25 January 2006, Volume 26 Issue 01

    

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研究报告
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  Study on inner structure controllable large volume 3-D scaffolds formation method

  China Biotechnology. 2006, 26(01): 1-5.

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  AbstractObjective: To develop a novel method for preparating large volume threedimensional scaffolds, and controlling the pore structure, uniformity, and interconnectivity of the scaffolds to meet the requirement of pore structure for tissue engineering. Methods: Spherical porogen (a pore generating materials made from sodium chlorate) and bonding reagents (developed by our laboratory) were mixed uniformly, the mixture was put in a polypropylene mold (cylindrical vial with microholes on bottom for solution leaving off), the mold containing mixture was centrifuged at a chosen force for 5min to get ride of unwanted solution, then the bonded porogen assembly was cut into halves with a razor blade after completely dried in dessicator at room temperature. The upper, middle and bottom sections of assemblies were observed by optical microscope to detect the bonding uniformity and degree. A chosen polymer (PDLLA) was dissolved in chloroform to prepare a solution of a desired concentration, the polymer solution was cast onto the assembly, and additional casting was repeated after the solvent was evaporated. The dried porogen/polymer discs were removed from the mold, and the top and bottom layers were cut away to obtain flat surfaces. The discs was immersed in distilled water to remove porogen, dried under vacuum, then the scaffolds was harvested for structure characterization. Results: Optical micrographs clearly displayed that the porogen spheres remained spherical appearance and the bonding areas between spherical particles were homogeneous in large dimensional bonded assembly, and there was no statistical difference in bonding extent. In addition, the bonding extent could be controlled by variety of bonding reagent concentration as well as centrifugal force. For instance, the bonding extent was 33.78±5.56 (134) μm and 42.89±5.87 (132) μm respectively, when reagent concentration was 20% and 40% with centrifugal force of 161g and porogen size range of 100~220μm. SEM imagines revealed that the pore size and the diameter of interconnected openings of the scaffolds equaled separately to porogen size and bonding extent in bonded assembly. For example, the diameter of openings was 33.34±5.21(12)μm, when the bonding degree was 33.78±5.56 (134)μm in bonded assembly. Conclusion: With the newly developed bonding reagent and bonding technique, large dimensional biodegradable polymer scaffolds with high porosity as well as with controllable and homogeneous innerstructure can be formed, the pore size of scaffolds as well as diameter of openings between pores can be controlled by adjusting the porogen size and bonding degree in bonded porogen assembly. In addition, the resulting completely interconnected scaffolds have implications for facilitating cell migration, nutrients or waste exchange, abundant cellcell interaction, and potentially improved neural and vascular growth within tissue engineering scaffolds.
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  Comparative study of human umbilical cord vein and adult bone marrow derived mesenchymal stem cells

  China Biotechnology. 2006, 26(01): 6-10.

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  Mesenchymal stem cells (MSCs) are promising candidates to develop new cellbased therapeutic strategies and bone marrow represents the main source of MSCs for experimental and clinical application currently. How to obtain enough MSCs is the biggest challenge faced by the experimental and clinical application. The difference of morphology, growth pattern, immunophenotype and multilineage differentiation of MSCs derived from human umbilical cord vein and their multiple differentiation capacity were investigated. The results showed that MSCs derived from human umbilical cord vein and normal adult bone marrow were similar in biological characteristics. They not only grew in fibroblastlike cells mode but had great expansion potency and multilineage differentiation ability in vitro. The MSCs derived from human umbilical cord vein may be an excellent alternative source for experimental and clinical application.
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  Prokaryotic Expression of Integrase Enzyme of HIV-1 and The Function Study of Intergrase Protein

  China Biotechnology. 2006, 26(01): 22-26.

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  The pol gene of HIV1 encodes mainly three enzymes: reverse transcriptase (RT), protease (PR) and integrase (IN). Currently, FDA approved drugs targeting RT and PR are available and administered in various combinations, while no antiIN drug was approved. HIV1 integrase is an essential enzyme for the viral replication and an interesting target for the design of new pharmaceuticals for multidrug therapy of AIDS. The 288 amino acids of IN (32kDa) recognizes specific sequences in the long terminal repeats (LTRs) of the retroviral DNA. The IN protein catalyzes the 3′processing step and the 5′strand transfer step reaction in vivo, which was called integration and this reaction could be analysed by ELISA Assay in vitro. It has been reported that F185K and C280S mutations of HIV1 integrase would improve the enzyme solubility, and the catalytical activity of the enzyme was the same as that of the wildtype enzyme in vitro. In order to build the platform of screening inhibitor against integrase of HIV1 virus, the IN enzyme was expressed and the function of integrase protein was assayed. The cDNA of clade B HIV1 genome was used as a template, overlapped PCR was used to construct site mutagenesis of F185K/C280S and NdeI/Xho I restriction sites were brought in. The PCR product was cloned into the prokaryotic vector pET28a(+) to form a recombined plasmid, transferred into the host cell E.coli(BL21 DE3). The recombined clones were identified by PCR and Nde I/Xho I digestion .The positive plasmid was sequenced, and the successfully recombined plasmid in the host cell was induced by IPTG. The expressed IN protein was purified by the Co+ affinity chromatography column and SDSPAGE was used to analyze the molecular weight and specificity. In addition, ELISA assay was used to analyze the function of the recombined IN protein. The recombinant protein was soluble, and expressed highly and stably in E.coli. The molecular weight of the expression product was identical to the expectation. The IN protein was proved to be functional in 3′ processing and 5′strand transfer by ELISA. It will be helpful to build the platform of screening inhibitors against HIV1 integrase.
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  Construction and expression of a fusion protein composed of cecropina-melittin and haFGF

  China Biotechnology. 2006, 26(01): 27-37.

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  A new type of fusion protein was composed of cecropin Amelittin peptide CA(17)M(512)NH2 and human acidic fibroblast growth factor ( haFGF ). The DNA fragment coding the fusion protein was cloned into the vector pET3c and constructed the recombinant expression vector pETaFCM.The recombinant plasmid was transformed into E.coli BL21(DE3). After induced with IPTG for 4h, the expression of the fusion protein was detected by 12% SDSPAGE and Western blot. The fusion protein was about 17% of total cellular protein and expressed as inclusion bodies. The fusion protein, which purified by affinity chromatography, was cleaved by Xa factor. The cecropin Amelittin was purified by gel filtration. The mitogenic activity of the fusion protein detected by MTT was about 1.471×106 IU/mg. The cecropin Amelittin peptide had the antibacterial activities against several bacteria including E.coli，Salmonella, staphylococcus aureus, Bacillus subtilis and Pseudomonas aeruginosa etc.
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  Activity Study on Antibacterial proteins Isolated

  China Biotechnology. 2006, 26(01): 33-37.

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  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. cationexchange 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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  Expression of Key Enzymes in Glycerol Synthesis of Candida Glycerolgenesis in Saccharomyces Cerevisiae W303-1A

  China Biotechnology. 2006, 26(01): 38-41.

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  Glycerol is an ideal osmotolerant medium. Two genes of GPD and GPP encoding glycerol-3phosphate dehydrogenase and glycerol-3phosphate phosphatase, key enzymes in glycerol synthesis, were cloned from Candida glycerolgenesis WL2002－5 using PCR method. In Escherichia coli JM109, enough GPD and GPP genes were amplified using Tvector, furthermore, expression plasmids pYX212－GPD containing GPD gene and pYX212－GPP containing GPP gene were constructed, which were introduced into Saccharomyces cerevisiae W303－1A using LiAc transformation method successfully. Primary results showed that the biomass of pYX212－GPD/S. cerevisiae W303－1A was higher than those of pYX212－GPP/S. cerevisiae W303－1A and the wild type during the fermentation. After 72h fermentation, introduction of GPD gene could increase glycerol production to about 12mmol/L, while GPP gene had little effect on glycerol production (only 4mmol/L) in comparison with the wild type.
技术与方法
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  Evaluation of Two Different Sample Labeling Methods on Background Signal Intensities for 60 mer Oligonucleotide Microarrays

  China Biotechnology. 2006, 26(01): 42-45.

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  The effects of two different sample labeling methods on background signal intensities for highdensity 60mer oligonucleotide microarray were investigated. Peripheral blood samples from five disease and five control subjects were collected. Total RNA targets from peripheral blood mononuclear cells were extracted and labeled with RDPCR protocol, which were hybridized to Agilent Human 1B oligonucleotide microarrays in a twocolor comparative format. The positive control targets were labeled with the directly incorporated fluorescentlylabeled dNTP labeling. The SPSS program was performed to test normality of the dataset, variance homogeneity between the groups, coefficients of variation (CV) and analysis of variance. The results showed that the background signal intensities of Cy3 channel were higher than those of Cy5 channel. The differences of background signal intensities between the RDPCR approach and the directly incorporated fluorescentlylabeled dNTP labeling were extremely significant (PCy3<0.01, PCy5<0.01). It is concluded that the RD-PCR is a potential and useful sample labeling method with lower background signal intensities for studying highdensity long oligonucleotide microarray.
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  The compare of structure disruption of BSA by ultrasonication and French Press

  China Biotechnology. 2006, 26(01): 46-49.

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  To investigate the structure disruption of BSA (1mg/ml, dissolved in PBS) induced by ultrasonication and the French press. The BSA solution was passed through the French press and received ultrasound irradiation, and then detected by HPLC（Highperformance liquid chromatography）,DLS（Dynamic Light Scattering）,CD（Circular Dichroism）and nondenaturing SDSPAGE. Detection results showed that BSA was polymerized after ultrasound irradiation and the polymerization can be reduced by adding mannitol (free radical scavenger). This means that the free radical play an important role in this process. However, the BSA passing through the French press for several times wasn’t polymerized, and the secondary structure was somewhat destroyed. These results suggested that ultrasound irradiation and French press destroy the molecular structure in different manners, so that the suitable cell lyses methods should be selected according to the characteristics of the protein.
研究简报
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  Expression and Bioactivites of Recombinant Glucagon-like Peptide 1

  China Biotechnology. 2006, 26(01): 50-55.

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  The synthesized fulllength hGLP-1 gene was cloned into pET-32a(+) to get the recombinant plasmid pET32-GLP-1, which could express a fusion protein containing thioredox， hexahistidine， and rhGLP-1 consecutively. The recombinant plasmid containing hGLP-1 was transformed into E.coli BL21 (DE3) and expressed by IPTG induction. The fusion protein was purified from lysates with Ni·IDA His·Bind affinity chromatography. rhGLP1 with the purity of 90% was achieved after enterokinase digestion, Ni·IDA His·Bind affinity chromatography again, then was concentrated by ultrafiltration. The purified rhGLP1 showed a single band on IEF gel with an isoelectric point between pH5.2 and pH5.85. ESI mass spectrometry showed that the molecular weight was 3355.0kDa as expected. rhGLP-1 was digested with trypsin followed by mass analysis and the peptide mapping produced two expected fragments with the molecular weights of 2097.7kDa and 1005.5kDa, respectively. The purified rhGLP-1 also showed obvious biological activity for both lowering plasma glucose and stimulating insulin secretion in mice.
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  The Changes of Saccharomyces cerevisiae's Glutathione and Ergosterol Under High Pressure

  China Biotechnology. 2006, 26(01): 56-59.

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  The growth changes of glutathione (GSH) and ergosterol in Saccharomyces cerevisiae (CICC1447 and CICC1339) were detected under 0.5Mpa pressure with compressed highpure air (O2∶N2=21∶79). The results showed that logarithmic phases of the two strains were delayed; their biomass and special growth rate were lower than those of control sample (0.1MPa) and the double time were prolonged under 0.5MPa. High-pressure could increase the content of GSH obviously, compared to ambient atmosphere control samples. When the holding time was 3h, the content of GSH and ergosterol in CICC1447 increased 42.6% and 20.1%, respectively. However, the content of GSH in CICC1339 increased 58.7% when the holding time was 6h, while ergosterol content reduced. The results indicated that different yeast strains have different stressresponse mechanism to copy with highpressure shock.
综述
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  Gentic Manipulation on Biosynthesis of Terpenoids

  China Biotechnology. 2006, 26(01): 60-64.

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  Terpenoids constitute a chemically diverse family of naturally occuring compounds with pharmaceutical significance, such as artemisinin and taxin. They are synthesized in many kinds of microorganisms and plants with very low yield. Metabolic engineering, modifying metabolic pathways of terpenoidssynthesized cells by recombinant DNA techniques, has been developed and applied to improve the production of terpenoids, as well as create a novel biosynthesis pathway of terpenoids in terpenoidfree organisms. The biosynthetic pathway of terpenoids was sumed up, and recent progress of metabolic engineering aiming at raising content of terpenoids in microorganisms and plants was described.
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  Progress on the study of sex-specific membrane proteins

  China Biotechnology. 2006, 26(01): 65-68.

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  The expression of specific genes in sex chromosomes is the basis of sexspecific membrane protein in mammalian spermatozoa. The gene expression products are shared among spermatozoa through intercellular bridges, however, the phenomena of male transmissionratio distortion and sex ratio distortion proved that differential proteins exist between X and Y spermatozoa. In addition, the existence of sexspecific proteins was confirmed by the separation experiment of X/Y chromosome bearing spermatozoa and the detection result of sex specific proteins. At the same time, it was also confirmed that the difference of the sexspecific protein is weak . The advance of separation techniques as well as the integration and optimization among these techniques has made it possible to separate sexspecific membrane proteins in mammalian spermatozoa.
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  Biosorption of Heavy metals by Saccharomyces cerevisiae: a review

  China Biotechnology. 2006, 26(01): 69-76.

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  Heavy metal pollution has become one of the most serious environmental problems today. Biosorption, regarded as a costeffective biotechnology for treating heavy metal of low concentration in wastewater, has not been utilized at large scale successfully. It’s helpful to increase the knowledge of biosorption mechanism and decreasing the costs of biosorbents for the biosorption application. The yeast of Saccharomyces cerevisiae is an ideal biomaterial to be used for exploring the mechanism and for actual utilization because of its unique characteristics in spite of its relatively mediocre capacity of metal uptake to other fungi. The yeast can grow easily in cheap media, and is widely used in food and beverage manufacture. It’s also a safe byproduct in large quantity as a waste of the fermentation industry, and easily manipulated at molecular level. The metal uptake specifically by S. cerevisiae was addressed. Firstly, it was discussed to use dead or live cells in biosorption . The yeast can absorb toxic heavy metals (Pb, Hg, Cd, etc), precious metals (Au, Ag, Pd, etc) and radionuclides (U, Am, etc). Secondry, metalbinding capacity of various heavy metals by S. cerevisiae in different conditions were compared. Lead and uranium, for instances, can be effectively removed from dilute solutions, while copper is not easily removed. Thirdly, various mechanism of metal uptake by S. cerevisiae were summarized in details according to the position in which metals are located. Metal uptake process is influenced by the ratio of the initial concentration of metal ions and the concentration of biomass. Cellular wall and its components are important for metal uptake. Functional groups for metallic ion fixation have been identified. Uptake is typically accompanied by ion exchange and complexation, sometimes with precipitation (for Pb) and redox (for Au or Ag). Intracellularly accumulated metal is associated with the cell membrane, vacuole and GSH, but may also be bound to other cellular organelles and biomolecules. The equilibrium and kinetic models used in the metalyeast biosorption systems were also introduced. In most cases, classic Langmiur model and Freundlich model, widely used to describe single metal biosorption system of equilibrium, fit the experimental data very well. Pseudosecond order equation is often employed to describe biosorption process by S. cerevisiae. Finally, futher researches in metal biosorpiton by S. cerevisiae were proposed.
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  Studies on RAPD Molecular Marker Applied in Edible Fungi

  China Biotechnology. 2006, 26(01): 77-80.

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  RAPD molecular marker was widely applied to the studies of edible fungi due to it’s simplicity , rapidity and economy. The principle of RAPD molecular marker and its applications to edible fungi were summarized. The applications of RAPD to edible fungi were introduced in species and parental strain identification,genetic diversity, gene clone, gene isolation and the construction of gene linkage map. RAPD molecular marker provids a powerful tool for the studies of edible fungi.
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  The Development of Red Recombination Applied in Knockout of Bacteria Chromosomal Gene

  China Biotechnology. 2006, 26(01): 81-86.

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  Traditional recombination technology of bacteria chromosome and its limitation were introduced. The definition of Red recombination technology is put forward: a method of homologous recombination between foreign linear DNA and the target gene in chromosomes mediated by λ phage Red system. The linear DNA referred here is general PCR product or oligonucleotide, which has a 36~50bp homologous sequence with the target gene in chromosome at both flanking. Red recombination technology leaves out the in vitro DNA restriction enzyme digestion and link process, which makes the knockout and alternation of target gene in bacteria chromosome relatively easier, and becomes an effective method to exploring genes and constructing new strains gradually. The gene inactivation and alternation method aiming at bacteria chromosome applied to Red recombination system was summarized by the structure element, action mechanism, and strategy of recombination, advantage and developing prospect. The Red system includes three genes: bet (akaβ), exo and gam (aka γ). Exo is a 5′→3′ exonuclease, which degrades the 5′ ends of linear DNA molecules. Bet is a singlestranded DNA binding protein that binds to the single stranded 3′ ends generated by Exo and promotes annealing to complementary DNA. Gam binds to the host RecBCD complex and inhibits its exonuclease activity. Red recombination system may be constructed in such plasmids as pKD20 and pKD46 or in chromosome of bacteria. Most bacteria are not readily transformable with linear DNA because of the presence of intracellular exonucleases that degrade linear DNA. But when bacteria cells are transformed with pKD20 or pKD46 plasmid, or integrated with a detective λ prophage, Red recombination enzymes may be expressed in host cells, which make linear DNA with 36~50bp extensions that are homologous to both flanking of target genes transform E.coli readily and knockout or alternate target gene. The Red recombination method is not only useful in chromosomal gene inactivation in E.coli, but also in other bacteria or virus, such as Salmonella, Shigella flexneri and virus HaSNPV. With the proceeding research, Red system will be applied for more and more purposes, and contribute a lot for gene improvement and gene function investigation in the coming Postgenome Era.
产业发展
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  The Globe Development Of the Biopharmaceutical Industry

  China Biotechnology. 2006, 26(01): 92-96.

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  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.