25 July 2006, Volume 26 Issue 07

    

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研究报告
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  Inducement, Purification and Characterization of β-mannanase from Armillariella tabescens EJLY2098

  China Biotechnology. 2006, 26(07): 0-0.

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  Armillariella tabescens EJLY2098 was capable of secreting β-mannanase by induced with konjac. A 34 orthogonal design was applied to determine the optimum medium of inducing mannanase by Armillariella tabescens EJLY2098. The results suggested that Armillariella tabescens EJLY2098 secreted the high activity enzyme by the optimum medium, which composed of 2% konjac, 1% peptone,25% potato juice.，0.3% KH2PO4,15% MgSO4·7H2O, 0.01％ VitB1。Purified by DEAE-anion exchange chromatography, two eluting peaks(P1 and P2)had activity of β-mannanase were obtained, and one of them(named β-mannanase P2) was a single band on the SDS-PAGE, and the molecular weight of β-mannanase P2 was 78.9kD.The isoelectric point of β-mannanase P2 was estimated to be 4.0~4.1. The optimum activity for the enzyme was found at 60℃ and pH4.0～6.0, and the enzyme was stable between pH4.5~6.0. The activity ofβ-mannanase P2 were enhanced by Na+ and Ba2+.Thisβ-mannanase can be used in feed industy and in this works, the authors obtained a new fungi secreting β-mannanase, the work improved an important base for cloning mannanase gene and constructing recombined microbe expressed β-mannanase .
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  Construction, expression and Internalization study of human anti-HBsAg single chain antibody/EGFP fusion proteins containing Arg9

  China Biotechnology. 2006, 26(07): 1-6.

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  To construct，express and purify ScFv14/EGFP fusion proteins which containing Arg9,and to study their binding activities and internalization function. METHODS： Arg9 gene were recombined into 5'terminal、3'terminal of ScFv/EGFP gene or between them before they were cloned into the expression vector pET32a. After induced in E.coli.BL21 and purified, the binding activity and internalization of them were respectively analyzed heir binding activities and internalization function by indirect ELISA and indirect immunofluorescence analysis. RESULTS： DNA sequencing and Restriction endonuclease digestion proved that the four fusion genes were correctly constructed. SDS-PAGE analysis and Western blot showed that they were successfully expressed and purified. Indirect ELISA confirmed that the expressed products had antigen specific binding activity. Indirect immunofluorescence analysis revealed fusion protein which containing Arg9 at its N terminal had much better internalization function，but never internalized into the cells which do not express HBsAg. CONCLUSION：The four fusion genes constructed, expressed and purified successfully. And the purified fusion proteins maintained the binding activity to HBsAg and fusion protein which containing Arg9 at its N terminal had internalization effect much better.
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  Fetal membrane derived adherent cells：a novel source for mesenchymal stem cells

  China Biotechnology. 2006, 26(07): 7-12.

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  Objective: To established a method for isolation and purification of fetal membrane derived adherent cells (FMDACs), and investigate the biological characteristics of FMDACs. Method: FMDACs were isolated with trypsin and cultured in vitro. Inducing FMDACs in vitro differentiate into osteoblasts and adipocytes. FACS and immunocytochemistry technique was used to examine the cell surface antigen. The genetic stability was verified by karyotype analysis. Results: Fetal membrane derived adherent cells were successfully isolated and expanded in vitro. It had strong proliferative ability. FMDACs were positive for CD44 and CD29, but negative for CD34, CD14 and CD45. FMDACs were differentiated into osteoblasts and adipocytes after inducement. The karyotype was stable in the sixth-passaged FMDACs and the tumorigenicity was not found. Conclusion: FMDACs have the possibility of a multipotent stem cells, which have strong capacities of self-renewal and multidirectional differentiation. The genetic background of FMDACs was stable. FMDACs may be used as a kind of novel seed cells for tissue engineering.
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  Secretory expression of the fusion protein IFNβ-HSA in Pichia pastoris

  China Biotechnology. 2006, 26(07): 13-18.

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  Overlapping PCR technology was employed to splice IFN β and HSA genes in vitro. The spliced gene was inserted into Pichia pastoris secretory vector pPIC9K. The IFNβ-HSA gene was designed to be secretory expression under the control of promoter AOX1 and Mat α signal peptide in pPIC9K. The recombinant plasmid pPIC9K/IFNβ-HSA was linearized by restriction enzyme SalI and transformed into Pichia pastoris KM71 by electroporation. The recombinant strains identified by G418 selection and conformed by PCR analysis were induced by methanol to express fusion protein IFNβ-HSA. SDS-PAGE and Western blot analysis of the fusion protein showed that the expressed fusion protein IFNβ-HSA with an apparent 90kDa molecular weight had the antigenicity of HSA. The specific activity of culture supernatant was about 640IU/ml assayed by the standard antiviral activity test on WISH cells challenged with VSV virus.
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  Prediction of the Secondary Structure and B Cell Epitopes of DMO and DMT Protein in Oreochromis aureus

  China Biotechnology. 2006, 26(07): 19-24.

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  The secondary structure of the protein of DMO and DMT in Oreochromis aureus were predicted by the methods of Garnier-Robson, Chou-Fasman and Karplus-Schulz based on the amimo acid sequences of DMO and DMT. And Hydrophilicity plot, Surface probability and Antigenic index for DMO and DMT protein were obtained by the methods of Kyte-Doolittle, Emini and Jameson-Wolf, respectively. Combined the results according to these methods, the B cell epitopes for DMO and DMT protein were predicted. The results demonstrated that there were some centers of α-helix in the DMO protein`s N- terminal No. 80~112, 144~147, 193~194, 251~255, 260~269 and No. 279~283, and in the DMT protein`s N-terminal No. 61~86, 98~105, 140~146, 239~241 and No.269~273. And there are some centers of β-sheet in the DMO protein`s N-terminal No. 59~61, 69~70, 148~150 and No.383~390, and in the DMT protein`s N-terminal No.125~129, 207~213, 255~264 and No.281~284. Furthermore, the DMO protein`s N-terminal No. 40~41, 44~45, 50~51, 128~129, 189~192, 204~207, 216~222, 226~233, 244~246, 298~299 and No.323~326, and the DMT protein`s N-termianl No.12~13, 26~27, 43~44, 58~60, 93~95, 115~120, 136~139 and No.149~151 may be the flexible regions. Moreover the B-cell epitopes possibly localized in or nearby the DMO protein`s 1~5, 41~51, 65~67, 86~89, 98~110, 154~170, 183~203, 205~248, 258~264, 284~291, 293~298, 270~375, 389~392 and No.402~410, and DMT protein`s N-termianl No.1~9, 17~28, 77~84, 114~123, 131~139, 157~184 and No.96~207.Theses results are helpful for studies on sex-control mechanism of DMO and DMT in Oreochromis aureus.
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  DNA shuffling of Arabidopsis thalianna K+ uptake transporter gene

  China Biotechnology. 2006, 26(07): 25-30.

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  The DNA fragment sized 2139bp which sequence is the same with AtKup1 gene from Arabidopsis thalianna was used as the templates to do DNA family shuffling. The shuffled AtKup1 gene library was expressed in the mutant of S. cerevisae in which potassium transporter gene TRK1 and TRK2 were knocked out by homologous recombination. Then the screening was carried out in the low potassium media containing 5.0 mM KCl and no histidine in it. it was found that both of diverse and wild AtKup1 genes can rescue the trk1 trk2 yeast mutant strain on low [K+] medium. The growth of 2 clones yeast contained diverse AtKup1 were beter than that of AtKup1 wild gene transformant. The sequencing results of the shuffled Atkup1 showed that there were 2 nucleotides had changed and resulted in 2 amino acid variations in it compared with original AtKup1. The potassium uptaking capacity of shuffled Atkup1 gene increased significantly when it was transformed into tobacco.
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  Cloning chitinase gene of the entomopathogene fungus Metarhizium anisopliae HN1 and high-level expression in Escherichia . coli

  China Biotechnology. 2006, 26(07): 31-36.

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  Chitinases genes from Metarhizium anisopliae which is an important entomopathogenic fungus were considered one of the key factors to invade their hosts. One Metarhizium anisopliae HN1 strain was isolated and screened by our lab. A chitinase gene was amplified by RT-PCR from Metarhizium anisopliae HN1, The whole length of this gene was 1275bp,and the nucleotide sequence of the gene was 96% similarity to that of the M. anisopliae E6 accessed in GenBank（AF02749）.The gene has been registered in GenBank and its accession number is DQ011865.The gene was subcloned into prokaryon expression vector pET-22b(+),transformed this recombinant expression plasmid into E. coli strain BL 21 and effective expressed. The SDS-PAGE analysis indicated that the recombinant protein was 42kDa which is same to the reported article.The expression level of recombinant protein was about 63.3% of whole expressed proteins ,And when recombinant E.coli were crushed by freeze and supersonic wave , the activity assay indicates that the chitinase expressed in bacteria possesses biological activity.
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  Expression of Recombinant Nematode Anticoagulant Peptide in Pichia Pastoris

  China Biotechnology. 2006, 26(07): 37-41.

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  Objective: To acquire recombinant nematode anticoagulant peptide (NAP) with high anticoagulant activity to lay a foundation for the development of novel anticoagulant agent. Methods: Pichia pastoris GS115 strain was transformed with recombinant yeast expression vector pPIC3.5K-rNAP. Expression of rNAP was induced with methanol after the identification of positive strains. NAP expressed in the collected yeast culture supernatant was confirmed with SDS-PAGE and Western blot. The biological activity of the products was validated with PT (prothrombin time), INR (international normalized ratio) and APTT (activated partial thromboplastin time), respectively. Results: The yeast strains stably express NAP were identified. The rNAP was secreted into culture supernatant with a molecular weight of about 10 kD due to glycosylation, which is a little bigger than that predicted (8.7kD). The anticoagulant efficiency of rNAP was confirmed with the in vitro assays. Conclusion: The recombinant nematode anticoagulant peptide with high biological activity was successfully expressed in Pichia pastoris and can be used in the future development of novel anticoagulant agent.
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  Cloning, sequence analysis and expression of dhaT gene from Citrobacter freundii and purification and property of corresponding recombinant enzyme

  China Biotechnology. 2006, 26(07): 42-47.

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  1,3-propanediol (1,3-PD) is an important material for chemical industry, therefore, there is much interest in the production of 1,3-PD. The gene dhaT encoding 1, 3-propanediol dehydrogenase (PDOR) of Citrobacter freundii was amplified by PCR. Sequence analysis of the similarity at the nucleotide and amino acid level between the gene encoding C. freundii PDOR and that of C.freundii (U09771) were 78% and 90%, respectively. The recombinant plasmid pSE-dhaT was constructed by inserting dhaT gene into expression vector pSE380 and then transformed E. coli JM109. The recombinant strain was induced by IPTG to express dhaT. Further more the recombinant enzyme was purifed from recombinant E.coli by Ni-nitrilotriacetate affinity chromatography followed by Sephacral S-300 gel filtration. A single obvious protein about 42kDa could be obtained by the sodium dodecyl sulfate-polyacrylamide gel electrophoresis of recombinant enzyme. The purified enzyme was used to determined enzyme property on the substrate of propionaldehyde and 1, 3-PD. The optimal temperature and optimal pH of the purified enzyme were 37℃, 8.0 for reduction and 25℃, 10.5 for oxidation, respectively; and the kinetic property of PDOR about Km and Vmax were 10.05mmol/L, 37.27umol/min/mg for propionaldehyde and 1.28mmol/L, 25.55umol/min/mg for 1,3-PD, respectively; The deduced dhaT gene product (388 amino acids) showed a specific reduction activity of 49.50U/mg and oxidation activity of 79.92U/mg. There also have a putative iron-binding motif (G-XX-H-X-X-A-H-X-X-G-X-X-X-X-X-P-H-G) as a fingerprint pattern in our recombinant enzyme, the motif is fully conserved among these 1,3-propanediol dehydrogenase. This study is beneficial to the researches of high producing 1, 3-propanediol by gene engineering strain.
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  Cultural Conditions for Production of Glutathione by Mutant Saccharomyces J-X25

  China Biotechnology. 2006, 26(07): 48-51.

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  The production conditions of Glutathione with shaking flask fermentation by the mutant Saccharomyces J-X25，a methionine-defected strain were studied, and the optimum culture conditions are as follow: initial pH6.0，temperature 30℃，100ml in 500ml flask，the inoculum size 10% and agitation rate 220r/min. Our study emphasizes on the stimulating effect on the cells by dioxogen and the sodium lactate as surfactant both of which were added in the logarithmic phase of fermentation, total producing GSH 0.253g/L , 52% higher than the control experiment without the additions. Compared with the initial strain，the production of GSH by the mutant in optimum conditions was increased by 79%.
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  SECRETED EXPRESSION OF MANNANASE GENE IN PICHIA PASTORIS AND ANYLYSIS OF ENZYMIC PROPERTIES

  China Biotechnology. 2006, 26(07): 52-56.

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  In this study a PCR method was used to amplify the sequence encoding the mature peptide of β-mannanase of Bacillus subtilis. The gene was inserted into the Pichia pastoris vector pPIC9K, downstream of α-factor signal peptide sequence. The resultant recombinant plasmid pPIC9K-MAN was lineared by BglII digestion and introduced into the host Pichia pastoris GS115 by PEG method. After screen, the recombinant P. pastoris strain MAN22 was obtained and fermented in large scale 5L fermenter. The recombinant mannanase activity could reach to 1102IU /mL . The properties of the recombinant mannanase were characterized.
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  Disruption and Compensation of dnmV gene from Daunorubicin-producing strain Streptomyces coeruleorubidus SIPI-1482

  China Biotechnology. 2006, 26(07): 64-68.

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  DnmV, encoded by dnmV gene, is a TDP-4-ketohexulose reductase in the biosynthesis pathway of daunorubicin. The biosynthesis of daunosamine, as well as daunorubicin, can be blocked if dnmV was disrupted. dnmV, along with its upstream dnmU gene, was amplified by PCR from genomic DNA of daunorubicin-producer S. coeruleorubidus SIPI-1482. For the disruption of dnmV gene, a recombinant plasmid pYG817 was constructed and transformed into SIPI-1482. The successful disruption of dnmV in SIPI-1482 was confirmed by daunorubicin absent from metabolites of the resulting transformant, which can also be a host for further introducing genes to modify the glycon structure of antibiotics. The daunorubicin biosynthesis can be reconstituted after dnmV expression plasmid was introduced into the disruptant, although daunorubicin yield is lower than wild-type SIPI-1482.
研究简报
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  Cloning and characterization of swamp Buffalo SRY gene

  China Biotechnology. 2006, 26(07): 69-73.

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  The SRY gene from Buffalo (Bubalus bubalis) genome was amplified by the polymerase chain reaction (PCR) with primers based on the sequence of Hostein SRY gene. The amplified fragment was 2005 bp include 5’ UTR(1~504bp) and 3’UTR(1196-2005bp). And The amplified fragment was cloned and sequenced. Sequence analysis showed that the coding region of SRY gene(505~1195bp) from Buffalo was highly homologous with those of other Bovine counterpart genes (96% homology), especially in the HMG box region (99%homology). It was found that there were only signal on male Buffalo genome on Southern blot,which indicate SRY gene are highly conservative on evolves.
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  Mouse-bovine somatic cell interspecies nuclear transfer

  China Biotechnology. 2006, 26(07): 74-79.

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  simplified interspecies nuclear transfer in mouse and bovine and the possibilities of the embryos reprogramming were developed in this study. The zona and nucleus of the bovine oocyte were removed by pronase and bisection method respectively. When fibroblast cell of the mouse skin fused with the nucleus-free oocyte of the bovine using 40μsec and 1.5 KV/cm direct current pulse，the rates of fusion and cleavage were 67.44% and 30.23% respectively. Reconstructed heterogenous embryos finally developed to 8-cell stage when they were previously activated by ionomycin and 6-DMAP and subsequently cultured in press-made micro-well in the drop of the medium. Zona-free bisection method could be used for mouse heterogenous embryos cloning, and the micro-well culture method could be used to avoid zona-free embryos aggregation.
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  Cloning and Sequencing of Suaeda hetroptera Kitag CMO cDNAand Construction of its recombinant Plant Expression Vector

  China Biotechnology. 2006, 26(07): 80-83.

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  Total RNA was extracted from leaf of Suaeda hetroptera Kitag, then the CMO(choline monooxygenase)cDNA was amplified using the Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) method and cloned into pMD-T-simple vector. The positive clones from the Blue/White Screen were sequenced. After confirming its validity, we subcloned the CMO gene fragment into pBI121 vector. Double enzymes restriction and PCR analysis indicated that the pBI121/CMO recombinant plasmid was successfully constructed.
综述
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  Development and application of RNAi interference libraries in functional genomics

  China Biotechnology. 2006, 26(07): 84-89.

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  RNA interference (RNAi) is a process in which double-stranded RNA (dsRNA) induces specific postranscriptional silencing of homologous transcripts. Systematic silencing of genes on a genome-wide scale by using RNAi library targeting many thousands of genes has been providing a powerful research tool in functional genomics. RNAi libraries, which may be derived from plasmids cloning, virus packaging, PCR amplification, chemically synthesis or enzymatic digestion, have been successfully used to gene function identification, signaling pathway dissection and drug target discovery, etc. As promising progresses have been made in this field, the development, application and problems of RNAi library methodology and future prospects were reviewed in this paper.
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  ADVANCES IN THE ISOLATION METHODS OF FUNGAL POLYKETIDE SYNTHASE GENES

  China Biotechnology. 2006, 26(07): 90-93.

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  Fungal polyketide synthases are responsible for the biosynthesis of secondary metabolites such as pigments, mycotoxins, which are very important in pharmacology ,food science and agriculture.The recent advances in the methods for the isolation and manipulation of multiple classes of polyketide synthase genes from fungi were introduced in this article.It is useful for discovery of novel fungal polyketide synthase gene clusters. These methods can also be useful for revealling the genetic potential of fungi to produce multiple types of bioactive polyketide.
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  Natural Anti-infectious Molecule: Bactericidal/ permeability- increasing Protein

  China Biotechnology. 2006, 26(07): 94-98.

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  Endotoxins are lipopolysaccharides (LPS), major component out wall in Gram- negative bacteria. And they often are considered as "prime criminals" of triggering systemic inflaming reaction such as sepsis or bacteremia and so on. It was found in recent years that bactericidal/permeability-increasing protein, a 55kD member of lipid-related protein family, was a kind of natural molecule of anti-infection and it has special endotoxin-neutralising activity and antibacterial activity. Comprehensive Phase Ⅱ/Ⅲ clinical trials demonstrated the feasibility and safety of recombinant BPI. Above all attract pharmaceutical corporations to try to apply it to therapy.
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  Research Process of Anaerobic Fermentative Hydrogen Production and its Development Future

  China Biotechnology. 2006, 26(07): 99-104.

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  Hydrogen is an ideal energy due to its high conversion efficiency, recyclability and nonpolluting nature. Compared with conventional methods, biological hydrogen production process is found to be less energy intensive and more environmental friendly, and nowadays more and more attentions are being paid on its fermentative way. The paper is a survey of fermentative bio-hydrogen production process, followed by its new approaches in the future development.
论坛
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  Status and Strategies for Sustainable exploitation of Marine Bioresources

  Chang-Yun Wang

  China Biotechnology. 2006, 26(07): 105-111.

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  The status of marine bioresources and the marine eco-environment issues were summarized and discussed, and the strategies for the development of Chinese marine bioresources resources in the future were proposed. The degradation of marine eco-environment and unreasonable exploitation of the resources resulted in acute decline of Chinese marine bioresources. The feasible stratagies to realize the goal of sustainable use of marine bioresources resources should be to intensify the basic research on marine bioresources science, to strengthen the protection of the marine environment and conservation of marine living resources, and to exploit and utilize marine bioresources scientifically and reasonably by using high-technology including marine biotechnology.