25 February 2006, Volume 26 Issue 02

    

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研究报告
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  Molecular Cloning and Characterization of mBolA1, a Novel Mouse Secreted Protein

  China Biotechnology. 2006, 26(02): 0-0.

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  AbstractUsing bioinformatical tools and experimental approaches, a novel secreted protein gene, mouse BolA1 was obtained . mBolA1 spans 730bp of cDNA and locates on mouse chromosome 3F2, which encodes a protein with 137 amino acids, mBolA1 has a conserved BolA domain and the PI is 9.05.The coding sequence of mBolA1 was cloned by RTPCR method from cDNA pool composed of mouse tissues cDNA. Western blot showed that mBolA1 was secreted out from the transiently transfected COS7 cells. Subcellular localization showed that mBolA1 protein located in cytoplasm and didn’t colocalized in Golgi, which indicate that mBolA1 is a unconventional secretion/nonclassical secretory protein. RTPCR result showed this gene was abundantly expressed in mouse tissues. The particular functions of this gene need further investigation.
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  Comparison of recombinant human β-NGF from E.coli and CHO in biochemical characters

  China Biotechnology. 2006, 26(02): 8-12.

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  AbstractThough prokaryotic cells could hardly express recombinant human beta nerve growth factor (rhNGF-β) with a proper threedimensional conformation, using of E. coli as a host for industrial production of rhNGF-β is controversial. Recombinant human beta NGF was expressed in E. coli and was refolded in vitro. The isolated products was shown to be consistent with those expressed and secreted by CHO cells in biochemical characters by SDS-PAGE, RP-HPLC, mass spectrometry, N terminal analysis and bioassay determined using DRG and PC12 cells. The products can be acquired with 95% purity, 1.8 ng/U biological activity from both expression system，which remain invariable biological activity when lyophilized in excipient and store at 37℃±2℃, RH 75%±5% for 3 months. Moreover, the product refolded from inclusion bodies of E. coli shows the predominance in homogeneity and the lower cost.
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  A study on construction of RGD motif in human interleukin-18 and its inhibitory activity on B16 melanoma

  China Biotechnology. 2006, 26(02): 13-19.

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  Using sitedirected mutagenesis and DNA recombinant technology, GGC fragment was inserted into human IL-18 cDNA. The mutated IL-18 cDNA was constructed into plasmid pPIC9K, then transformed into Pichia pastoris GS115 and efficiently expressed. A Glycine residue was inserted into the recombinant IL-18 between Arg39 and Asp40 to form a RGD motif. The mutated IL18 was termed IL-18-RGD. The protein was purified with Sephadex G100. The cell culture of melanoma B16 showed that IL-18-RGD efficiently inhibited B16 tumor growth, IC50 = 8.10μmol/L, but the inhibitory effect of IL-18 was not detected. Both IL-18-RGD and IL-18 showed inhibitory activities on mice loaded B16 in vivo, the inhibitory efffct of IL-18-RGD was stronger than that of IL-18. The inhibitory activities of IL-18-RGD and IL-18 on bFGF induced angiogenesis on chorioallantoic membranes were detected. There was no differences between IL-18-RGD and wild IL-18 in the activity of inducing IFN-γ in human PBMC.
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  Characteristics of HEK293 Cells Growth and Metabolism under Carrier-Free Immobilization Culture Mode

  China Biotechnology. 2006, 26(02): 20-24.

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  By using the cell density, cell viability, size distribution of cell aggregates，specific consumption rate of glucose (qglc), specific production rate of lactate (qlac), lactate transform rate (Ylac/glc) and amino acids utilization as the evaluation indexes, the growth and metabolism of HEK293 cells under carrier-free immobilization culture mode were examined and compared with those of HEK293 cells cultured in static tissue flasks. It was found that HEK293 cells grown as suspended cell aggregates in spinner flasks maintained the basic growth and metabolism characteristics of HEK293 cells in stationary anchored culture, and HEK293 cells under carrier-free immobilization culture mode as suspended aggregates in stirred bioreactor facilitate perfusion performance and increase unit productivity. Cultivation of HEK293 cells in carrier-free immobilization culture mode is a potential and to be further improved mammalian cells culture technique.
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  Study on the CXXC Activity Motif of Human Augmenter of Liver Regeneration

  China Biotechnology. 2006, 26(02): 25-28.

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  Human augmenter of liver regeneration protein (hALRp) had a fragment of Cys-Xaa-Xaa-Cys(CXXC) amino acid sequence. In order to study the CXXC activity motif ALRp, the amino acid at the position of 65 and 88 in hALRp was exchanged against alanine and cysteine respectively, and then expressed and purified mutagenesis protein. The sulfhydryl oxidase activity of ALRp and its mutagenesis in vitro were detected. The concentration of thiol groups in ALR-FAD and ALRQ88C-FAD group were decreased, and they had significant difference when contrasted to control (P<0.05), but ALRC65AFAD group had no significant difference when contrasted to control. The mutation of C65A that changed the CXXC motif of hALRp deprived all of its activity of sulfhydryl oxidase. The mutation of Q88C that added another CXXC motif in hALR couldn’t improve its activity of sulfhydryl oxidase. At the same time, FAD was a cofactor that was indispensable to the activity of sulfhydryl oxidase of ALR, and it maybe helps the mutation protein at the time of renaturation.
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  Purification and Refolding of Recombinant human insulin-like

  China Biotechnology. 2006, 26(02): 29-33.

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  To obtain Insulin-like growth factor (IGF-1) with high purity and activity; Methods: His-tag-beta-galactosidase-IGF-1 fusion protein was expressed in Escherichia coli as inclusion body with IPTG induction. Cells were then harvested, sonicated and centrifuged , and the inclusion bodies were isolated and purified by Ni2+-high performance affinity chromatography. After cleavage of the fusion protein with hydroxylamine, the released IGF-1 was purified by Ni2+- high performance affinity chromatography again and refolded in the presence of GSH/GSSH. Results: The purity of the released IGF-1 was more than 90% after Ni2+-high performance affinity chromatography, and the refolded IGF-1 was with high biological activities. Conclusion: The procudure of fermentation, simple purification and renaturation of recombinant IGF-1 could build the foundation for the large-scale production of IGF-1.
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  Reduced incidence and severity of collagen-induced arthritis in mice lacking LFA-1

  China Biotechnology. 2006, 26(02): 34-37.

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  Lymphocyte function associated antigen1 (LFA-1) is a member of integrin family, that plays an important role in the adhesion of lymphocytes with other cells and matrix. To investigate the role of LFA1 in collageninduced arthritis (CIA), the incidence of CIA, histological and radiological assessments in the LFA-1 deficient (LFA-1 -/-) mice and control mice were examined. LFA-1 -/- mice and control mice were immunized with 100μg collagen type II(CII) emulsified with an equal volume of Freund’s complete adjuvant (CFA), followed by the booster injection of the same amount of CII in CFA on day 21. Then, clinical, histological and radiological assessments were done. It showed that 57% control mice developed arthritis and apparently changed in the histological and radiological assessment, whereas the all of LFA-1-/-mice had the normal histological and radiographic response and none developed arthritis. These results suggeste that LFA1 is indispensable for the onset of CIA.
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  Transfection and Expression the EGFP Gene in the Primarily Cultured Cells from Chinese Shrimp Fenneropenaeus chinensis

  China Biotechnology. 2006, 26(02): 38-43.

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  The study tried to develop the technology related to introducing the eukaryotic expression plasmid into the primarily cultured cells from Chinese shrimp (Fenneropenaeus chinensis), one of the most commercially important aquaculture marine invertebrates in China. The primary cell cultures included the adherent or suspension cultures from the haemocytes and the cell adherent cultures from the lymphoid organ（Oka organ）and ovary of the shrimp. The methods of gene transfer tried in this paper included calcium phosphate mediated transfection, liposome mediated transfection（lipofection）and electroporation. Lipofectin could be used to introduce the plasmid into the cultured cells and to express the reporter gene, EGFP（enhanced green fluorescence protein）gene, in the haemocyte suspension cultures and the cell adherent cultures from the Oka organ and ovary. The present work is to facilitate the study of shrimp immunology and transgenic studies by developing a primary culture system of the shrimp.
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  Expression of Helicoverpa armigera Cathepsin B in Pichia pastoris

  China Biotechnology. 2006, 26(02): 44-48.

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  Cathepsin B from Helicoverpa armigera (HCB) belongs to the group of cysteine proteinases. HCB is proved being involved in the degradation of yolk proteins during embryonic development，which is an acidic preferring enzyme and is resistant to SDS. The expression of the proenzyme may offer a model for investigating the activation of the enzyme. The HCB gene was constructed into pPIC9K and expressed in Pichia pastoris KM71 strain . After induction by methanol, HCB was expressed and secreted into the medium. The molecular weight of the recombinant procathepsin B was determined as about 38 kD. The expressed product was confirmed to be HCB by immunoblotting assay using specific rabbit anti-HCB polyclonal antibody. The activity of the product was assayed by in situ hydrolysis （gelatin-SDS-PAGE）. These results showed that HCB with proteolytic activity was expressed in P. pastoris KM71. This proenzyme can be used for further research on the activation of the proenzyme or industrial production.
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  Cloning of a Cellulose Synthase Gene from Aspen PtoCesA1 and Construction of it's Plant Expresstion Vector

  China Biotechnology. 2006, 26(02): 49-52.

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  The DNA fragment was cloned from the cDNA that was synthesized using the RNA from Chinese white poplar and verified by sequencing This cDNA clone ,designated PtoCesA1, was 3215 bp long with an opening reading frame of 2934bp extending from nucleotides 52~2985.Comprision of the nucleotide sequence of PtoCesA1 with PtrCesA1 showed 97% identity.For construction of the PtoCesA1 binary expression vector, the BamHI site was made by synonymy mutation, the full length PtoCesA1 cDNA was subcloned into pBI121 between the BamHI site and XbaI site. The PtoCesA1 binary vector，containing PtoCesA1,was verified by digestion and PCR.
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  Genome-shuffling Improved Acid Tolerance of

  China Biotechnology. 2006, 26(02): 53-58.

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  Lactic acid fermentation is a typical productinhibited bioconversion. The strains with improved performance of growing at low pH are desirable because low pH of the medium could increase the proportion of product in the freeacid form and contributes to the economics of the lactic acid purification process. The use of genome shuffling to improve the acid tolerance of an industrial strain of Labtobacillus casei was described. Two populations were obtained with subtle improvements by UV and NTG mutagenesis and were taken as the starters for genome shuffling. Parental protoplasts were inactivated and fused by polythyiene glycol 6000. The fusants grew at the RM plates were the shuffled strains. The shuffled strains after screening on low pH plate and CaCO3 plate were identified. The mutant strains were incubated in YE liquid medium at pH 3.8 and 3.4 as well as the wildtype. The mutant strains grew obviously better than the wildtype strain. Only the shuffled strains can grow under the condition of pH 3.4. The mutant strains and wildtype were both fermented at 37℃ for 48 h at pH 38. The production of Llactic acid of the mutant strain was 2.4 folds of the wildtype.
技术与方法
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  Prokaryotic Expression of the Partial gBGene of the Marek's Disease Virus

  China Biotechnology. 2006, 26(02): 62-65.

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研究简报
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  PREPARATION AND IDENTIFICATION OF MONOCLONAL ANTIBODY AGAINST CLOXACILLIN

  China Biotechnology. 2006, 26(02): 66-68.

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  CX-conjugated antigen(BSA-CX) were produced by by methods of EDC.The spleen cells of BALB/C mouse immunized by BSA-CX were fused with SP2/0 plasmacytoma cells using PEG4000,and selected cultured with HAT and HT medium.A hybridoma cell line of 2H8-B1-F4 was secreened for specificity to CX,and cloned by limited dilution method for 3 times,which secrete high affinity and stable monoclonal antibodies against CX with indirect ELISA. The antibody was characterized by IgG1 isotype, the titers of antibody in cell supernatant and ascites were 1:1000 and 1:4×105,respectively.The mAb against CX was selective toward Dicloxacillin、Oxacillin with cross-reactivity of about 19.6％ and 21.3％,which can be used to detect CX residues by competitive ELISA.
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  In CNE2 cell after bax gene transfection analysed by mRNA defference to demonstrate

  China Biotechnology. 2006, 26(02): 69-73.

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  bjective ：To look for the correlated gene of CNE2 cell after bax gene transfection. Method: In CNE2 cell after bax gene transfection analysed by mRNA defference to demonstrate, and DNA sequencing was performed, and small amount of PCR products were used as probes for Northern blot after labeled with radioisotope. Result: By mRNA differential display technique, ten differential expression bands were found in CNE2 cell, four bands of which were induced by pSFFV-bax-neo and the others inhibited. The four cDNA framents were to reamplify by PCR selected from the inhibited bands and expressions in CNE2 cell were found by Northern blot. Conclusion：Bax gene can induce the expression of some genes in CNE2 cell, whereas it can also inhibit the expression of some genes. By selecting two lengthened primers and adding T7RNA polymerase promoter to the nondT anchoring primers, it can overcome the disadvantags of excessive PCR products and pseudopositive bands in mRNA differential display technique, Thus PCR products can be sequenced directly.
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  Effect of foreign plasmid DNA on material and energy metabolism of spleen in mice

  China Biotechnology. 2006, 26(02): 74-78.

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  To study the influence of foreign plasmid DNA on spleen metabolism in immunestimulated mice via the gastrointestinal tract. Mice were oral administered by pipette with 200μg of plasmid pcDNA3 and spleen were isolated at 4h and 18h after oral administration. Total RNA were extracted from spleen and spleen gene expression profile of Balb/c mice was analyzed by using Affymetrix oligonucleotide genechip after oral plasmid pcDNA3 administration. Functional cluster analysis was conducted by Genmapp and MAPPFinder software. By functional cluster analyzing the genes which were upregulated more than twofolds, Genmapp results showed that plenty of metabolic pathway were induced in spleen after oral administration of plasmid pcDNA3. These metabolic process included purine metabolism, pyrimidine metabolism, protein synthesis, cholesterol synthesis, fatty acid synthesis, Glycolysis, TCA cycle and mitochondria oxidative phosphorylation pathway. The similar results also took place at 18h after oral administration. The result indicated that foreign plasmid DNA can modulate metabolism process in spleen of mice via the gastrointestinal tract, and may help understand the mechanism of action of foreign plasmid DNA uptaked via the gastrointestinal tract.
综述
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  Advances in the Research of Long Life

  China Biotechnology. 2006, 26(02): 79-82.

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  The recombinant protein drug usually has a short half-life after intravenous (IV) or subcutaneous (SC) administration. The methods of prolonging the half-life of recombinant protein drug in common use are mainly based on three principles: 1, Amplifying the molecule weight of protein drug; 2, Making use of drug balance in the blood; 3, Reducing Immunogenicity. This review focuses on three methods: Analogy construction、PEGylation and Albumin fusion technology. The characteristics, half-life and immunogenicity problem of their products in the market and under development are summarized.
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  The Application of Countercurrent Chromatography with Aqueous Two Phase System in the Separation of Proteins

  China Biotechnology. 2006, 26(02): 83-88.

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  Aqueous two phase system(ATPS) provides a gentle, non-denaturing separation environment for proteins, enzymes. While high-speed countercurrent chromatography (HSCCC) is a liquid-liquid partition chromatography which uses centrifugal force to hold the stationary phase and facilities the mobile phase partitioning through the stationary phase, it can produce high separation efficiency with large sample loading capacity. However, the ordinary HSCCC apparatus (Type J ) fails to retention a satisfactory stationary phase of ATPS because of its high viscosity and low interfacial tension. Nevertheless, the later designed cross-axis planetary centrifuge system(X-CPC) can produce a greater lateral force field and enhances significantly the retention of the ATPS stationary phase. This paper gives a review of the application of these CCC techniques with ATPS in the separation of proteins. Meanwhile, new techniques such as pH－peak focusing CCC and dye-ligand affinity CCC and some new CCC column design for improvement of separation efficiency and retention of ATPS stationary phase are introduced in this paper.
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  Research Progress in Cofactor Engineering of Xylose Metabolism in Recombinant Saccharomyces cerevisiae

  China Biotechnology. 2006, 26(02): 89-94.

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  Cofactor engineering, a vital part of metabolism engineering, changes the redox cofactor regeneration approach. Its main goal is to rebuild the components of metabolic products. The bioconversion of xylose for the production of ethanol is being studied intensively because ethanol is an alternative energy source and a potential liquid fuel. Saccharomyces cerevisiae has been traditionally used in producing ethanol from fermentable sugars but it cannot utilize xylose, only its isomer xylulose. Introduction of the xylose fermentation pathway from Pichia stipitis into S. cerevisiae enables xylose utilization in recombinant S. cerevisiae, but the ethanol yields of xylose fermentation with recombinant S. cerevisiae has been low and large amounts of the byproduct xylitol are produced. The major reason is that the catabolism of xylose with the fungal pathway leads an imbalance of redox cofactor. The process of the catabolism of xylose requires NADPH and NAD+, both of which have to be regenerated in separated processes. More and more attention has therefore focused on the redox cofactor balance in S. cerevisia. The research progress of cofactor engineering to solve the imbalance of redox cofactor in xylose metabolism recombinant S. cerevisiae was introduced. This included expression of transhydrogenase, increasing the utilization of NADPH, and achieving the anaerobic reoxidation of NADH. Reversing the cofactor specificity of enzymes is another effective way.
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  Denitrification using biodegradable polymers (DBPs) as carbon source and biofilm carrier

  China Biotechnology. 2006, 26(02): 95-98.

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  Raw waters polluted with nitrate present serious problems to drinking water source of many countries. Heterotrophic denitrification is one of the main techniques to remove nitrate from water. Adopting economic solid organic carbon sources, such as cotton, wheat straw and so on, can cut down the running cost of denitrification. But it is possible to deteriorate the quality of water when removing nitrate from drinking water. Denitrification system using biodegradable polymers (BDPs) as carbon sources and biofilm carriers has perfect ability to adapt to the shock of influent water quality and it is impossible to pollute the water for the reason that they are no harm to human health. With the development of new kinds of BDPs material and the decline of BDPs cost, BDPs may get more and more attention in drinking water denitrification.