25 April 2006, Volume 26 Issue 04

    

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研究报告
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  Investigation of apoptosis of the SGC7901 cells induced by the expression of the recombinant gene of anti-HER2 ScFv/tBid

  China Biotechnology. 2006, 26(04): 1-6.

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  Objetive: To investigate whether apoptosis of SGC7901 cells can be induced by the expression of the recombinant gene of anti-HER2 ScFv/tBid. Methods: The recombinant anti-HER2 ScFv/tBid gene was cloned into vector pCMV and the recombinant plasmid was transfected into SGC7901 cells. The gene expression was detected by RT-PCR and immunofluorescent staining. Cell counting was carried out to show the effect of the gene transfection on cell growth. At the same time, significant apoptotic peak was detected by flow cytometry in recombinant anti-HER2 ScFv/tBid gene transfected cells. Results: The fusion protein of anti-HER2 ScFv/tBid was observed in the cytoplasm of transfected SGC7901 cells. The transfected cells displayed typical cell growth inhibition and apoptosis. Conclusion: Fusion protein of anti-HER2 ScFv/tBid can induce apoptosis of SGC7901.
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  Immunogenicity of different genetic type rotavirus NSP4 in mice

  China Biotechnology. 2006, 26(04): 7-11.

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  NSP4, as the diarrhea-related protein of rotavirus, is becoming an attractive candidate for vaccine development. To compare the immunogenicity of NSP4 from different genetic groups, we constructed eukaryotic expression plasmids comprising the NSP4 genes from four different genetic types using the pCI vector. The recombinant vectors were designated as pCI-97B6, pCI-97S36, pCI-97S34 and pCI-97SZ8, respectively. Following the conformation of the transient expression of the constructs in 293 cells, the plasmids were respectively subjected to the 5 round i.m. inoculation of BALB/c mice. The specific antibodies against NSP4 as well as the IgG1/IgG2a subclasses of immunoglobulin in mice sera were examined with indirect ELISA after each immunization. The results showed that the immunization of plasmids expression NSP4s could elicit not only humoral but also cellular immunity, but the humoral immune response is dominant. There is a difference of immunogenecity among the NSP4 of different genetic type. Further studies were needed to focus on the relationship between the immunogenicity and protection effect.
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  Increasing the productivity of recombinant TNFRp75:Fc by specific nutrient compensation and metabolism control strategy in continual perfusion process

  China Biotechnology. 2006, 26(04): 12-19.

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  To increase the productivity and yield of recombinant protein in continual perfusion processing，we analyzed every amino acid consumption rate in continual perfusion culture of engineering CHO cell line which expressed recombinant TNFRp75:Fc fusion protein. Then rational amino acids were accordingly added to improve its comprehensive utilizing. At the same time, glucose supply was controlled to make the concentration of glucose below 0.5 g/L for ameliorating the toxicity of lactate accumulation in order to decrease the perfusion rate. The result showed that the productivity of recombinant protein was 3.1 times (388mg/L) and the total yield was 4.7 times (244.4g) that of control cultures after nutrient compensation and metabolism control in 30 liter working volume, and the fermentation period was prolonged one week longer. The sialic acid content and bioactivity in vitro of recombinant TNFRp75:Fc were not changed after nutrient compensation and glucose control supply. Nutrient compensating and metabolic control in continual perfusion fermentation could significantly increase the productivity and yield of recombinant TNFRp75:Fc, and thus reduced relative industrialization costs.
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  Antisense Sites Screening of Fas Gene mRNA and its Validation in vitro

  China Biotechnology. 2006, 26(04): 20-26.

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  Three candidate antisense target sites of mouse Fas gene were screened by PARASS(poly-A anchored RNA accessible sites screening) technology. They were target at Fas gene 297nt-317nt, 618nt-638nt and 662nt-682nt. Antisense oligos (A1, A2 and A3) and DNAzymes (D1, D2, and D3) for every target site were designed and synthesized. In vitro, the validation of the sites were judged by antisense oligos included RNase H splicing and the DNAzyme degradation. The results indicated that A1, A2 and A3 introduced RNase H degradation. DNAzymes D1, D2 and D3 cleaved Fas mRNA effectively. Neither degradation observed in antisense oligo RNase H group in non-target site (1211-1231nt) and 2 bases mismatched of A3, nor splicing occurred in DNzyme group in non-target site (1211-1231nt) and 2 bases mismatched of D3. Site 2 and 3 were at the same positions with those of ISIS Pharmaceuticals. The effective antisense oligos and DNAzymes for Fas gene could be used for the research subsequently.
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  Effect of recombinant N-segment of Thrombospondin-1 on the proliferation of ECV304

  China Biotechnology. 2006, 26(04): 27-31.

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  Objective To construct an expression system containing the N-terminal segment of Thrombospondin-1(TSP-1) in E.Coli.. To investigate the activity of TSF. Methods thbs1 gene fragment was amplified from human fetal cord vein endothelial cells and inserted into plasmid pET32c(+) which was then transfected and expressed in E.Coli.. Investigate the effect of recombinant TSF to the proliferation of ECV304 in vitro with MTT. The expression level of CD36 was assayed by FACS. Results Recombinant TSF was purified. TSF could restrain the proliferation of ECV304 with CD36 low-expression. Conclusions Low dose of rTSF was a latent assistant treatment of anti-cancer.
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  Expression , Purification of Human Endostatin in Pichia pastoris and the Detection of Its Anti-angiogenic Activity

  China Biotechnology. 2006, 26(04): 32-35.

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  Endostatin is a potent, endogenous inhibitor of angiogenesis, which corresponds to the C-terminal fragment of collagen ⅩⅧ. The human endostatin gene was amplified from a human fetal liver cDNA library by means of PCR and was cloned into pPIC9K vector. Soluble endostatin was superexpressed in Pichia pastoris (50.5mg/L) . The recombinant protein was purified by cation exchange chromatography with the final yield of more than 95%. Western blotting analysis showed positive immunoreactivity to the expressed product. Recombinant endostatin was able to suppress the angiogenesis on the CAMs. Also, the paper showed that endostatin inhibited specifically of the migration of HMECs stimulated by bFGF, with EC50 being about 0.4 μg/ml.
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  Design and functional investigation of a novel anti-coagulative fusion protein by hirudin with a recognizing sequence of FXa

  China Biotechnology. 2006, 26(04): 36-39.

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  Hirudin (HV) is known as the most potent and specific inhibitor of thrombin. Although hirudin has many advantages ,it has the bleeding side effect and this is the great shortage of hiudin for clinical application. In order to alleviate bleeding side effect of hirudin, fusion protein, named as FHV(fusion hirudin linked with FXa recognition peptide) was designed. The fusion protein gene(fhv) was cloned into plasmid pPIC9K. FHV engineered Pichia pastoris containing high copies was chosen for fermentation and purification at 30 L fermentor scale, finally, FHV with purity of above 97% was obtained. To investigate the function of FHV in vivo, mouse tail thrombosis model was used. In the mice thrombus tail model induced by carrageenan, FHV decreased the length of tail thrombus significantly, similar to that of HV control, and had no obvious effects on the TT, PT and APTT. In conclusion, FHV is constructed and expressed in yeast. FHV fusion proteins is obtained by fermentation and purification. FHV has antithrombotic effects not influencing TT, PT and APTT after administration immediately in animal models. Therefore, FHV is a promising anticoagulant and antithrombotic drug.
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  Cloning, Sequence analysis and Proeukaryotic Expression of Peroxisomal Membrane Protein Allergen Gene from Chaetomium Globosum

  China Biotechnology. 2006, 26(04): 40-45.

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  The full length cDNA encoding peroxisomal membrane protein from Chaetomium globosum was cloned using RACE technology and the sequence in cDNA library of C. globosum in GenBank（Accn:BP099709）. The 747bp full length cDNA encoding peroxisomal membrane protein allergen (pero) gene was assembled with 412bp 3′and 508bp 5′RACE products. The open reading frame was 501bp encoding 166 amino acids. The molecular weight of the protein was 17.5kD and its theoretical isoelectric point was 5.75. The pero gene was amplified using specific primers of cDNA 5′and 3′untranslated region, sequence analysis indicated that the gene have 3 exons and 2 introns. ClustalX analysis revealed that amino acids sequence of pero gene from C. globosum and Neurospora crassa shared 83% high similarity. To construct pET28a-pero expressive plasmid, pero gene was inserted into pET28a expressive vector. Escherichia coli BL21 transformed by pET28a-pero plasmid was induced with IPTG. The protein expression was analyzed with SDS-PAGE.A 21kD pero fusion protein representing the pero gene was expressed in recombinant E. coli BL21. The sequences of cDNA，DNA and deduced amino acid of the pero gene from C. globosum were submitted to GenBank (Accn: AY555771，AY584753，AAS66898).
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  Expressing Purification and Identification of Neuritin Gene in the E.coil

  China Biotechnology. 2006, 26(04): 46-50.

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  Neuritin is a new neurotrophic factor found rectently.In order to identify the function of Neuritin clearly . the coding sequence of human neuritin was amplified by PCR from neuritin cDNA ,this fragment digested by NOcI and NotI was inserted into pET32a by T4 ligase and transformed into E.coil BL21 then the recombinant plasmid named pET32a-neuritin was constructed successfμlly .neuritin was expressed distinctly after inducing by IPTG.The product was identified as neuritin by SDS-PAGE and Wester blot analysis .The expression production was purified on Ni2+-NTA column .
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  DETECTION OF SEGREGATION OF FOREIGN GENES IN TRANSGENIC WHEAT LINES BY MULTIPLEX POLYMERASE CHAIN REACTIN

  China Biotechnology. 2006, 26(04): 51-57.

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  The method of multiplex PCR was set up to identify two or three transgenes in one reaction such as uidA and bar; uidA and 1Dx5 or uidA, bar and 1Dx5 genes. Three sets of primer pairs which was specific to each of these three genes respectively were designed and synthesized. Recombinant plasmids pAHC25 and p1Dx5 harboring uidA+bar and 1Dx5 gene separately were used as template DNA in the process of optimizing an multiplex PCR reaction. The optimal annealing temperature for uidA and bar MPCR is range from 57.1℃ - 62.3℃, for uidA and 1Dx5 is range from 60℃ to 60.6℃, and for uidA、bar and 1Dx5 range from 57.0℃-58.4℃. The amount of template for MPCR is twice as much as that for simplex PCR, while the concentration of primers is the same with simplex one. Less than 50bp MPCR products can be separated clearly by 10% non-denaturalized polyacrylamid gel electrophoresis. Fourteen transgenic wheat lines were tested by multiplex and simplex PCR respectively, which shows the same results and hence presents that MPCR is the reliable, rapid and high-effective approach to detect foreign genes from transgenic plant.
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  Cloning, Expression and property analysis of Arabinosidase in Pichia pastoris

  China Biotechnology. 2006, 26(04): 58-64.

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  SMART-RACE was performed after Isolating the total RNA of Armillariella tabescens to amplify the full-length cDNA of arabinosidase（GenBank Accession No. AJ620046）. Bioinformatics analysis was used to analyze the code frame of arabinosidase, to predict its structure and function. Recombinant plasmid pPIC9-AF was constructed and then electroporated into methylotrophic yeast Pichia pastoris GS115. The secreted 6×His fusion protein was purified to analyze its enzymology property. This arabinosidase had high activity at 30℃-35℃ under acid condition, and was stable within wide range of pH and temperature. It maintained about 80% activity at the range of pH4.0-8.0 and 20℃-40℃，which was wider than many other cloned arabinosidase. So it was worthy to go step further to study this enzyme. And recombinant expression provided a chance of highly expressing arabinosidase.
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  Kinetics of fed-batch cultivation on recombinant Escherichia coli containing human-like collagen cDNA

  China Biotechnology. 2006, 26(04): 65-69.

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  The kinetics of batch and fed-batch cultures of recombinant Escherichia coli to produce human-like collagen were investigated. Through examining the density of substrate and the amount of mushrooms and the density of products during the process of fermenting, a set of kinetic models are set up. In the paper, the influence of cell without plasmid was considered. The results show that the kinetic model may well simulate the fermenting process.
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  Isolation Bacteria by Use the Principle of Magnetic Separation

  China Biotechnology. 2006, 26(04): 70-74.

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  There are similarities between magnetotactic bacteria and Acidithiobacillus ferrooxidans(A.ferrooxidans) [1-2] which isolated from Acid mine drainage(AMD).The weak magnetotaxis of some bioleaching bacteria isolated were found by microscope. A magnetophoresis apparatus was designed based on these weak magnetotaxis and be used to analysis the movement of these strains. The physiological properties of the anear magnetic field strain and removed magnetic field strain which isolated successfully by magnetophoresis apparatus have large difference. The nanometer magnetic particles was extract from the Acidithiobacillus ferrooxidans which purified by spread plate method from AMFS and its' main elements are Fe and O by energy spectrum analysis. The results show that A. ferrooxidans have weak magnetotaxis and can be isolated by magnetophoresis. With the development of this new isolating method, the research of magnetotactic bacteria and bioleaching will get more benefit from it.
技术与方法
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  A speedy extraction method for bacterial chromosomal DNA

  China Biotechnology. 2006, 26(04): 75-80.

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  How to get functional gene from uncultured-microbiology is the hotspot content of microbial ecology. What the most important is how to obtain the pure and integrated genomic DNA. This article had showed an efficient, nonselective extraction method to gain chromosomal DNA from eight kinds of bacteria. Amount DNA released by hot-detergent gave the highest DNA yields from different G+ and G- bacteria. Running 20 hours by PFGE mode, the size of total DNA is over 23kb. The pure DNA could be digested by Hind III and used in PCR. The total environmental DNA also can be extracted from soil by the same method. As a result it showed a new way for the environmental DNA extraction.
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  Study on photolysis of Vitamin B12 and New Method to Determine the Contents of Vitamin B12 in Fermentation Broth

  China Biotechnology. 2006, 26(04): 81-85.

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  The photolysis of deoxyadenosylcobalmin and methylcobalamin in water was studied. The results showed that the speed of photolytic cleavage is related to photolytic energy.The more photolytic energy ,the more photolytic speed. The content of hydroxycobalamin, which is the photolytic product of dexyadenosylcobalmin, is according to the contents of vitamin B12 in ferment broth. An new method measures the contents of vitamin B12 in fermentation broth is established.
简报
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  Inhibition of K-RASAsn12 Expression by Vector-based RNA Interference in Human Pancreatic Cancer Cell Line

  China Biotechnology. 2006, 26(04): 86-90.

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  To silence the expression of K-RASAsn12 in human pancreatic cancer cell line by vector-based RNAi(RNA interference) technique,two single-strand DNA sequences encoding mutant-specific shRNA (short haipin RNA) for K-RASAsn12 were synthesized and then inserted into pSilenCircle. The recombinant plasmid was called pSC-K-RASAsn12.According to the same method, pSC-GFP encoding shRNA for GFP was gained.Both recombinant plasmids were transfected into human pacreatic cancer cell line AsPC-1 and BxPC-3.The expression level of K-RASAsn12 was detected by semi-quantitative RT-PCR and Western Blot. The result indicated that the recombinant plasmid edcoding mutant-specific shRNA for K-RASAsn12 can inhibit significantly the expression of K-RASAsn12 without affection of wild-type K-RAS（K-RASWT）in Human Pancreatic Cancer Cell Line.
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  2D-PAGE Analysis of Chinese Rose Leaf Protein Under Heat Shock Stress

  China Biotechnology. 2006, 26(04): 91-94.

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  Proteins extracted from two varieties of Chinese roses' leaves were separated by two- dimensional polyacrylamide gel electrophoresis (2-DE) with immobilized pH gradient (IPG). Many difference proteins were isolated with molecular weights ranging 10-30 kD and pI5-6. Three proteins of high levels observed in a gel were excised and identified using peptide mass fingerprinting and MS-MS. A summary of the identified proteins and their putative functions are presented. They are identified as eIF-5A、LEA protein and Hsp17.5. Functions of these proteins in plant tolerance to high temperature were discussed in this paper.
综述
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  Interaction of virus with the interferon system

  China Biotechnology. 2006, 26(04): 95-100.

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  Interferons are potent cytokines with antiviral activity that have been founded earliest. Different types of interferons have similar bioactivity , e.g. anti-viruses activity, anti-tumor activity and immune modulation. They are induced by virus infection and trigger the host defense by different mechanisms. Firstly, IFNs directly induce the expressionof effector proteins with antiviral activity, thus establishing a first line of defense. Secondly, they help to shape adaptive immunity, leading to long-lasting protection. Due to this key position of IFNs in antiviral defense, viruses have evolved effective countermeasures in order to successfully invade the host. By expressing so-called IFN antagonists, viruses interfere with either IFN induction, IFN signaling, or the action of IFN effector proteins.
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  Proceeding of Xenotransplantation

  China Biotechnology. 2006, 26(04): 101-105.

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  Xenotransplantation is a feasibility way of solving the shortage of human organs for transplantation. But there are multiple hurdles existed to clinical application, such as the immune rejection between human body and the xenografts, the infection of pathogens, and so on. In this paper, we first give a brief historical retrospect of xenotransplantation, and then probe into the strategies according to the main problems and the actualities. Finally, we show the prospect in the field of xenotransplantation.
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  LITERATURE ANALYSIS OF ION BEAM BIOENGINEERING FOR 10 YEARS IN CHINA

  China Biotechnology. 2006, 26(04): 106-106.

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  This paper statistics the ion beam bioengineering study articles for 1994-2003 years, and analyses from the literature quantity, the research material and the level of the research, magazine, the ion source and the fund source .The result indicate that the ion beam bioengineering of China get a fast development under the support of the nation and the local government and college, the development of the microbe is the most quick; in 21 century, the local government and college gradual enlarge the support of the ion beam bioengineering, some articles are finished with the support of enterprise researcher and fund; the contents of these articles mainly is an application study, the fundamental research is less. After the analysis data，forecast the future of ion beam bioengineering of China.