25 May 2006, Volume 26 Issue 05
    

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    研究报告
  • China Biotechnology. 2006, 26(05): 1-6.
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    The secondary structure of Capsid protein was predicted by the methods of Chou-Fasman, Garnier-Robson and Karplus-Schultz based on the sepuence of capsid protein gene of Swine Vesicular Disease Virus (SVDV) and hydrophilicity, surface probility plot and antigenic index for capsid protein were obtained by the methods of Kyte-Doolittle, Emini and Jameson-wolf respectively, Combining the results according to these methods, the B cell epitopes for capsid protein of SVDV were predicted. The results showed that there are much flexible region such as coil region and turn region in capsid protein of SVDV and there are more predominant B cell epitopes in VP1 than in VP2 and VP3. This study would be helpful for identification of B cell epitopes for capsid protein using experimental methods and research of reverse vaccine of SVDV.
  • China Biotechnology. 2006, 26(05): 7-10.
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    Objective: To obtain the bFGF mimic peptide binding to FGFR via phage display, and to provide the base for developing peptide agonist of bFGF. Methods: Using Balb/c 3T3 cells as the target cells and COS-7 cells as the subtractive panning, the phage display heptapeptide library was biopanned for 4 rounds to obtain the single phage clones. The affinity and the specificity of the clones were assessed by ELISA.DNA sequencing was applied to further analyze the positive clones. Results: Twelve positive clones were selected from the enriched phages. A group of hydrophobic peptides containing a conserved motif, PR, was identified. Conclusion: Two bFGF mimic heptapeptides binding to FGFR were selected, which may be used as the candidates for bFGF agonist.
  • China Biotechnology. 2006, 26(05): 11-16.
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    The expressing cassette, LoxP-CMV-gpt-IRES-LoxP( about 2.9kb), was amplified by PCR from a plasmid, pIRES-gpt, by use of the primers , which contained the loxP sites in 5' terminals, respectively. The loxP sites were designed into primers by the software of Primer primer 5.0. Then the cassette was cloned into the site of BalⅠin pBUS10 to obtain pUS-gptIRES(L). The sequencing analysis for pUS-gptIRES(L) indicated that two loxP sites with the same direction were correctly inserted into pUS-gptIRES(L).The gpt gene in pUS-gptIRES(L) was replaced by a fragment including the full length GFP gene as well as SV40 poly A sequence to get pUS-GFPIRES(L). pUS-GFPIRES(L) was transiently transfected into CHO cell lines, and then the green fluorescence could be seen, the result showed that GFP gene could be expressed correctly. Moreover , pUS-GFPIRES(L) was transfected into the CEF infected MDV CVI988 strain and recombinant virus was selected by the green fluorescence. In vitro, the growth curve of virus showed the characteristic of recombinant virus was the same as that of CVI988. These results give the basis for further studying the characteristic of MDV in vivo and the application of the Cre/LoxP system to MDV genome.
  • China Biotechnology. 2006, 26(05): 17-21.
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    In this study, RT-PCR were employed to amplify the cDNAs of HA gene and NA gene of A pigeon avian influenza isolate with two pairs of primers, the cDNAs were cloned to pMD18-T and then sequenced. The results of sequence analysis shows that the cloned HA gene consists of 1707 nucleotides and encodes 568 amino acids residues and contains whole open reading frame. There are 7 Potential glycosylation sites and 6 basic amino acids(R-R-R-K-K-R) insert at the cleavage site in HA of the influenza virus isolate which is a symbol of highly pathogenic avian influenza , amino acids of receptor-binding site are YWIHELY, amino acids of left edge of receptor-binding site are SGVSSA, amino acids of right edge of receptor-binding site are NGQSGR; The cloned NA gene consists of 1350 nucleotides and encodes 446 amino acids residues,There are 3 Potential glycosylation sites in NA of the influenza virus isolate.
  • China Biotechnology. 2006, 26(05): 22-26.
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    According to bias in codon choice of Pichia pastoris ,The phytase phyA gene from Aspergillus niger N25 was mutated without changing its amino acid sequence. The expression plasmid pPIC9k-phyAm was constructed and transformed into GS115 strain. Positive clones,of which the chromosomes were integrated with phyA gene,were identified by the phenotype and PCR. SDS-PAGE analysis suggust that the size of enzyme protein of the expression product was about 70.15KD.Southern blotting analysis to the yeast transformants showed that phyA gene was intergrated into the chromosome genome. The phytase activity of PP-NPm-4-4 with codons optimized reached 136000Uoml-1 in malt wort culture medium after being induced with 36h, which was the 2.8 times of the original strain PP-NPm-8.
  • China Biotechnology. 2006, 26(05): 27-32.
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    OBJECTIVE To facilitate the functional analysis of chromosomal genes and their products, we adapted the recombineering technique to epitope tagging of chromosomal genes of Y. pestis. METHODS The epitope tag was generated by primer annealing and then fused with resistance gene by fusion PCR. The epitope-resistance cassette was inserted into pBluecript, resulted in the template plasmid, pBS-MH. The tagging cassette for rpoS was obtained by PCR amplification from pBS-MH with primers containing homology specific to the target gene. PCR products were transformed into recombination competent cells and recombinants were selected. PCR and DNA sequencing were used to confirm the correct tagging event. The expression of the tagged protein was detected with Western blot by using monoclonal antibody to the epitope. RESULTS The template plasmid containing fusion of epitope and resistance gene was successfully constructed. The sigma factor gene, rpoS, was tagged with a myc-his tag at the COOH terminus. Expression of the tagged rpoS was successfully detected indirectly by the antibody against His tag. CONCLUSION The chromosomal gene tagging by recombineering technique represents a powerful tool in the functional study of bacterial genes and their products.
  • China Biotechnology. 2006, 26(05): 33-37.
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    Recombinant human BMP-2 was compounded with chitosan/gelatin/hydroxyapatite(HCG) scaffold and the complex was sterilized by 60Co radiating. Osteoblast isolated from cranial bones of newborn rat was primary cultured and seeded onto the complexes. 3 days after culturing, scanning electron microscope(SEM) was applied to detect the compatibility of the cell with the complex. SEM showed osteoblast attached closely with the complex and grew well in its pores. Then the complexes with osteoblast modification were implanted into athymic nude mice subcutaneously. 8 weeks after implantation, X-ray photograph and histological observation were applied to detect the bone formation of the complexes. Under X-ray a high-density areas consistent with the shape of the implanted complex could be seen. Histological observation also proved there was bone formation in the interspace of the complex. A conclusion was drawn that rhBMP-2 compounded HCG scaffold had good osteogenesis ability in vivo.
  • China Biotechnology. 2006, 26(05): 38-43.
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    Objective: To study expression of the gene of n-6 fatty acid desaturase fat-1 in human breast cancer cell, composition change of fatty acids of cell membrane, and effect of the gene on apoptosis of breast cancer cell. Methods: Construct recombinant adenovirus vector (Ad.GFP.fat-1) containing fat-1 gene, the recombinant adenovirus was produced in 293 package cell, then it infected the breast cancer cells MCF-7; total RNA of the cells was isolated and hybridized with antisense RNA probe of fat-1 mRNA by Northern to analyze the expression of fat-1 in MCF-7; the effect of fat-1on the proliferation of MCF-7 cell was analyzed by MTT method,apoptosis of the cells were detected by apoptosis kit; content of n-6 PUFAs/n-3 PUFAs was analyzed by Gas Chromatography. Results: The proposed recombinant virus was got through DNA recombinant technique; fat-1 gene effectively expressed in human breast cancer cell MCF-7; the fat-1 mRNA band appeared 2 days after infection of virus Ad.GFP.fat-1; compared with the control cell (Ad.GFP), proliferation of MCF-7 cell was markedly inhibited by the gene fat-1( 23%, p<0.05) and apoptosis of the cells was promoted; moreover, fat-1 gene decreased ratio of n-6 PUFAs/n-3 PUFAs of MCF-7 cell membrane. Conclusion: The fat-1gene transfer mediated by adenovirus could heterologously express in human breast cancer cell MCF-7, inhibit proliferation of the cell. The mechanism analysis shown that fat-1 decreased ration of n-6 PUFAs/n-3 PUFAs of cell membrane.
  • China Biotechnology. 2006, 26(05): 44-48.
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    Change in the neuronal nitric oxide synthase (nNOS) is a key factor in pathogenesis of a number of clinical disorders. In order to find specific molecule with ability to modify nNOS activity, the nNOS protein fragment contains the sequence around Calmadulin binding region was expressed in E. Coli. The purified recombinant protein was used as target protein and a 12-mer phage-displayed peptide library was screened to identify potential nNOS inhibitor. 10 phage clones were shown to specifically inhibit nNOS activity. DNA sequencing of the clones revealed a common amino acid sequence KPHHHPMPPQVS, we named this sequence NPI. The NPI was synthesized and showed inhibition effect on activity of freshly prepared nNOS from mice brain (inhibition rate is 25% ~ 35%). Howerever, to nNOS that lost part activity , the peptide recovered the activity in a dose-dependent fashion. Our results suggest an approach to develop new drugs to treat diseases based on nNOS disorder and understanding the process of nNOS in vivo.
  • China Biotechnology. 2006, 26(05): 49-53.
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    Objective To obtain for the high expression of Herpes Simplex Virus Type 1(HSV-1)Glycoprotein D gene. Methods The Herpes Simplex Virus Type 1(HSV-1)Glycoprotein D(gD1) gene fragment containing dominant antigen epitopes s confirmed by computer analysis was cloned by PCR technical and inserted into plasmid vector pTrxA, then the recombinant plasmid was transformed into Rosetta 。The expressed product was analyze by SDS-PAGE.  Results 930 bp gene fragment was amplified by PCR as anticipated. Nucleotide sequencing showed a 100 % homology with that of the published sequence in GenBank. The molecular weight of the expressed protein was about Mr48000(Dalton) , Western blotting indicated that the antigenicity of the protein was good. Conclusion The plasmid pTrxA-gd1 was constructed and a high efficiency expression of the gd1 gene from Herpes Simplex Virus Type 1(HSV-1)strain was made. The expressed product shows a good antigenicity.
  • China Biotechnology. 2006, 26(05): 54-57.
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    Objective To study the expression and the function of survivin. Results The expression of survivin in embryo spleen, embryo kidney, embryo liver and in many cancer tissue was determined by RT-PCR. Construct the engineered Escherischia coli expressing human survivin and identify the expressed human survivin by Westen-blot. The combination activity of Survivin and RhSmac was determined in vitro. L929 cells transferred with Survivin can survive longer than which transferred with BSA.
  • China Biotechnology. 2006, 26(05): 58-62.
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    Objective Using the recombinant staphylokinase there is a lack of ten amino acids in the N end to construct engineering bacterium expressing soluble protein. Research the content of recombinant staphylokinase gained at different induced conditions and the way of purification. Methods The aim protein of recombinant staphylokinase induced using the activation and cultivation of bacteria and its content measured with SDS-PAGE. The protein was purified with chromatography. Result The content of induced recombinant staphylokinase was about 50% of total protein. After purification, the rate of recollected aim protein was 60% and its purity was over 99%. Conclusion The engineering bacteria of induced aim protein of recombinant staphylokinase were successfully constructed. The high expression and purified protein was acquired.
  • China Biotechnology. 2006, 26(05): 63-68.
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    In this paper the influence of cell preparation methods of Ralstonia solanacearum on the cell viability and cell surface properties was studied. The results showed that the batter method was as follows: bacterial cultures harvested by centrifugation at 5000×g for 10 min, washed and centrigugated twice, resuspended in millipore water (>16MΩ) ). Using this method could not only avoid the interference of medium, but also keep the cell viability and cell surface properties.
  • 研究简报
  • China Biotechnology. 2006, 26(05): 69-73.
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    A prokaryotic expression plasmid containing VIP (vasoactive intestinal peptide) and sTNFRⅡ(soluble tumor necrosis factor receptor II ) genes was constructed. The sTNFRⅡ was cloned by PCR by using special primers which contained VIP gene ORF and a linker in its forward primer. The amplified fragment was inserted into the expression vector pET32a between Bam HI and Hind Ⅲ restriction sites. Transformed E coli DH5 by pET32a-VIP- sTNFRⅡexpressed the fusion protein. After being identified, the protein was purified by ion exchange chromatography and by hydrophobic interaction chromatography. The reconstructed protein showed high bio-activity and could be applied for further use.
  • China Biotechnology. 2006, 26(05): 74-80.
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    The complete genomes of more cyanobacterial strains have been completed for sequence, and genetic engineering of cyanobacteria has evolved in the post-genome era. Since Kaneko and colleagues had completed the sequence for the complete genome of Anabaena sp. PCC 7120 in 2001, functions of some genes in this genome, including fructose-1,6-bisphosphate aldolase (FBA) gene, have been predicated using the method of bioinformatics. However, little information is available regarding whether this gene can encode FBA and its product characteristics of related enzyme. Here, to explore this information, the predicted II-FBA gene-encoding region in Cyanobase database was cloned by PCR method and then ligated into pET-32a to generate the expression vector, pET-FBA-II. The results of SDS-PAGE indicated that the expression level of the expected target polypeptide was approximate 23.4 percent as compared to total protein and the molecular weight is about 40 kDa as compared to the protein molecular marker. The results of enzyme activity analysis showed that the activity of II-FBA was ~11.8 U per mg protein and owned a standard activity of II-FBA. Taken together, the results not only prove the functional prediction of this II-FBA gene from the Cyanobase database, but also provide the important conditions for further studying its physiological and biochemical characteristics and functions of the gene expression product.
  • China Biotechnology. 2006, 26(05): 78-80.
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    Counting the number of bacteria、antinomyces and fungi in the soil after different years of planting Bt cotton.The results showed as follow:(1)the trend of influence of planting Bt cotton to the bacteria、antinomyces and fungi number in the soil is basically similar.(2)After one year of planting Bt cotton the number of bacteria、antinomyces and fungi in the soil would increase. With 4 successive years of planting,it reaches to the peak and then begins to fall. After 7 successive years the number even is close to that of 1 year.(3)After one year of planting Bt cotton and then one year of non-Bt cotton,the number of microbes in the soil is smaller than that of planting one year of Bt cotton which is close to the number of planting non-Bt cotton. So, there are some effects on the number of microbes in the soil, while planting Bt cotton for more years.
  • China Biotechnology. 2006, 26(05): 81-84.
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    Three enzymes,pepsin,trypsin and catalase were selected to research the effects and mechanisms of ultrasound on protein.A series of experiments, taking advantage of the change of the biological activity of their solutions under sonication as the index, were done to figure out the main factors influencing the effects of ultrasound on protein. The results indicate that there exist some rules and mechanisms related with the effects of ultrasound on the three enzymes. The three enzymes displayed different mode or degree of activity change under sonication.The degree of the destruction of ultrasound on them rises with the increase of the ultrasound output power and the extension of the time.In addition, the effects of ultrasound on the three enzymes vary with the concentration of their solutions,which means that the degree of the destrcuction of ultrasound could be reduced by adjusting the concentration.The mannitol,a strong free radical scavenger,and Tween-80,a kind of non-ionic surfactant,could protect the activity of the three enzymes under sonication,implying that free radicals produced via the thermal dissociation of water induced by ultrasound cavitation and the cavitation bubbles disturbing the hydrophobic conditions around the enzyme molecules play important roles during the process of the destruction of sonication.However,for different enzyme,different factors dominate the critical role.
  • 综述
  • China Biotechnology. 2006, 26(05): 85-88.
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    Disuse-ralated osteopenia is mainly due to decrease of bone formation which relates closely to the low level of osteoblast activity. Mesenchymal stem cells which are multipotent cells present in bone marrow can differentiate into osteoblast. The factors which regulate the osteogenic differentiation of bone marrow mesenchymal stem cells were introduced. This will increase the understanding and aid in the prevention of bone loss.
  • China Biotechnology. 2006, 26(05): 89-92.
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    Adipose-tissue mesenchymal stem cells, is one kind of multipotent stem cells, can differentiate into adipogenic, osteogenic, chondrogenic, myogenic cells and so on in vitro in lineage-specific culture media. Human adipose tissue is plentiful, easily harvested in large quantity with little patient discomfort. adipose-tissue derived mesenchymal stem cells may be an alternative stem cell source for mesenchymal tissue regeneration and engineering without ethical consideration of embryonic stem cells and severe pain resulted by bone marrow procurement. This review highlights research work on adipose-tissue mesenchymal stem cells and future clinical perspectives.
  • China Biotechnology. 2006, 26(05): 93-100.
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    During the latest fifteen years a great progress has been achieved on transgenic research of the main crops including soybean, maize, rice, and wheat. Almost all the transformation techniques have been successfully used in these main crops. Especially for the Agrobacterium mediated transformation, not only successes it in soybean, a recalcitrant dicot plant to transformation, but also in rice, maize and wheat of monocot plants. At the same time, some useful genes related to the improvement of genetic characteristics, such as insect resistance, herbicide resistance, disease resistance, stress tolerance, quality modification, developmental regulation, nutrition absorption, have been transformed into these plants. Since 1996 transgenic rapeseed, cotton, soybean, and maize have been planted commercialized more and more year by year worldwide, and have produced huge economic benefit. In 2004 GMO crops covered 81.0 million hectares in the world. The status of research and commercialization on transgenic crops was summarized, and some possible problems on this field were discussed in this paper.
  • China Biotechnology. 2006, 26(05): 101-106.
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    Na+/H+ antiporter plays an important role in plant salt toletance. There are two types of Na+/H+ antiporter in plant. One is the SOS1, which localizes in plasma menbrane, the other is AtNHX1, which localized in tonoplast. The plasma menbrance Na+/H+ antiporter is responsible for the transport of Na+ out of the cell, while the tonoplast Na+/H+ antiporter is responsible for the transportion of Na+ from cytoplasm into vacuole. Therefore, these two types of antiporters were considered as key proteins for plant salt tolerance. Recently, many evidences have shown that overexpression of a plasma menbrance Na+/H+ antiporter SOS1 and a tonoplast Na+/H+ antiporter AtNHX1 can increase salt tolerance of plants. This review mainly focused on the latest progress in the study of Na+/H+ antiporter and its relationship with plant salt tolerance.
  • China Biotechnology. 2006, 26(05): 107-112.
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    As is a flexible energy carrier that can be produced from a variety of resources and with comprehensive uses, hydrogen is one of the most promising substitutes for fossil. It is certain that renewable-based hydrogen will be quite important in the future especially hydrogen from biomass which has series of unique merits. Technologies of hydrogen production from biomss mainly contains two kinds of processes, one is thermochemical route, including biomass/waste gasification, biomass pyrolysis, hydrogen from biomass-derived methane/ methanol/ethanol; The other is biological route, including direct biophotolysis, indirect biophotolysis, photo fermentation, hydrogen synthesis via the water-gas-shift reaction of photoheterotrophic bacteria, dark fermentation and microbial fuel cells etc. This article introduces and updates the developments of various hydrogen production processes from biomass, and tries to indicate the trends of these processes.
  • China Biotechnology. 2006, 26(05): 113-119.
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    The conventional nitrification/denitrification process can be significantly improved through the introduction of new microbial treatment technologies ? completely autotrophic nitrogen removal processes. They're mostly based on partial nitrification combined with anaerobic ammonium oxidation, which involves in SHARON+ANAMMOX, CANON, OLAND and NOx processes. The mechanism, characteristics and differences among these processes were reviewed in this paper, and the future development was discussed.