25 July 2010, Volume 30 Issue 07
    

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  • Progress on the Cryodamage of Frozen-thawed Boar Spermatozoa
    China Biotechnology. 2010, 30(07): 0-0.
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    The morphological changes of acrosome, plasma membrane and mitochondrion of frozen-thawed boar spermatozoa and their effect on the fertilization ability of sperm were reviewed, and the changes of biomacromolecules of frozen-thawed boar spermatozoa such as, protein, DNA, etc, and their influence on the quality and fertility of frozen-thawed boar spermatozoa were also analysed. As a result, the conclusion was drawn that the effect of quality and quantity of biomacromolecules such as protein, DNA, are the cryodamage essence of frozen-thawed spermatozoa in the process of cryopreservation. The application of the advanced proteomics on study of the cryodamage mechanism of frozen-thawed boar spermatozoa will contribute to the elucidation of the molecular mechanism of freezing injury, and will provide rationale support for the technique breakthrough of the cryopreservation of boar semen.
  • Prokaryotic Expression of human FBXO31 and preparation of its polyclonal antibody
    China Biotechnology. 2010, 30(07): 0-0.
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    Abstract FBXO31 is the chromosome 16q24.3 senescence gene, a candidate breast tumor suppressor, and a component of an SCF complex. In order to obtain rabbit anti-FBXO31 polyclonal antibody, the complete coding sequence was cloned into pET-28a. Prokaryotic expression plasmid pET-28a-FBXO31 was constructed and expressed in the strain of E. coli BL21（DE3）. Fusion protein mainly existed in inclusion body. Fusion protein was purified and immune rabbit to obtain rabbit anti-FBXO31 polyclonal antibody. The specificity confirmed by western blot and immunoprecipitation. These results showed that rabbit anti-FBXO31 polyclonal antibody could identify FBXO31 protein and be a useful tool in FBXO31 function analysis.
  • Point Mutation of Nucleoside Diphosphate Kinase A and Preparation and Bioactivity Determination of its C4S Mutant
    China Biotechnology. 2010, 30(07): 0-0.
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    Objective: To construct nucleoside diphosphate kinase A mutant (NDPK-A C4S), and detect its phosphotransferase activity and DNase activity to study the influence of the disulfide bond on the bioactivities of NDPK-A. Methods: A synthetic oligonucleotide primer for site-directed mutagenesis was designed with the template pBV220-nm23-H1, where the codon for the residue cysteine-4 was replaced with that of serine. Expressed by BL21 host cell and purified by DEAE-sepharose Fast Flow and Cibacron Blue 3GA Sepharose CL-4B, the homogeneous recombination protein was obtained with the purity of 98%. The DNA sequencing and MALDI-TOF mass spectrography analysis demonstrated the success of constructed NDPK-A C4S mutant. The diversities of the phosphotransferase activity and DNase activity from NDPK-A C4S mutant and wild type NDPK-A were measured by reverse high performance liquid chromatography (HPLC) and DNA cleavage (DNase) analysis respectively. Results: After site-specifically mutating the critical residue cysteine-4 contributing to the disulfide bond isomerism, not only the phosphotransferase activity but also the DNase activity of NDPK-A C4S are higher than that of wild type NDPK-A. Conclusion: forming the intrachain disulfide bond in NDPK-A is possibly one of the negative functional regulation modes, which put a solid foundation for the research of NDPK-A’s bioactivity, and it will be an alternative practical application for the more active function of NDPK-A C4S.
  • Secreted Expression of a Thermostable α-galactosidase gene in Pichia psatoris and Analysis of Enzymic Properties
    China Biotechnology. 2010, 30(07): 0-0.
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    cDNA sequence（GenBank No. DQ234280）of the α-galactosidase from Absidia ramose WL511 was cloned and inserted into the Pichia pastoris expression vector pPICZαA，and integrated into the genome of P. pastoris GS115 cells by electricity pulse. The culture supernatant showed 32U/ml of enzymatic activity at 30℃ with 0.5%（V/ V）methanol added. The specific activity of purified recombinant enzyme at the grade of electrophoresis pure was 137 U/ mg. The α-galactosidase expressed by P. pastoris was glycosyled protein with a 6kDa larger molecular mass than native enzyme, and a tetramer with the molecular weight of 348 kDa estimated by gel filtration and the subunit molecular weight of 87 kDa by SDS-PAGE. The pI value, optimal pH and temperature of the enzyme are 5.2，6.8 and 73 ℃. With pNPG as the substrate, the Km, Vmax, and kcat are 0.42 mmol/Lol/L, 413 U mg-1 and 64531 min-1, respectively. This enzyme showed a stable activity at pH5.5-9.0 and below 60℃, still remained 54% of activity for 2 h at 75 ℃, and completely inactivated at 85℃.the secret-expressed α-galactosidase from A. ramose WL511 have better enzymatic properties of high thermostability, high specific activity and wide pH range.
  • The Application and Mechanism of Electrofusion for Developmental Biology and Stem Cells Research
    China Biotechnology. 2010, 30(07): 0-0.
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    As a method of cell fusion, electrofusion has played a great role in monoclonal antibody, gene transfer and tumor immunity. Now electrofusion has been applicated in developmental biology to elucidate the process of embryonic development and raise excellent strain. After combination with somatic cell nuclear transfer and somatic cell nuclear reprogramming, this technology can establish pluripotent stem cells which consistent with ethics principle and individual to avoid the rejection,and is available in the mechanism research.
  • Agrobacterium Tumefaciens-mediated Transformation of Trichoderma atroviride for Improvement of Laccase Activity
    China Biotechnology. 2010, 30(07): 0-0.
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    Trichoderma atroviride T23 was transferred with linear pBHT1 by agrobacterium tumefaciens-mediated transformation, and 6 mutants were obtained and further identified by conventional PCR technique. Guaiacol method has been used to screen mutant strains and found that 3 mutant strains produced laccase was significantly higher than original strain T23. In the limit carbon medium, the laccase activity of the mutant strain TA5 is 25.3 U/mL. It was significantly higher than the original strains. Study found that the mutant strain TA5 produced laccase is more appropriate for acidic conditions in the context of the measured pH. When the pH is 3.5 and the temperature is 18℃, the laccase activity is highest.
  • Strategic Alliance of Technology Innovation in Bio-pharmaceutical Industry
    China Biotechnology. 2010, 30(07): 0-0.
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    Abstract: The definition of “strategic alliance of technology innovation (SATI) in bio-pharmaceutical industry” is interpreted in detail in this paper. It is followed by the analysis of its international advanced experience in the triangle relationship of cooperation by production, study and research, and the illustration of significant role model of SATI in American bio-pharmaceutical industry and its operation. Then, the domestic status of SATI in bio-pharmaceutical industry is evaluated. In the end, development ideas and counter measures are suggested from four levels of governments, enterprises, universities and research centers, and alliances themselves.
  • Effect of Terpenoid in Plants Indirect Defense
    China Biotechnology. 2010, 30(07): 0-0.
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    Plants indirect defense releasing volatile terpenoids which attract herbivore enemies after attacking is a novel and environmentally-friendly insect-resistant pattern. Induced by insect damage and other factors, plants regulate themselves to release a blend of volatiles, including terpenoids, with which the herbivore enemies could locate their preys and hosts. Cooperated this indirect defense with direct defense would play a role in pest control.
  • The progress of Chloramphenicol and tetracycline inhibit bacterial protein secretion
    China Biotechnology. 2010, 30(07): 0-0.
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    Both chloramphenicol and tetracycline can inhibit bacterial protein secretion. A protein that is destined for secretion possesses a signal sequence at its amino terminus,which directs it to a translocator complex that is formed by secretion protein complex SecY, E, G, and A proteins. Protein translocation also depends on the folding characteristics of the fusion protein, misfolding of the protein after its translocation into the periplasm can lead to the formation of toxic aggregates. Fast-folding fusion protein can jam the translocator complex, which also leads to cell death (because all protein secretion is inhibited). Antibiotics chloramphenicol and tetracycline treatment bacteria caused the translocation complex in the degradation of SecY, then caused deadly protein jam. In this paper, we overview a new model of the interference mechanism of antibiotics chloramphenicol and tetracycline in bacteria protein synthesis and lead to the degradation of SecY to play inhibit bacterial protein secretion activity, in order to provide a scientific basis for studying new treatments that target the bacteria
  • The applications and progress of genome shuffling
    China Biotechnology. 2010, 30(07): 0-0.
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    Genome shuffling combined traditional random mutagenesis with cell fusion was a novel approach for whole genome shuffling. Genome shuffling based on recursive protoplast fusion, has been applied in the rapid improvement of multi- complex and desired cellular phenotypes without the necessity for Genomic and metabolomic background in detail. In this review, we attempted to introduce the process of genome shuffling, and present the advantage of this technology. This study also given the perspective on the development and application of this approach in future.
  • Expression Of The Hemagglutinin And Neuramidinase Gene Of Influenza A Virus H9N2 in P.methanolica
    China Biotechnology. 2010, 30(07): 0-0.
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    Objective To clone,express and characterize the HA（Hemagglutinin）and NA（Neuramidinase，NA）protein of avian influenza virus H9N2. Methods On the basis of successful clone the full length HA and NA gene and sequence analysis of avian influenza virus H9N2, part of the gene were ligated into pMET A.An expression vector pMETA/HA (52bp-1545bp)，pMET A/NA(121bp-1254bp)were constructed and expressed in pMAD16 induced by methanol.recombinant protein was purified through Ni2 affinity chromatography column. Western Blotting and ELISA were used to determine the antigenic of the recombinant protein. Results The recombinant capsid gene can be overexpressed in P.methanolica. SDS-PAGE showed that the product is a dimer.The purity of the protein is greater than 95%.ELISA and Western Blotting showed that the recombinant protein has good antigenic. Conclusion the HA and NA Protein of avian influenza virus H9N2 has been successful cloned and expressed, which could be useful for developing diagnose reagents or vaccine of H9N2.
  • Prokaryotic Expression, Purification of human 14-kDa phosphohistidine phosphatase and its interacting protein study in vitro
    China Biotechnology. 2010, 30(07): 0-0.
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    Objective: To express and purify human PHP14 protein and explore its interacting proteins in vitro. Methods: The cDNA sequence encoding PHP14 protein was amplified and subcloned into pGEX4T vector to construct the prokaryotic expression plasmid. The recombinant plasmid was transformed into E. coli BL21 strain and exogenous protein expression was induced by IPTG. After purification using Glutathione Sepharose 4B affinity chromatography, the GST-PHP14 fusion protein was separated by SDS-PAGE. GST-Pull down assay was performed to explore the interacting proteins in vitro and then subject to MALDI-TOF-MS identification. Results: GST-PHP14 fusion protein was expressed effectively in E. coli BL21 strain and successfully purified using Glutathione Sepharose 4B beads. Furthermore, GST-Pull down assay indicated that PHP14 could bind HSP90 in vitro. Conclusion: GST-PHP14 fusion protein was expressed and purified successfully and discoveried the interaction of PHP14 and HSP90 in vitro.
  • Prokaryotic Expression and RNase activity analysis of Rice XIOsPR10
    China Biotechnology. 2010, 30(07): 0-0.
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    It has been regarded that PR10s played an important role in systemic acquired resistance (SAR). Furthermore, many PR10 proteins have been reported to exhibit RNase activity and predicted to be responsible for its antibiotic activity. So we constructed a Prokaryotic Expression Vector pET28a-XIOsPR10. The recombinant protein XIOsPR10 was obtained by Prokaryotic expression and purification of magnetic microbeads, and showed the RNase activity.
  • Preliminary Research on the Interaction between SSX2IP and 14-3-3η
    China Biotechnology. 2010, 30(07): 0-0.
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    SSX2IP(SSX2 interacting protein) was identified as a tumor associated protein in recent studies. Here, the human liver cDNA library was screened with pDBLeu-SSX2IP as bait plasmid by yeast two-hybrid system and 14-3-3η was identified as a SSX2IP interactive protein. The interaction was confirmed by GST Pull-down and co-immunoprecipitation assays. Immunofluorescent localization experiments showed that SSX2IP co-localized with 14-3-3η in the cytoplasm and perinuclear area when co-transformed in HeLa cells. The CCK-8 assay which was employed to detect the effect on cell proliferation when SSX2IP was co-transformed with 14-3-3η in HEK293T cells showed that the proliferation of cells co-transformed with SSX2IP and 14-3-3η was higher than that of both control cells and cells transformed with SSX2IP or 14-3-3η. It seems that to do further research on the interaction of SSX2IP and 14-3-3η may be very important and useful.
  • The Inhibition Efect of RNA Interference Silence NRP2 Expression inTransplantation Tumor of Gastric Carcinoma in Athymic Mouse
    China Biotechnology. 2010, 30(07): 0-0.
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    Abstract AIM：To Investigate the effect on tumor growth and lymphangiogenesis by RNA interference to Specifically inhibit NRP2 gene expression in SGC7901 gastric cancer cells in nude mice .METHODS：To construct shNRP2 plasmid by the small RNA interference. To detect NRP2 protein expression before and after transfection byWestern blot. To establish the human gastric cancer cell SGC7901 xenografts in nude mice ,The modols were randomly divided into shNRP2 group (experimental group), shCon Group (HK negative control group) and normal control group to observe the growth of transplanted tumors.All mice were sacrificed after 6 weeks．The volume and Weight of the tumors were measured The distribution of NRP2 and MLD in the tumors was detected by immunohistochemistry. RESULTS ： ShNRP2 plasmid was constructed successfully , compared with the other two groups, NRP2 protein expression decreased significantly in shNRP2 group, and the transplanted tumor tissue growth, NRP2 expression and MLD significantly were inhibited. CONCLUSION：Inhibition of NRP2 gene expression, can inhibit the growth of gastric cancer xenografts in nude mice and the formation of lymphatic vessels, NRP2 gene may be a potential target for biological therapy of gastric cancer.
  • A Combinatorial Megaprimer PCR Strategy for Adjacent Multi-site-directed Gene Mutagenesis
    China Biotechnology. 2010, 30(07): 0-0.
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    Abstract A novel combinatorial multi-site directed mutagenesis strategy was developed, which performed in three-stage PCR in one tube. The multi-site mutagenic megaprimer is amplified in the first stage PCR, then the megaprimer are extended in the second stage PCR, and the full length mutant sequence is generated in the third stage PCR. In this strategy, based on combinatorial megaprimer reactions of the annealing temperatures and thermo-cycles, the multi-site directed and adjacent mutagenic DNA fragments were successfully amplified with the small annealing temperature difference (lesser than 10°C) between three stage PCRs. It is shown that this method is simple and high efficient.
  • Improved GLGI Method to Identify Tags from LongSAGE of Silkworm，Bombyx mori
    China Biotechnology. 2010, 30(07): 0-0.
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    Generation of Longer cDNA Fragments from SAGE tags for Gene Identification（GLGI）being as a new tag identification technique was brought about with the serial analysis of gene expression (SAGE). Its result is an EST in the cDNA isolated by the 3 'end and with a ployA tail. Their steps are complicated and the cost is relatively high. Based on the improvement details reported, some new improvement was conducted to aim directly at two LongSAGE libraries of silkworm constructed by our laboratory. 30 differentially expressed tags were selected to improve the GLGI method：(1) Total RNA was used to synthesis dsDNA instead of mRNA; (2) The order of beads adsorption of DNA was adjusted, so as to diminish loss of template and simplify process; (3) DNA ordinary polymerase in PCR reactions was used instead of the high-fidelity enzyme Pfu. Then sequence results were analyzed BLAST against Bombyx mori database on NCBI. Annotation result of 3 tags was conformity with known annotation, it provided a verification proof that improved test is reliable. There are also some tags without annotation, which can be taking as a foundation for further study of novel genes. This try provides a valuable reference system for other tags and novel genes research of SAGE library.
  • Proteomic analysis of ischemic postconditioning on rabbit myocardium with Two Dimensional Polyacrylamide Gel Electrophoresis.
    China Biotechnology. 2010, 30(07): 0-0.
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    【Abstract】 Objective: To analyse the differential expression of proteome in postconditioning and ischemic reperfusion rabbit myocardial tissue. Methods: six New Zealand white rabbits were randomly assigned toI/R group and ischemic postconditioning group. I/R groups underwent ligation of the left anterior descending coronaryartery(LAD) for 30 minutes and reperfusion for 180 minutes. Ischemic postconditioning group, four circles of myocardial ischemia(30s) and reperfusion(30s) followed the index ischemia/reperfusion protocol. Results: Differential analysis using the Student’s t-test(P<0.05) showed that the expression of 11 protein spots changed, Conclusions: Ischemic postconditioning resulted in the changes of protein expression profiles in the myocardium. The differential proteins may be involved in the cardioprotection of Ischemic postconditioning
  • Establish an ELISA Method for Screening Agonists of the Rattus Norvegicus LXRβ
    China Biotechnology. 2010, 30(07): 0-0.
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    Objective：Nuclear receptor LXR ligand-binding domain was cloned and heterogeneously expressed in E.coli, followed by Ni-affinity chromatographic purification. Purified protein was used to establish an ELISA method for screening LXR activators. Method: A DNA fragment encoding LXR ligand-binding domain was amplified by PCR using pSG5/rLXRβ as template. The DNA fragment was sequenced and cloned into an E.coli expression vector pET28a(+) which was transformed into E.coli strain BL21(DE3) for expression. Expressed protein was purified by Ni-affinity chromatography and analyzed using SDS-PAGE and Western blotting. A synthetic peptide FITC-EAEEPSLLKKLLLAPANTQ was used along with HRP-conjugated anti-FITC polyclonal Fab antibody in an ELISA method to detect ligand-dependent receptor/co-activator interaction. Result: rLXRβ-LBD was successfully expressed in E.coli in high yield (30% of total protein). After affinity purification, full-length target protein was more than 90%. Using ELISA-based co-activator recruiting assay, LXR activator hyodeoxycholic acid dimethyl amide had an apparent Kd of 2.5μM, which was in good agreement of a cell-based dual reporter gene assay. Conclusion: An in vitro LXR ligand screening method was established which may be further improved for high-throughput drug screening.
  • Organophosphorus Pesticides (Ops) Operate to Arabidopsis Thaliana by the Different Expression Protein and Inhibiting the Activity of Esterase Isoenzymes
    China Biotechnology. 2010, 30(07): 0-0.
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    Plants can tolerate or even degrade organophosphorus (OPs), yet with little known about the mechanisms. Employing Arabidopsis thaliana, a model plant, this paper studied the response of the esterase isozymes and the total soluble proteins to OPs stress. The results showed that OPs could be uptooke by Arabidopsis thaliana roots rapidly into the plant, repressed the activity of esterase isoenzyme without reduction in the enzyme content, promoted the production of one different protien highly homologous to the large-subunit sequence of its own ribulose-1,5-bisphophate carboxylase/oxygenase (RuBisCO) and finally, markedly inhibited the plant development. Also, the negative effects of OPs on the plant enhanced with the increase of OPs amount and treating time. This strongly suggested that esterase isoenzyme. probably was the target enzyme of OPs, like in animal, and the different expression protein may also be involved in the response to OPs.
  • Current Status and Trends Analysis of Thailand Bioindustry Development
    China Biotechnology. 2010, 30(07): 0-0.
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    Abstract: Thailand is a world leader in the production and export of many agricultural products，the government place importantce on bioindustry development, and has made great progresses in bioagriculture, biomedicine and bioenergy industry, through establishing high level administration, launching privileges policies, formulating development framework. Thailand will emphasize more on the government guideline, continue to modifying related laws, strengthen the attraction of excellent talents, implement industry privileges policies, strengthen protection of bioresource and intellectual property rights, prioritize bioagriculture, biomedicine and bioenergy development.
  • Research in Bacterial Ghost as DNA VaccineDelivery System
    China Biotechnology. 2010, 30(07): 0-0.
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    Low immunization response is a major impediment for DNA Vaccine development. Bacterial ghost (BG) is an empty gram-negative bacterial envelope generated by the controlled expression of the PhiX174 lysis gene. Since the native cell wall components are preserved during the formation of ghosts, BG keeps the natural intrinsic adjuvant properties and the structural integrity of bacteria that can elicit both humoral and cellular immune response. Besides, the sealed periplasmic space of BG can be used for loading DNA, which enables targeting delivery of DNA vaccine into antigen presentation cells. Furthermore, BG loaded with DNA vaccine can be administrated through various means, such as intramuscularly, subcutaneously, orally and mucosally. Therefore, BG is a promising carrier system to highlight the use of DNA vaccine. In this paper, the characteristics of BG and the role of BG as delivery carrier of DNA vaccine are discussed.
  • Enhancement of Microbial Transglutaminase by Streptomyces hygroscopicus CCTCC M203062 Using Tripsin
    China Biotechnology. 2010, 30(07): 0-0.
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    Effects of Tripsin addition on the accumulation of microbial transglutaminase(MTG) by Streptomyces hygroscopicus CCTCC M203062 were investigated. The results showed that the addition of Trypsin enhanced MTG accumulation, and the optimal addition time and concentration of Trypsin were at 0 h and 200 U/mL. The maximum MTG activity in the culture broth was 6.61 U/mL and increased by 27.1% compared with the control. With the addition of Trypsin, pro-TGase was cut to MTG, thus we presume adding Trypsin into the culture broth can enhance the production of pro-TGase by relieving the product inhibition, and the accumulation of MTG was improved.
  • Status and Trends Analysis of Thailand Bioindustry Development
    LI Rui-Guo, YIN Jun-Xiang, ZHANG Da-Lu
    China Biotechnology. 2010, 30(07): 116-120. https://doi.org/Q81
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    Thailand is a world leader in the production and export of many agricultural products.The government place importantce on bioindustry development,  and has made great progresses in bioagriculture, biomedicine and bioenergy industry, through establishing high level administration, launching privileges policies, formulating development framework. Thailand will emphasize more on the government guideline, continue to modifying related laws, strengthen the attraction of excellent talents, implement industry privileges policies, strengthen protection of bioresource and intellectual property rights, prioritize bioagriculture, biomedicine and bioenergy development.

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