25 September 2006, Volume 26 Issue 09

    

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研究报告
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  Eukaryotic expression of βig-h3 gene and its effects on secretion of MMPs in the human hepatoma 7721 cells

  China Biotechnology. 2006, 26(09): 1-4.

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  βig-h3 was first identified as a transforming growth factor-beta1-inducible gene in human lung adenocarcinoma cell line. It encodes for a secreted extracellular matrix (ECM) protein, which is thought to act on cell attachment and ECM composition. Our previous study showed that Objective: To construct eukaryotic recombinant expression plasmid βig-h3/ pEGFP-C2 and βig-h3 highly expressed in human hepatoma cell lines such as human FHCC-98 with high metastatic potentials but several others such as human 7721 hepatoma cells. The present study aimed to transfect βig-h3 into 7721 cells to investigate its effect on secretion of MMPs in the transfected human hepatoma cells. Full-length βig-h3 gene，cloned by reverse transcription polymerase chain reaction (RT-PCR) was inserted into the eukaryotic expression vector pEGFP-C2. The recombinant plasmid was transfected into 7721 cells with Lipofectamine2000 and Gelatin-Zymography were adopted to detect the production of MMPs in the transfected cells. Results showed that βig-h3/pEGFP-C2 recombinant expression plasmid was successfully constructed and achieved high transfection efficiency. MMPs expression of the transfected cells was promoted significantly. These results suggest that overexpression of βig-h3 promoted the production of MMPs, indicating that βig-h3 may play roles in the invasive and metastatic processes of hepatoma.
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  Construction of Prokaryotic Expression Vector for Soluble HLA-A*0203-BSP and Its High Yield Expression in Escherichia coli

  China Biotechnology. 2006, 26(09): 5-10.

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  Objective: To clone the cDNA of HLA-A*0203 heavy chain and to construct the prokaryotic expression vector for the ectodomain of HLA-A*0203 fused with a BirA substrate peptide (BSP) at its carboxyl terminus ( HLA-A*0203-BSP ) and express the recombinant protein in Escherichia coli (E．coli). Methods: The cDNA for HLA-A*0203 heavy chain was cloned by RT-PCR from the PBMC of three HLA-A2+ donors and confirmed by DNA sequencing. DNA fragment encoding HLA-A*0203-BSP was amplified by PCR with the sequence-verified cDNA as a template. It was then cloned into pET-3d vector. After confirmed by DNA sequencing once again, the prokaryotic expression vector was transformed into BL21(DE3) strain. The recombinant protein was expressed in E. coli after IPTG induction. SDS-PAGE and Western blot analyses were employed to detect the expressed fusion protein. Results: DNA sequence analysis showed that the cDNA encoding the heavy chain of HLA -A*0203 was cloned from donor 2, which had been confirmed to be HLA-A2+ by flow cytometry. The expression vector for the recombinant HLA-A*0203-BSP fusion protein was then constructed and verified by DNA sequencing. SDS-PAGE analysis revealed that the fusion protein was highly expressed in E. coli and accounted for 30％ of total bacterial proteins. Furthermore, Western blotting showed that all the recombinant protein existed in the inclusion bodies. The fusion protein had a molecular weight of about 34 kD, which is in accordance with the theoretical value. Conclusion: The cDNA of HLA-A*0203 heavy chain was cloned and the prokaryotic expression vector for the fusion protein of HLA-A*0203-BSP was constructed. The recombinant protein was highly expressed as the form of inclusion bodies in E. coli, which would facilitate the preparation of soluble HLA-A*0203 tetramer.
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  High Cell Density Cultivation of Recombinant Escherichia coli for Production of Fusion Protein Staphylokinase-Hirudin

  China Biotechnology. 2006, 26(09): 11-15.

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  Recombinant fusion protein staphylokinase-hirudin has been produced by adopting the dissolved oxygen feed-back fed-batch with the high cell density cultivation strategy in E.coli BL21(DE3). After selection of the strain and the preliminary optimization by flask cultivation, the fermentation was performed with comparing the differences between defined medium and complex medium in 5L fermentor. With the optimization of the cultivated conditions, the recombinant fusion protein staphylokinase-hirudin was over-expressed in E.coli BL21(DE3), and the final cell density reach 115g wet cell weight per liter, the target protein is about 1.1~1.2g/L accounting for more than 30% of the total protein. The culture process in 5L fermentor was performed on 40L fermentor successfully, and indicated that the process was easy scalable.
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  Expression of Spodoptera litura Multicapsid Nucleopolyhedrovirus VP39-GST Fusion Protein in Escherichia.coli

  China Biotechnology. 2006, 26(09): 16-19.

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  The ORF of Spodoptera litura multicapsid nucleopolyhedrovirus（SpltMNPV）vp39 gene was obtained by PCR method from SpltMNPV genomic DNA. The PCR product was cloned into pMD18-T vector to get the recombinant plasmid（pMD18-vp39）.The vp39 gene was recombined in vitro with prokaryotic expression vector pGEX-4T-1 and transformed into Escherichia coli BL21(DE3). The BL21(DE3) strain, containing vp39 recombinant plasmid, expressed a 60 kD fusion protein after the induction with 1 mmol/L IPTG (Isoprophylthio-β-D-galactoside). The expression of the VP39-GST fusion protein was suitable for further analysis of the inter-action between baculovirus and its host.
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  Generation of a recombinant fowlpox virus co-expressing Foot-and-Mouth Disease virus P1-2A gene and porcine interleukin 18

  China Biotechnology. 2006, 26(09): 20-23.

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  AObjective：In order to prevent the O-FMDV, we constructed and screened a recombinant FPV which can co-expressing Foot-and-Mouth Disease virus P1-2A gene and porcine interleukin 18 as the vaccine. Methods：We designed two primers for amplifying the O-FMDV P1-2A gene：The Kozak sequence was inserted into the upstream primer；the linker was inserted into the downstream primer. Then pIL-18 gene was connected to P1-2A gene in order to construct the expression gene box, which named as P1-2A-IL-18. The gene box was inserted into the FPV transfer vector pUTA-16-LacZ-3C to construct recombinant transfer vector plasmid pUTAL-3C-P1-2A-IL-18.The recombinant transfer vector plasmid was cotransfected with 282E4 fowlpox viruses into the CEF. The recombinant fowlpox viruses-3C-P1-2A-IL-18 (rFPV-3C- P1-2A-IL-18) was selected 3 passages by culturing in CEF with MEM medirum containing BrdU. The selected viruses were plaque-purified in CEF without BrdU. After RT-PCR and IFA analysis, the recombinant FPVs expressing gene box P1-2A-IL-18 was obtained. Results：he rFPV-3C-P1-2A-IL-18 strains could express 3C-P1-2A gene and pIL-18 gene correctly. Conclusion：This research indicated that a recombinant fowlpox viruses rFPV-3C-P1-2A-IL-18 which can co-expressing Foot-and-Mouth Disease virus P1-2A gene and porcine interleukin 18 was successfully obtained.
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  F protein B Cell Epitopes between Comparison of Immunogenicity of NDV F48E9 and La Sota strains

  China Biotechnology. 2006, 26(09): 24-31.

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  To identify and compare predicted the B cell-epitopes between NDV F48E9 and La Sota strain, we cloned the predicted 4 poly-epitopes genes segments of each strain, then constructed into Prokaryotic vector separately. The expressed 8 recombinant proteins were analysed by SDS-PAGE, molecular weight of four peptides segments P1,P2,P5and P6 are 8.0KD, 10.8KD, 13.5KD, 10.5KD respectively.The Antigenicity of expressed peptides was identified with anti-F48E9 and anti-La Sota strains serum. The result showed that peptides P1, P2, P5 and P6 of NDV F48E9 and La Sota could reacted with both anti-serum. However, the western blot of peptide P5 against anti-F48E9 and anti-La Sota serum showed a strain specific reaction. Then we purified P5 ploypeptide and mChIL-18 protein by Ni-NTA Resin. Using mChIL-18 as adjuvant, immunized SPF chickens with P5 peptide of F48E9 or La Sota，serum were collected and NDV specific antibody titers were detected with ELISA. The results indicate that F48E9 P5 peptide (FP5) in combination with mChIL-18 protein has the highest antibody titer among all immunized groups, and it is the only group that could protect some of immunized chicken from dead (20%). Finding of immunogenicity difference between FP5 peptide and LP5 peptide, suggested that there are strain specific epitopes exist in F48E9 and La Sota F protein.
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  The Research on Expression and Purification of a Chimeric anti-p185 antibody

  China Biotechnology. 2006, 26(09): 32-37.

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  TThe Her-2 proto-oncogene encodes a 185kD transmenbrane glycoprotein p185 which has intrinsic tyrosine kinase activity. It is overexpressed in several malignant human tumors like breast cancer. We have constructed a chimeric antibody by assembling a single-chain Fv antibody and a human IgG1 Fc fragment. This chimeric antibody reacts with tumor surface antigen p185c-erbB-2 specifically. In order to put the antibody into clinical application, we use two steps purification method to attain the antibody's purity more than 95%. Then we found both the lyophilized pharmaceutical formulations of the antibody. The formulations can keep the stability and activity of the antibody for at least 1 year. These results were the foundation of the chimeric antibody for cancer therapy.
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  Induction of Bone Mesenchymal Stem Cells to Form Osteoblast on Alginate Gels

  China Biotechnology. 2006, 26(09): 38-42.

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  Bio-material is a key factor in bone tissue engineering. In this article, the biological effects of a new bio-material, alginate gels, on bone mesenchymal stem cells (bMSCs) were investigated. bMSCs were induced to form osteoblasts on alginate gels. Cell growth, appearance and calcification were detected by MTT, toluidine blue stain and von Kossa stain, respectively. osteoblast related genes were examined by RT-PCR. We found that bMSCs on the alginate gels (experimental group) grew well and formed obvious colony. The genes related with osteoblast (alkaline phosphatase, collagen I and osteocalcin) were positive both in the experimental group and the control group (bMSCs on tissue culture plastic), moreover, the expression levels in the experimental group were higher than those in the control group. The results show that alginate gels can promote bMSCs to differentiate into osteoblasts. This suggests that alginate gels may act as a favourable scaffold in bone tissue engineering.
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  Cellular Compatibility of Blends Consisting of Poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) and Polyethylene Glycol

  China Biotechnology. 2006, 26(09): 43-50.

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  Polyhydroxyalkanoates (PHAs) are a family of biodegradable polyesters, which are generally biodegradable and biocompatible and possess various plastic and elastomeric material properties depending on their monomer constituents. One PHA family member, namely, copolyesters of 3-hydroxybutyrate and 3-hydroxyhexanoate (PHBHHx) showed better mechanical properties compared with poly (3-hydroxybutyrate) (PHB) and poly (3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV). In order to extend the potential application of PHBHHx in vascular-related tissue engineering, surface modification of PHBHHx were made by blending with polyethylene glycol (PEG). When PHBHHx was blended with PEG in ratios of 3:1 and 2:1, the two materials were miscible. When the ratio of PEG in the blends increased, phase separation appeared and the materials became partially miscible. Blending with PEG increased the hydrophilicity and surface energy of PHBHHx, leading to significantly enhancement of attachment and proliferation of RaSMCs and HUVECs in a PEG dose dependent way. Specially, PHBHHx blending with PEG in ratio of 1:1 promoted RaSMCs' proliferation compared with HUVECs'. These results suggest that PHBHHx blended with PEG in this way is promising in constructing complex multilayer vascular grafts.
技术与方法
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  Development of Monoclonal Antibodies of Chicken Infectious Anemia Virus VP3 Protein

  China Biotechnology. 2006, 26(09): 51-55.

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  Monoclonal antibodies (McAbs) specific to CIAV VP3 proteins were developed by fusing SP2/0 myeloma cells and spleen cells of BALB/c mice immunized with the recombint VP3 protein purified by Ni-NTA colomn. Three hybridoma cell lines were examinated by indirect ELISA and cloned for three times by limited dilution. Three of them were named as AG2, BC11 and FG1 which ELISA titers of supernatant culture and ascites were 2.5ⅹ102, 1.5ⅹ102, 1ⅹ10 and 2ⅹ106, 1ⅹ106, 2.5ⅹ105 respectively. The numbers of chromosome of all McAbs are between 97 and 104. In this study, the McAbs against VP3 proteins of CIAV did not show cross-reacting to avian viruses (NDV、MDV、IBDV、REOV) and cells of MSB1 and Sf9 by ELISA indicating the specify of these McAbs and can be used to study the structure and function of VP3 protein.
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  Medium Optimization for the Novel Antifungal Material by Burkholderia cepacia CF-66

  China Biotechnology. 2006, 26(09): 56-60.

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  Statistics-based experimental design was used to investigate the effect of medium components on CF66I production by Burkholderia cepacia CF-66. A fractional factorial design augmented with center points revealed that sodium citrate and yeast extract were the most significant factors, which influenced CF66I production positively, while the other factors were not important with the levels tested. The method of steepest ascent was used to approach the proximity of optimization. This task was followed by a central composite design to determine the optional concentration of sodium citrate and yeast extract. The optimized medium allowed the activity of CF66I to be increased from 2.12 U/ml to 6.24 U/ml，about 200% higher than the original medium.
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  The study on separating the protein of bovine sperm by two-dimensional gel electrophoresis

  China Biotechnology. 2006, 26(09): 61-66.

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  Two-dimensional gel electrophoresis is a protein separation technique and it can be used for separating sperm proteins. The objectives of this study were to develop an optimal two-dimensional gel electrophoresis, and the bovine sperm proteins were isolated with the developed technique. In this study, isoelectric focusing electrophoresis program was optimized, and various sample preparation methods, different loading quantities and strip length were studied. The results indicated that the better proteins map was obtained when the bovine sperm were lysed by two step method of urea-guanidine hydrochloride and the 13cm N-linear IPG strip was used to conduct the isoelectric focusing electrophoresis. More than 800 protein spots could be detected by the imaging analysis system, and the analysis result indicated that molecular weight and isoelectric point of most proteins were from 10 to 100 KD and 4.0 to 9.0 respectively. The study provided a basis for detecting and analyzing the differential proteins of the X，Y sperm.
研究简报
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  The Formation of Recombinant Strain Producing Catechol and the Optimization of Fermentation Conditions

  China Biotechnology. 2006, 26(09): 67-71.

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  A complete aniline dioxygenase gene cluster cloned from an Acinetobacter sp. strain, which could utilize aniline as the sole carbon, nitrogen and energy, was sequenced. Sequence analysis showed that the gene cluster had six intact ORFs, and the whole sequence had high similarity with that of Acinetobacter sp. YAA at amino acid level. A recombinant strain was formed with the gene cluster ligated to vector pLAFR6 and transferred to E.coli. After optimizing, the fermentation conditions of this strain for producing catechlo, we confirmed LB as the final medium, pH7.0, aniline concentration 0.5mg/mL, E.Coli DH5αas the host, incubation temperature 37℃, amount of inoculum 3%. Under above conditions, the yield of catechol could get to 0.546mg/mL, and the converting rate of substrate at molecule level could get to 92.4%.
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  Effects of Surfactants on Neutrophilic-granulocytes

  China Biotechnology. 2006, 26(09): 72-75.

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  A new method is discussed to select a reagent which reacts to blood corpuscle in this paper. After the neutrophils are separated and purified, the effect of the surfactant on other blood corpuscle was avoided, so it reflects the exact effect caused by surfactant. It is an ideal method of selecting a reagent which reacts to blood corpuscle.
综述
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  Metagenomics: cultutre-independent genomic analysis of environmental microbial communities and its application

  China Biotechnology. 2006, 26(09): 76-83.

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  The biosphere is dominated by microorganisms which play an important role in the substance cycle of the earth, yet about 99﹪of these microorganisms have not been or can not be cultured by traditional methods, thus impeding the detailed study on them. With the development of molecular biology and its application in microbiology, a new research field, namely environmental genomics, has been developed．Without prior isolation and cultivation of relative microorganisms from environmental samples, all the genetic materials are directly extracted and an environmental genomic library is constructed. The library is further analyzed with similar strategies for functional genomics, either to screen active metabolites and their coding genes, or to analyze sequences of relative phylogenic anchors to establish the organization, evolution of relative microorganisms and their respective roles in substance cycle in a specific ecosystem．On the other hand, with more information about the physiological and biochemical characteristics of uncultured microorganisms revealed by environmental genomics, appropriate culturing conditions might be developed and pure cultures of relative uncultured microorganisms could be finally obtained. The strategies for the construction and analysis of environmental libraries and recent progress in this field are reviewed. The application of environmental genomics in microbial ecology is also introduced.
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  Advances on mechanism and application of RNAi function in plant cell engineering

  China Biotechnology. 2006, 26(09): 84-90.

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  RNAi, of whom the regulatory mechanism is being investigated deeply, has become a kind of powerful research tool to regulate the expression of target genes for expectant phenotypes. There are at least three RNA silencing pathways in plant, and the silencing signals involved in these pathways can be amplified, transmitted and self-regulated. In order to construct an effective and commercial technical system, the problems must be solved: efficient delivery, enhanced stability, minimization of off-target effects and identification of sensitive sites in the target RNAs. The advances on mechanism and application of RNAi function in plant cell engineering up to date were summarized in this review, and the practical technical system was also discussed in details.
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  Progress in elimination of elemental sulfur by the acidophilic sulfur-oxidizing bacteria

  China Biotechnology. 2006, 26(09): 91-95.

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  Metal sulfides are chemically attacked by Fe3+ and H+, resulting in the formation of elemental sulfur via polysulfides or thiosulfate pathway. Elemental sulfur may aggregate and even form a layer on the metal sulfide surface, which will inhibit leaching metals from the sulfides minerals. Elimination of inert elemental sulfur in a typical acidic environment can exclusively be by way of oxidation of acidophilic sulfur-oxidizing bacteria, such a way includes the attachment, transport and oxidation process of elemental sulfur by acidophilic sulfur-oxidizing bacteria. On the basis of analysis on the pertinent researches, this paper showed that the molecular mechanism of sulfur elimination by the acidophilic bacteria is far away from elucidated, and to attain that target, there are still much work to be done for elucidating the molecular mechanism on the attachment, transport and oxidation process of elemental sulfur by the acidophilic sulfur-oxidizing bacteria.
产业发展
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  Peanut cotyledon bioreactor-a potential new force in peanut industry

  Haiyan Yan

  China Biotechnology. 2006, 26(09): 96.

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  The product from bioreactor of peanut cotyledon are organ-specific, enrichment in content, orally taken directly, etc. these property made it a better bioreactor than others. This paper introduced current research background and future perspective in this area. Summarized the techniques need un peanut bioreactor. The key of peanut bioactor is construction of transformation vector. The promoters of major storage proteins are best choice for construction of transformation vectors. The promoter of glycinin genes from soybean can also be used in construction of transformation vector.