25 February 2013, Volume 33 Issue 2

    

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  Construction of Bovine Lactoferricine Transgenic Mice

  ZHANG Jin-xia, FAN Zhi-qiang, LIU Ye, ZHANG Shou-feng, YANG Yang, WANG Ying, ZHANG Fei, HU Rong-liang

  China Biotechnology. 2013, 33(2): 9-13.

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  Purpose: To investigate the expression of bovine lactoferricine in mammary gland of the transgenic mice and the antibacterial activity. Methods: Mammary gland expression vector PI-bcp-LfcinB containing the goat β-casein promoter and bovine lactoferricine gene was digesting with Xho I and Nru I. DNA fragment containing the expression elements was gained for microinjection. About 500 fertilized eggs of mice were injected using classic microinjection technique,and about 430 living eggs were transplanted into 13 pseudopregnant mice,among which 6 pregnant mice produced 13 offspring.These offspring were identified by PCR,1 mice was bovine lacteferricine transgenic mice.Target gene can be transferred by the transgenic mice,which was proved by PCR. The expression of bovine lactoferricine of transgene mice was detected by RT-PCR and Kirby-Bauer. Results: These tests showed that the mammary-tissue-specific expression vector driven by the goat beta-casein gene promoter could efficiently direct the expression of bovine lactoferricine in mice milk. And the expressed product exhibited evident bactericidal activity to Escherichia coli. Conclusion: It is feasible to get bioactive bovine lactoferricine through the mammary gland of animal and it provides a basis for disease-resistant animal breeding and production of antibacterial peptides through transgenic animals.
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  Effects of Humoral Immune Response Vaccinated by Sequential Inactivated-live Rotavirus Vaccine

  ZHANG Biao, TONG Lin, YI Shan, ZHANG Guang-ming, LI Hong-jun, SUN Mao-sheng, CHEN Dong

  China Biotechnology. 2013, 33(2): 14-20.

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  Objective: To evaluate the humoral immune responses vaccinated by sequential inactivated-live rotavirus vaccines, primary immune with inactivated rotavirus vaccine (IRV) and boost immune with live rotavirus vaccine. Methods: The mice were randomly divided into four groups (oral vaccine group, sequential vaccine group, oral control group and sequential control group) and immunized by respective protocols. Then, the levels of serum rotavirus specific IgG, IgA and fecal rotavirus specific IgA were assessed by ELISA, the levels of serum neutralizing antibody were measured by microneutralization assay, the rotavirus sheddings in fecal following live rotavirus vaccine vaccination were assessed by ELISA. Results: Compared to sequential control groups, the levels of serum IgG, IgA, neutralizing antibody and fecal IgA were higher in sequential vaccine groups. Compared to oral vaccine groups, sequential vaccination induced the higher levels of serum IgG, IgA, neutralizing antibody, but there was no significant difference for the levels of fecal IgA between two groups. At the same time, it was found that there were no significant differences for the amounts and time of rotavirus shedding after first vaccination of live rotavirus vaccine between sequential vaccine groups and oral vaccine groups. However, after second vaccination of live rotavirus vaccine in sequential vaccine groups, the amounts and time of rotavirus shedding rapidly decrease, which were similar to the rotavirus shedding after third vaccination of live rotavirus vaccine in live vaccine groups. Conclusion: The systemic and mucosal humoral immune responses were induced by sequential inactivated-live rotavirus vaccines in mice. The protocol of vaccination with sequential inactivated-live rotavirus vaccines may be used as a candidate protocol for clinic application of rotavirus vaccine in the future.
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  Effects of RUNX2 on BMP9-induced Osteogenic Differentiation of C3H10T1/2 Mesenchymal Stem Cells

  ZHAO Yan-fang, SONG Tao, LIU Yue-liang, LUO Jin-yong

  China Biotechnology. 2013, 33(2): 21-26.

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  Objective: To investigate and validate the effects of RUNX2 on BMP9-induced osteogenic differentiation of C3H10T1/2 mesenchymal stem cells.Methods: Expression level of RUNX2 was measured by luciferase activity assay, RT-PCR and Western blot, and the effects of RUNX2 on BMP9-induced osteogenic differentiation of C3H10T1/2 were detected by Alkaline phosphatase staining, Alizarin red S staining, immunocytochemical and entopic bone formation.Results: BMP9 increased the expression level of RUNX2.In vitro,RUNX2 increased BMP9-induced early osteogenic marker ALP activity and late osteogenic marker calcium deposition, however, inhibited OCN expression.In vivo, RUNX2 enhanced BMP9-induced entopic bone formation.Moreover,RUNX2 gene silence was showed to inhibit ALP activity,calcium deposition and entopic bone formation.Conclusion: In a word,RUNX2 may promote BMP9-induced osteogenic differentiation of C3H10T1/2.
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  Effects of High Magneto-Gravitational Environment on Pseudomonas aeruginosa N1207

  ZHAO Jian-feng, XIN Xing, WEI Pei-pei, QIAN Ai-rong, Akateh Tazifua Alfred, SHANG Peng, YANG Shu-lin

  China Biotechnology. 2013, 33(2): 27-33.

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  The aim is to investigate the effects of a High Magneto-Gravitational Environment (HMGE) produced by a superconducting magnet on Pseudomonas aeruginosa N1207. A specially designed large gradient superconducting magnet was used to produce gravity levels from hypo-gravity (0 g) to hyper-gravity (2 g) and relevant magnetic field strengths, namely (0 g, 12 T), (1 g, 16 T) and (2 g, 12 T). After Pseudomonas aeruginosa N1207 was mutated in HMGE for 24h, 48h, 72h, respectively, the mutant M14808 with the highest yield of rhamnolipids was isolated from the strongest magnetic field (16 T). Results showed that the rhamnolipid production of M14808 was dramatically increased by over 30%, and maintained a constant yield for 6 generations. Comparing the growth cycle of the original strain and mutants, it was observed that mutants reached the exponential phase earlier than the original strain. The mutation result indicated that the high magnetic field was primarily responsible for the production of rhamnolipids and the growth rate. The antitumor cytotoxicity experiment showed that crude di-rhamnolipids could inhibit the growing of four cancer cell lines, MCF-7, H460, HepG2 and A549, with the IC₅₀ of di-rhamnolipids to MCF-7 as low as 125.13μg/ml.
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  Effects of Temperature Stress on the Accumulating of Triterpenes and Defense Enzyme Activity in Suspension Cells of Birch (Betula platyphylla Suk.)

  ZHAO Wei, YIN Jing, ZHAN Ya-guang, REN Chun-lin, WANG Yan, MA Hong-si, SU Xin

  China Biotechnology. 2013, 33(2): 34-40.

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  After 7-d-suspending-culture cells of Birch (Betula platyphylla Suk.) which were treated with two kinds of temperature stress: the high (50 ℃ for 1h) and low temperature (4℃ for 3h),respectively, they were continuously suspended culture for 0 h～7d, and during that time, the growth, accumulation of total triterpenes and oleanolic acid, and the activities of defense enzyme were investigated. The results showed that: for the total triterpenes, its accumulation significantly increased in the cells suspending-cultured for 24～3d after both the high and low temperature treatments; it was higher in these cells treated with high temperature than low temperature; and its highest was in the cells suspending for 48 h after high temperature treatment, higher 36.4% and 12.87% than those after control(25℃) and low temperature treatment, respectively. The accumulation of total triterpenes in the 4d～7d suspending-culture cells after low temperature treatment, also obviously increased, and higher 22.1% in the 7-d suspending-culture cells than that in control. For the oleanolic acid, its accumulation in the cells suspending-culutre for 15h～4 d was significantly improved after both the high and low temperature treatments, and they in the cells suspending-culture after high and low temperature treatments were higher 38.1% and 39.65% than control, respectively. The activities of defense enzyme: SOD, CAT, PAL and POD in the cells treated with the high and low temperature, were accelerated, and the accumulation of MDA (malondialdehyde) was higher. But their activities changes were different. Those results suggested that the interaction of these defense enzymes in cells relieved the free radical hurts, which made cells adapt to tremperature stress, and promote the secondary metabolism materials.
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  Optimization of Fermentation in Shake Flasks for the Xylanase in Recombinant E. coli

  HE Jie, SU Ling-qia, WU Jing

  China Biotechnology. 2013, 33(2): 41-46.

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  In order to achieve high production of the xylanase, the optimization of culture medium was investigated in E. coli BL21(DE3) harboring the plasmid pET20b(+)/coe/xynA. The optimized medium and induction condition were as follows: 11 g/L glycerol, 24 g/L yeast extract, 8 g/L peptone, 89 mmol/L PO₄^3-, 4 mmol/L Mg²⁺, induced by 0.4 mmol/L IPTG at 6h of culture. Under this condition, the enzyme activity increased from 346.8 U/ml to 780.2 U/ml, which was 2.2 times as high as that when it was not optimized.
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  Asymmetric Reduction of 2-Octanone with the Yeast Carried on Macromolecule Membrane

  XIONG Long-bin, ZENG Hong-yan, HE Ping, TAN Yi-zhe, LIU Xue-ying, ZHANG Cun-ying, CAI Xi-ling

  China Biotechnology. 2013, 33(2): 47-53.

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  The baker yeast DX213 (Saccharomyces cerevisiae DX213) was immobilized on the activated woven nylon-6 membrane using glutaraldehyde as a cross-linking agent. In the process of asymmetric reduction of 2-octanone to 2-octanol by the immobilized yeast cells, the effect of organic solvent, reaction time, pH, 2-octanone, co-substrate and heating treatment on the yield and enantiomeric excess(e.e. value) of (R)-2-octanol was studied. The function mechanism of the influence factors with enzymatic activity of the cells was presented in the reaction. The results showed that all the mentioned factors had effects on the reaction. Dichloromethane was found to be the optimal organic solvent for the reaction. Under the conditions of 57 g/L immobilized cell preheated at 50℃ for 50 min, 4/1 volume ratio of water phase to organic phase, pH7.0, 60 mmoL/L initial 2-octanone concentration added in three separate doses at 0, 10 and 27 h, 5.7 g/L sucrose concentration, 28℃ and reaction time 48 h, the yield and enantiomeric excess of (R)-2-octanol were 89.3 % and 96.8%, respectively.
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  Enhancement of Transglutaminase Production in Streptoverticillium mobaraensis as Achieved by Treatment with NaCl

  CHEN Jia, JIN Ming-fei, TAN Yu-jing, LIU Ying, TIAN Pei-lin, CHANG Zhong-yi, BU Jian-guo

  China Biotechnology. 2013, 33(2): 54-58.

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  The effects of three inorganic salts on the production of transglutaminase (TGase) in Streptoverticillium mobaraense were tested. It indicated that enzyme activity increased in the presence of 0.5% MgCl₂ or 0.5% NaCl compared with control, and 0.5% NaCl functioned better. The SDS-PAGE results showed that the total amount of pro-TGase and TGase in each sample points of experimental group was more than that of control group, and the increase of TGase of experimental group was faster than control. So NaCl had a significant effect on the TGase production by promoting the secretion of proenzyme (pro-TGase) and the maturation of TGase. Furthermore, the optimum addition of NaCl was 0.5% so that the TGase activity increased by more than 12%, and the fermentative period shortened 15 hours.
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  Effect of Carbon, Nitrogen Sources on Lipid Accumulation and Fatty Acid Composition of Monoraphidium sp. FXY-10 under Heterotrophic Cultivation

  HUANG Li, HE Ci-an, ZHAO Peng, YU Xu-ya

  China Biotechnology. 2013, 33(2): 59-64.

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  Effect of carbon and nitrogen sources on heterotrophic cultivation of microalgae Monoraphidium sp. FXY-10 is studied. The biomass, oil accumulation and fatty acid composition of microalgae cell are analysed comparatively by selecting various types and setting different concentrations of carbon and nitrogen sources with BG-11 as basic medium. The results show that microalgae lipid accumulating with glucose as carbon source and sodium nitrate as nitrogen source is an ideal candidate for biodiesel production. The lipid production is not significantly impacted (P>0.05) by different sodium nitrate concentration which was 1.00, 3.00 and 5.00 g/L, respectively. Moreover, when glucose concentration is 10.00 g/L using sodium nitrate as nitrogen source, lipid production can be obtained the highest experiment value 0.84 g/L. And the fatty acid of microalgae lipid is mainly composed of short chain saturated and monounsaturated fatty acids, such as C16:0 and C18:1. So they result a lower degree of unsaturation (DU) value 61.98.
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  Study on Recombination and Preparation of PACAP-derived Polypeptide RHMP and Its Effects on Promoting Corneal Wound Healing

  XU Wen-na, MA Yi, YE Zu-lu, LUO Tian-jie, RAO Lei, HONG An

  China Biotechnology. 2013, 33(2): 65-69.

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  Preparing pituitary adenylate cyclase-activating polypeptide (PACAP) recombinant peptide RHMP using genetic engineering techniques and exploring its biological effects on corneal wound healing in vitro can provide novel candidate drug and method for the treatment of corneal injury and research. Using genetic recombinant technology to prepare recombinant peptide, expressed fusion protein were purified with Chitin-Beads columns, the released polypeptide were analyzed and identified with HPLC, SDS-PAGE and MS, then studied on its effects on corneal wound healing in mice. Experimental results showed that the molecular weight of RHMP is 3.4 kDa, its purity is 96%. In addition, the corneal repair rates of experimental group after injury are higher than control, the difference is very significant. Therefore, realized efficient synthesis of RHMP, and it can significantly promote corneal wound healing in mice, so it will likely to become a new drug for corneal injury. At the same time the corneal epithelial wound model in mice can be effectively used to study on the biological effects of the related drugs.
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  Studies on Purification and Properties of Rabbit Muscle Glycerol 3-Phosphate Dehydrogenase

  WANG Zhen-wei, LI Gang-rui, LI Lin-li, WANG Shuai-kun, MENG Yan-fa

  China Biotechnology. 2013, 33(2): 70-76.

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  Objective: To explore the purification and properties of glycerol 3-phosphate dehydrogenase(GPDH) from rabbit muscle and provide theoretical support for its applications on enzyme linked diagnosis reagents in vitro. Methods: Glycerol 3-phosphate dehydrogenase from rabbit muscle was purified by ammonium sulfate fractional precipitation, DEAE-sepharose, Blue-sepharose and Hydroxyapatite. Sephacryl S-200HR chromatography and gradient PAGE were used to determine its molecular weight, kinetic properties such as effect of pH, temperature, substrate concentration and chemicals were also determined. Results: The purified enzyme showed a prominent single band on PAGE, and its molecular weight was estimated to be 115～122 kDa. The enzyme was stable below 45℃ and pH 6.0～9.0 with maximal activity at 45℃, pH 9.0. K_m value of the enzyme was calculated to be 7.4×10^-3mol/L for substrate glycerol 3-phosphate, and to be 1.47×10^-4mol/L for substrate NAD⁺. Ba²⁺, Mn²⁺, Fe²⁺, Al³⁺, Cu²⁺, Ni²⁺, Ag⁺, Hg²⁺, NaN₃ and EDTA inhibited the activity of GPDH at various degree, whereas Mg²⁺, Ca²⁺, Co²⁺, Zn²⁺ could stimulate the enzyme activity, NaF had no obvious effect on glycerol 3-phosphate dehydrogenase.
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  Study on Generation of Bovine Induced Pluripotent Stem Cells Treated by piggyBac

  ZHAO Li-xia, ZHANG Jin-dun, ZHANG Jian, LI Yun-xia, SU Jie, SUN Wei, DAI Yan-feng, GUO Ji-tong, ZHAO Gao-ping, HU Shu-xiang, WANG Wei, LIU Pen-tao, LI Xi-he

  China Biotechnology. 2013, 33(2): 77-82.

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  Authentic bovine embryonic stem cells have not been established despite over two decades efforts. Recently, induced pluripotent stem cells (iPSCs) derived from somatic cells have been successfully generated from mouse, human, monkey, rat, pig, sheep, horse and cow. However, the pluripotency of large domestic animal iPS cells have not yet been tested by generation of chimera and germline transmission. Differently to virus vector, piggyback transposon was used to reprogram bovine fetal fibroblast (BFF) cells into iPS like cells. The piggyback transposon and transposase system is simple、virus-free and efficient. It just need transposase enzyme to catalyse insertion or excision events.It was reported that coexpress mouse Rarg(RAR-γ) and Lrh-1 (liver receptor homologue 1) with the four factors Oct4、Sox2、Klf4 and c-Myc can obtain bovine iPS like cells. These cells exhibited typical colony morphology, like mouse ES cells. They showed normal karyotypes. Bovine iPS like cells also express ES cell markers, including Oct4、Sox2、Dnmt3b、E-cadherin、Dppa4、Stella and stage-specific embryonic antigen-4(SSEA4), TRA-1-60, TRA-1-81. The cells were able to differentiate into all three germ layers both in vitro and in teratomas. The results showed that the piggyback is an efficient tool for iPS cell derivation.
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  Two-step Fermentation of Carotenoid by Blue Light Induction in Cordyceps militaris L.

  SHEN Jun-liang, JIN Hua-yan, GAO Ya, ZHA Mei-ling, FU Ming-jia

  China Biotechnology. 2013, 33(2): 83-87.

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  Utilizing the dark shake flask culture and the static culture of blue light irradiation in the two-step fermentation, the blue light induced the carotenoid production of Cordyceps militaris L mycelia. The results showed that after 2 days shake flask in the darkness and 5 days static culture in the blue light, Cordyceps militaris L carotenoid content reached a maximum value 558.4μg/gFW. But adopting the dark shake flask culture of 2 days and different times static culture in blue light, the results show that for the first 2 days of blue light irradiation, the increase of Cordyceps militaris carotenoids content was not obvious, then a rapid increase was obtained and a maximum 558.4μg/gFW was reached in the fifth day, subsequently the content of carotenoids did not significantly change. The problem about imposed blue light is solved in the carotenoid production of Cordyceps militaris L fermentation.
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  Fermentation Optimization Directed by Physiological Parameter Respiratory Quotient

  WANG Ping, WANG Ze-Jian, ZHANG Si-liang

  China Biotechnology. 2013, 33(2): 88-95.

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  Online measurement of process parameters play a fundamental role in optimization and control of fermentation process engineering. Respiratory quotient(RQ) is the response of intracellular microscopic flow on global metabolic parameters, which reflect the utilization of substrates, product and by-products synthesis, and the changes of metabolic pathways and flux in microbial cultivation. The optimal controlling strategies of fermentation process based on the combination of micro-intracellular metabolism and macro-physiological parameter RQ in several industrial products production were investigated. RQ, measured by exhaust gas analysis online, is a crucial factor in optimization of the intracellular metabolic pathway flux through the regulation of global metabolic parameters and improvement of the yields of target product.
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  RANTES Derivates and HIV-1 Entry Inhibitor

  NIE Lun, WU Wen-yan

  China Biotechnology. 2013, 33(2): 96-102.

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  HIV-1 entry inhibitor is a new hot spot in the area of AIDS drug development. The identification of CCR5 as the main HIV co-receptor have triggered a wealth of research about CCR5-targeting inhibitors. Natural ligands of CCR5, RANTES, MIP-1α, and MIP-1β are potent HIV-1 inhibitors. Lots of efforts have focused on RANTES engineering aiming at enhancing the antivirus ability of the native molecule, while reducing its side effect. Here an overview of RANTES derivates developed as HIV-1 entry inhibitors is presented.

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  Effect of Illumination on Microalgae Cultured at High Cell Density in Photo-bioreactor

  LI Yong-fu, MENG Fan-ping, LI Xiang-lei, MA Dong-dong

  China Biotechnology. 2013, 33(2): 103-110.

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  Photo-bioreactors (PBR) are important device for realizing high-density culture of microalgae. One of the key technologies for designing PBR is to choose the appropriate illumination mode. According to related research achievements domestic and international in recent years, the present research conditions of illumination technique in PBR were reviewed. An introduction is emphatically given of the effect of two factors as incident light properties (including light source, light intensity, light quality and light dark cycle etc.) and light energy distribution, on the microalgae growth. The future research directions are also put forward. It could provide reference for the efficient PBR system design and optimize.

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  Scanning International Bioeconomy Strategies

  CHU Zong-ling, ZENG Hai-yan, DENG Xin-an

  China Biotechnology. 2013, 33(2): 111-116.

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  The formation of the concept "bioeconomy" has been promoted by the rapid development of life sciences and biotechnology. On the basis of systematic survey to the origin and evolution of the bioeconomy concept, several common mainstream features of the bioeconomy strategies have been induced by panoramic scanning and comprehensive analyses to international bioeconomy strategies and policies. These features are concluded as below: the bioeconomy covers many areas such as agriculture, medicine & healthcare, biorefinery, environmental protection, etc., which all are confronted with green transformation; paying great attention to the sustainability of bioeconomy, which has been taken as a synthetic platform to a sustainable future; taking biomass as the underpinning resource that drives the bioeconomy, and strengthening R&D investment to innovative technologies for sustainable production and conversion of biomass.