the effects of such factors as reaction temperature, catalyst dosage，mole ratio of methanol to Kosteletzkya virginica oil，reaction time and the rate of rotation on oil transesterification ratio. The results indicated the order of factors that influence the oil transesterification ratio within the experimental range was as follows: the rate of rotation r＞catalyst dosage＞mole ratio of methanol to Kosteletzkya virginica oil ＞reaction time＞reaction temperature. The optimal technological parameters of oil transesterification from Kosteletzkya virginica oil should be as follows: the rate of rotation 1800r.min-1, catalyst dosage 0.6%, mole ratio of methanol to Kosteletzkya virginica oil 6/1,reaction time 60min and reaction temperature 65℃, and under such conditions, the total oil transesterification ratio after transesterification three times is 97.8%.
Objective A real-time PCR method for the detection of Aspergillus nidulans was developed. Methods Multiple nucleotide sequence alignment of 13 Aspergillus species shows high Variation in partial sequence of Glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH gene). Base on the Variation region the primers and TaqMan probes were designed for specific detecting Aspergillus nidulans specie. The specific and sensitivity of the real-time PCR was tested. Results 41 Aspergillus isolates and 12 others genus fungi were used，No cross-amplification was observed. The lowest detection limits of the real-time PCR are proved by the sensitivity testing ，4.03×10－12μg/ml of the A. nidulans DNA template can be amplified successfully. Conclusion Real-time PCR is a time-efficient，specifically method，it is a novel tool for identifying and determining the strain of A. nidulans. This method may be useful for detecting and identifying Aspergillus nidulans specie.
γ-glutamyltranspeptidase was detected from the cultured mycelia of Cordyceps sinensis (CSGT). Km and Vmax of CSGT was 2.54×10-4 mol·L-1 and 0.1808 mol·L-1·min-1 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.
In this article , rapid propagation of Panax ginseng in tissue culture was reported ; the process of morphogenesis through different path from shoots , stems , leaves and seeds as explants obtained regenerated plant . The callus was induced on MS medium supplemented with 4.0mg/L 2,4-D and 0.2mg/L BA .The callus was subcultured on MS medium supplemented with 2.0mg/L 2,4-D and 0.2mg/L KT. Embryogenic callus could be obtained and then lacking 2,4-D we can obtain embryoid. It can grow fast on MS medium with no growth regulators. Plantlets regenerated on 1/2MS. We assume that the origin of somatic embryo of Panax ginseng is from the single-cell. The origin of adventitious buds of Panax ginseng is from callus surface or inner. High sugar and starch involve in its embryoid callus and development. High activities of POD and PPO and protein level were necessary to earlier offer material and energy. The contents of IAA is higher in earlier embryo and ABA is higher in mature embryo. The ratios of ABA/IAA were the development of somatic embryogenesis.
Cyclodextrin (CD) is gradually applied in the nonviral gene vector system, due to its biocompatibility and flexibility of tailing via structural modification, polymerization or supramolecular combination. This paper reviews the ideas and research progress of the CD, its low molecular derivatives, CD polymers and CD supramolecular combination in the field of norviral gene vectros, and discuss their "structure - safety - transfection efficiency" relationships.
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. In this review, we summarize the knowledge that has been accumulated about the principles of miRNAs and target recognition. The currently available computational methodologies and software for prediction of miRNA and their target genes also have been discussed.
Peanut，which provide people vegetable fatty and protein, is one of the most important oilseed and cash crops. Genetic transformation has launched a new era in peanut breeding and germplasm creativity. It offers a direct method of creating varieties that selectively targets gene or a few heterologous traits for introduction into the peanut plant. Great advances have been made in peanut genetic transformation in the past years. This paper reviews the recent progress in genetic transformation of peanut on heterologous gene such as resistance to viruses, insects, abiotic stress, quality improvement and so on. It also summarizes the recent progress and the exploration of genetic peanut on transformational methods including A . t umef aciens mediated, particle bombardment and non-tissue culture.
As a model terpenoid, the ginsenoside is one of Panax ginseng's main effective components. This article has carried on an overview of biosynthetic pathway of terpenoids and the HMG-CoA reductases. The massive research materials indicated that the HMG-CoA reductases is the first key enzyme of the regulation of the mevalonic acid way, which has some reference value to promote the research on the biosynthetic pathway and the regulation of ginsenoside.
As a renewable clean energy, biodiesel has been the subject of much research in recent years owing to its excellent environmental performance. And the bio-enzymatic method would be the direction in biodiesel industrialization process. This paper summarized the progress and application of three biocatalytic approaches for biodiesel production including immobilized lipase, free lipase and whole-cell catalysis. Finally, the challenges of biodiesel industrialization in China are analyzed, and some effective measures are put forward.
Ever from FDA approved the Rituxan in 1997, an Anti-CD-20 chimeric antibody, there have been more than 20 antibodies approved and they have got into more than 56 countries. About half approvied antibodies are curing kinds of cancers and seven of them have got a excellent sale targets to be "Blockbuster Drug".As the cell culture technology improving, the large scale cell expression titer comes from 1g/L to 5g/L. At the same time, the culture scale is coming form 12,000L to 15,000L, even 20000L and it can run several bioreactors at the same time. All these are make the improvement of the antibody purification. The new edition of media, system, chromatography technology and filtration technology can product the protein to several tens kilogram antibody per cycle and tons per year, although the productivity is only several kilograms per cycle many years ago. Also the new combined technology makes a two-step chromatography production protocol come into been. This will highly improve the antibody productivity and save the investment cost. This paper will introduce this new production protocol particularly.
