25 March 2006, Volume 26 Issue 03

    

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研究报告
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  Studies of the Expression and Biologic Activity of an Anti- transferrin Receptor ScFv-BDNF Fusion Protein

  China Biotechnology. 2006, 26(03): 1-5.

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  The brain-derived neurotrophic factor had nutrition, repair and protection functions to neurons of central nervous system, but its utilization was restricted by the blood-brain barrier. This paper used the single-chain antibody (ox26-scFv) to transferrin receptor(TfR) as vector to transport BDNF. The ox26-scFv gene and BDNF gene were amplified by PCR from plasmid PUC19/ ox26-scFv and plasmid PUC19/BDNF, inserted into the prokaryotic expression vector pTIG-Trx which carried a thioredoxin (Trx) gene and a C-terminal His.tag separately, constructed a pTIG-Trx/scFv-BDNF recombinant plasmid. The ScFv-BDNF fusion proteins were expressed at 20℃, after 0.02mM IPTG induction in the strain E.coli BL21(DE3). The soluble ScFv proteins in cytoplasm suspension were purified by immobilized metal chelation affinity chromatography(Ni-NTA), a single band with molecular weight 41 kD appeared on SDS-PAGE gel. Immunocytochemistry staining showed that ScFv-BDNF fusion proteins could recognize and bind to transferrin receptors on the surfaces of rat GH3 cells, and the fusion proteins could promote growth of the chick embryo dorsal root ganglion (DRG), which indicated that the fusion proteins not only could combine with transferrin receptors specially but also had biological activity of BDNF. These results would lay groundwork to transport BDNF across BBB as a CNS therapeutics.
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  The Synthesis of Novel Protein Modification Reagent and its Application of Modifying Bovine Hemoglobin

  China Biotechnology. 2006, 26(03): 6-10.

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  A novel tetrafunctional reagent for protein modification was successfully synthesized by L-glutamic acid and adipic acid, and it was characterized by H1-NMR and FTIR. The new reagent was then applied to modify bovine hemoglobin, and the preliminary properties of modified protein were characterized by HPLC, SDS-PAGE and Hemox Analyzer. The results showed that the intra-molecular and inter-molecular cross-linking of bovine hemoglobin was formed simultaneously by using the new reagent, and the modified hemoglobin performed well for O2 delivery (P50: 21.7 mmHg, Hill coefficient: 2.01). Therefore, the new cross-linking reagent is promising in development of stroma-free hemoglobin based artificial blood substitutes.
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  Research on the Expression of Human Coagulation Factor IX in Tobacco

  China Biotechnology. 2006, 26(03): 11-16.

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  Hemophilia B is a bleeding disorder caused by deficiency or abnormality in the plasma coagulation protein factor IX. Currently treatment method of hemophilia B is based on protein replacement therapy by blood transfusion and intravenous infusion of condensate of coagulation protein factor IX with excellent therapy effect, which also has some problems including expensive costs, and high risks of virus infection by blood filtering. So how to obtain human factor IX more cheaply and safely is very important for the cure of hemophilia B. Plant expression system own advantage on cost and security, which showed good potential for production of human factor IX. Therefore, in this work, plant binary expression vector p35S-2300：：hFIX（2.8kb）：：noster was constructed and introduced into Agrobacterium tumefaciens strain EHA105 by freezing-melting method. The resulting vector was introduced into tobacco（Nicotiana tabacum L. cv. Bai Rihong）plants via Agrobacterium tumefaciens -mediated method. Total 4 independent transgenic tobacco plants confirmed by PCR and Southern blot analysis were obtained. The hFIX gene was introduced into genome of transgenic tobacco plants, ranging from 1 to 4 copies. The expression of the introduced gene in transgenic T0 plants was confirmed by RT-PCR and ELISA analysis at transcript and translation level, respectively. Content of hFIX ranged from 2.5 to 8.8ng/g·FW in fresh leaves of transgenic tobacco with immunocompetence. This work provides good foundation for further expression of hFIX and is also helpful for expression of other pharmaceutical proteins using plant expression system.
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  Setting up of the tumor angiogenesis model in vitro

  China Biotechnology. 2006, 26(03): 17-21.

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  We set up the tumor angiogenesis model in vitro by embedding the rat aortic rings in collegen and culturing them in serum-free MCDB 131 culture medium with or without lung cancer A549 cells. This model was based on the rat aortic ring model. In both control cultures and cultures supplemented with lung cancer A549 cells, most of the microvessels sprouted from the cut edges of the aortic rings. Microvessels usually appeared by the third day, at their maximal growth by days 6-10 and began to degenerate by the end of the second week, but cultures supplemented with lung cancer A549 cells could generate more microvessels than control. We concluded from current study that Lung cancer A549 cells could promote angiogenesis. The rat aortic ring model is simple and sensitive. It is a promising meathod to study angiogenesis and its mechanisms.
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  Comparison of Cellular Immunity Raised by a Novel Adjuvant DC-Chol with Different HBsAg Preparations

  China Biotechnology. 2006, 26(03): 22-25.

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  Objective: To screen candidate antigen for therapeutic HBV vaccine with a novel adjuvant DC-Chol. Methods: BALB/c mice were injected with DC-Chol liposome and HBsAg prepared from CHO and Yeast respectively. One week later, IL-4, IL-2, IFN-γwere measured by ELISA or ELISPOT. Results: The levels of IL-2, IFN-γof HBsAg from Yeast with DC-Chol liposome were 20 and 119 times higher respectively than those of HBsAg from CHO with DC-Chol liposome. ELISPOT assay showed that the counts of spot-forming cells of IL-4 and IFN-γof HBsAg from Yeast with DC-Chol liposome were 2.8 and 46.3 times higher respectively than those of HBsAg from Yeast with Al(OH)3. Conclusion: HBsAg prepared from Yeast together with DC-Chol liposome may be an appropriate candidate for therapeutic HBV vaccine .
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  Expression and Purification of Human Parathyroid Hormone Peptide（1-34） in Escherichia coli

  China Biotechnology. 2006, 26(03): 26-30.

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  Human parathyroid hormone peptide1-34(hPTH1-34) was highly expressed in Escherichia coli by inserting the synthesized hPTH1-34 cDNA into pThioHis, the prokaryotic expression vector. The expressed hPTH1-34 was purified by chelating sepharose immobilized metal ion affinity, reverse and filter chromatographic steps. Its purity was verified above 95% by HPLC. The quality was identified by N-terminal sequencing and MALDI-TOF-MS analysis. In vitro analysis showed the adenylate cyclase of ROS 17/2.8 cells was activated by hPTH1-34.
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  Soluble Expression and Purification of Snake Venoms Fihrino(geno)lytic Emzyme Alfimeprase in E.coli

  China Biotechnology. 2006, 26(03): 31-36.

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  Fibrolase is a non-hemorrhagic zinc metalloproteinase isolated from southern copperhead snake venom (Agkistrodon contortrix contortrix) and is capable of degrading fibrin clots resulting from purified fibrinogen or from blood plasma. Alfimeprase, a truncated form of fibrolase, as a clinical agent was successfully completed PhaseⅡclinical trials.The cDNA of alfimeprase was amplified by recursive PCR, digested with BamHⅠand HindⅢ, and cloned into pET43.1a, pMALp2X and pMALc2X vectors to generate fusions with NusA, MBP and sMBP(with signal peptide), respectively. Nus/alfimeprase was expressed in soluble form by co-expressing with chaperone FkpA and inducing with1mmol/L IPTG. The fusion protein accounted for about 25 % of total protein following cell lysis. Alfimeprase was successfully purifiesd by Ni-NTA affinity chromatography and cleaved by enterokinase. The results demonstrate the fibrinolytic activity of recombinant alfimeprase using fibrin plate assays and fibrinogen hydrolysis.
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  Expression of VP6 Gene from Group a human rotavirus in E.coli

  China Biotechnology. 2006, 26(03): 37-41.

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  The structural protein VP6 of rotavirus form the middle layer of the triple-layered viral capsid, playing a key role in the organization of the virion. The gene of structural protein 6 of rotavirus strain TB-Chen isolated from a clinic sample was amplified using PCR from the reverse transcription product of RV genome RNA, using pET as expression vector, a recombinant plasmid pET-VP6 containing coding sequence of VP6 protein was constructed. The results showed that the VP6 was highly efficiently expressed in E. coli BL21(DE3) cells which were transformed with the recombinant plasmid pET-VP6.The expressed VP6 protein possessed 27.4% of total cells protein, with an approximately 45kDa of molecular weight, and could be recognized by guinea pig anti-SA11 antibody on Western blot. The results obtained in this report provide important basis for further study on structure and function of the VP6 protein.
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  Modification，expression and purification of human endotoxin binding peptide gene

  China Biotechnology. 2006, 26(03): 42-46.

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  Objective To modify EBP(endotoxin binding peptide), clone and express the mutate of EBP gene and gain purified mEBP.Method mEBPgene was cloned by PCR site-directed mutagenesis. PinpointXa-3/mEBP expression vector was designed to express human mEBP as a fusion protein.in BL21 (DE3) pLysS, Digested engineering bacteria by lysozyme and collected inclusion bodies.Fusion protein was purified by pinpoint TM Xa purification system and cleaved by factorⅩa,mEBP was purified by RP-HPLC. Results. Mutations at residues 5 and 18(Gln Lys) was obtained by PCR site-directed mutagenesis,Expressed and purified mEBP successfully.Conclusions: Obtaining of purified mEBP lay a foundation for its biological activity research.
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  Study on the Characteristics of Nicotine Degradation by Strain DN2 and Its Application

  China Biotechnology. 2006, 26(03): 47-50.

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  In this work the kinetics of nicotine degradation by O.intermedium DN2 and its application in tobacco waste were investigated. The results showed that the optimum temperature of nicotine degradation by O.intermedium was 30 ℃, the pH value was 6.5 and a mount of inoculum was 5 %. Under above conditions, the kinetics of nicotine degradation of initial concentration 500 mg/L was studied. The results indicated that the degradation process of nicotine with no-induced strain DN2 followed inverse S-shaped curve, and degradation process of nicotine with induced cells of DN2 followed Eckenfelder mode. The half life of nicotine degradation was 17.43 h and 4.10 h, respectively. And the results also showed that tolerance of O.intermedium DN2 to nicotine was up to 5000 mg/L when 0.1 % of glucose was added. Nicotine (2220 mg/L) in extract of tobacco wastes degraded about 95.22 % by strain DN2 in 60 h incubation, indicating that strain DN2 was of application value in treatment nicotine pollution.
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  Strong Expression of Recombinant Human Morphogenetic Protein-4 in Escherichia coli and its Bioassay in vivo

  China Biotechnology. 2006, 26(03): 51-56.

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  Objective To produce rhBMP-4 with bioactivity in E.coli . Methods The full-length human BMP-4 gene was mutated by PCR without changes in amino acid sequence, then the synthesized gene was cloned into plasmid pET-3c, transducted into BL21(DE)plysS, and induced by adding IPTG to a final concentration of 1.0 mmol/L. The protein product was purified using ion-exchange chromatography method and then renaturated, bioactivity was checked by C2C12 differentiation in vitro and mouse ectopic bone formation in vivo. Results A 438 bp gene fragment encoding mature peptide of hBMP-4 was cloned , the protein product was mostly in the form of inclusion body, after renaturation, the engineering protein shows better bioactivity. Conclusion The mutant strategy can enhance the expression of bioactive rhBMP-4 in E.coli expression system.
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  The study of expression HBcAg and HBsAg preS1 epitope peptide fusion protein

  China Biotechnology. 2006, 26(03): 57-62.

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  To construct, express and purify fusion protein containing HBcAg and HBsAg preS1 epitope peptide for the purpose of investigating a novel HBV vaccine with both prophylactic and therapeutic functions. Using DNA recombinant technology, we constructed prokaryotic expression plasmid pBTcs1 expressing HBcAg and HBsAg pre-S1 epitope peptide fusion protein. After expressed in E.coli. HB101, the production BTcs1 was purified by sucrose density gradient ultracentrifugation and identified by SDS-PAGE, SEC, WESTERN-BLOT and electron microscope. The results indicated that expression plasmid pBTcs1 was constructed successfully, and 20-25 mg purified BTcs1 fusion protein was obtained from 1 L LB culture. Result of DOT-BLOT indicated that the distribution of BTcs1 was mainly in 30-50% sucrose, the purity of BTcs1 was greater than 95% by SDS-PAGE and SEC analysis. BTcs1 could probe with specific antibodies at 28 kD by WESTERN-BLOT, BTcs1 could also self assemble VLP by electron microscope analysis, its diameter was 30-34 nm approximately. The present study lay a foundation for further research functions and applications of BTcs1.
综述
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  A REVIEW ON GENE ENGINEERING AND TRANSGENIC EXPRESSION STRATEGY OF ANTIMICROBIAL PEPTIDES

  China Biotechnology. 2006, 26(03): 63-67.

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  Antimicrobial peptides distribute in a wide variety of organisms and show the multiple biological functions including broad-spectrum antibacterial, immune- modulating and inhibiting tumor activity, as well as exhibiting unique mechanism, which made antibacterial peptides become a research focus in the field of gene engineering. It was reviewed on advances of general properties and gene engineering research of antimicrobial peptides; And their transgenic expression strategy and theoretical foundation were also discussed.
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  Food-Grade Nisin Controlled Gene Expression System--NICE of Lactic Acid Bacteria

  China Biotechnology. 2006, 26(03): 68-72.

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  Lactic acid bacteria (LAB) are traditional dairy strains which have a long history of safe use. They are a kind of food-grade microorganisms that can be used in industry to produce lots of food and drink, such as yoghourt. In the last two decades the physiology and genetics of these bacteria have been thoroughly studied. Because of their genetic accessibility and easy to handle, LAB, in addition to their traditional applications, have been extensively developed and used for the expression of heterologous genes. So they have an important foreground in agricultural, medical and many other fields. People have developed a series of food-grade gene expression systems of LAB. This article will introduce LAB, especially their model strain Lactococcus lactis and their most useful food-grade induced expression system--nisin controlled gene expression system NICE and its food-grade inducer nisin, food-grade host, expressing vectors and applications in expressing heterologous proteins of NICE.
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  Plant G Protein and Plant Defense Response

  China Biotechnology. 2006, 26(03): 73-77.

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  The presence of plant G proteins and their signaling have become a hot topic regarding the research on cell signaling in plants In recent years. A number of genes encoding plant G proteins which exhibit some homologue to animal G proteins have been cloned and characterized from a variety of plant species, and the amount and types of plant G proteins show its unique features. Plant G proteins play an important role in transmembrane signaling in plants and are involved in the regulation of diverse cellular processes. In this paper, we mainly review the involvement of plant G protein on regulation of plant defense response.
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  Advances in Study on Lipase Bio-imprinting

  XiJin Song

  China Biotechnology. 2006, 26(03): 78-82.

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  Lipase is a kind of widely used hydrolase. Bio-imprinting is a new technology , it induces the lipase to exhibit exciting catalytic ability in nonaqueous solvents, such as higher activity and stability, and enhances lipase's industrial use as bio-catalysts. The characteristic of the lipase's structure, the process and the mechanism of the bio-imprinting were introduced. Influencing factors such as the selective principle of different kinds of bio-imprinting templates and the function of lyoprotectant through the process of the bio-imprinting were discussed. Meanwhile, the condition of the bio-imprinting, the reaction catalyzed by bio-imprinted lipase and the results were concluded. Finally, the problem and the prospect of the bio-imprinting technology were mentioned.
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  Progress in Mucosal Adjuvants

  China Biotechnology. 2006, 26(03): 83-88.

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  Mucosal adjuvants play important roles in vaccine development. By now, the common used mucosal adjuvants can be divided into three categories: the bacterial derivatives, cytokines and chemokines, and antigen delivery systems. Progresses of the three kinds of adjuvants were reviewed in this paper to give a reference to novel vaccine research.
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  The Action of Protease Activating factor Apaf-2/CytC on Apoptosis

  China Biotechnology. 2006, 26(03): 89-92.

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  This article has described the action of Apaf-2/CytC on apoptosis. It has mainly set forth the discovery of apoptosis-correlation element Apaf-2/CytC, the action of Apaf-2,Apaf-1 and Apaf-3 on apoptosis and their relations, the manner of CytC mediating the apoptosis. It has also discussed the mechanism of CytC leakage in the mitochondria, and set forth the MPTP hypothesis, specific tunnel hypothesis and mitochondria swelling. It has analyzed the signal transferring pathway of Apaf-2/CytC-mediated apoptosis, such as CytC pathway, Fas/FasL pathway, protein kinase pathway, apoptosis inducing factor pathway and their cross talking. Finally, it has analyzed the meaning of studying the molecular mechanism of CytC-mediated apoptosis, and put forward the problem existed in the research of molecular mechanism of apoptosis.