2010年06期目次

  • Construction and Identification of Recombinant Adenoviruses Expressing Chimeric Ubiquitin-ligase TrCP-CC and Its Mutant △F-CC

    LUO Hong-wei,YUAN Ying,JI Mao-sheng,et al(Department of Clinical Hematology,Key Laboratory of Laboratory Medical Diagnostics of Education Ministry,Chongqing Medical University,Chongqing 400016,China)

    Objective To construct a recombinant adenovirus expressing chimeric ubiquitin-ligase TrCP-CC and its mutant △F-CC and determine the expressed products.Methods Eukaryotic expression vectors Migr1-TrCP-CC-HA and Migr1-△F-CC-HA were constructed by PCR and overlap PCR,and used as templates for amplification by PCR.The amplified target gene fragments were cloned into shuttle plasmid pAd-Track-CMV,and the constructed recombinant plasmids pAdTrack-TrCP-CC and pAdTrack-△F-CC were used for homologous recombination with skeleton vector pAdeasy-1.The obtained recombinant adenovirus vectors pAd-TrCP-CC and pAd-△F-CC were transfected to HEK293 cells for package and amplification.The obtained recombinant adenoviruses Ad-TrCPCC and Ad-△F-CC were determined for titers,in which the transcription levels of target genes were determined by RT-PCR.Results Both restriction analysis and sequencing proved that recombinant adenovirus vectors pAd-TrCP-CC and pAd-△F-CC were constructed correctly.Green fluorescence was observed in the HEK293 cells transfected with the recombinant adenovirus vectors.Recombinant adenoviruses Ad-TrCP-CC and Ad--△F-CC reached titers of 2.5 × 109 and 1.0 × 109 pfu /L respectively,in which target genes were expressed effectively as proved by RT-PCR.Conclusion Recombinant adenoviruses Ad-TrCP-CC and Ad-△F-CC were successfully constructed and expressed in HEK293 cells,which laid a foundation of study on the mechanism of targeting fusion protein Bcr-Ab1 for ubiquitination and degradation.

    2010 06 v.23 [Abstract][OnlineView][Download 295K]

  • Establishment of Vero Cell Strain for Stable Expression of Fusion Protein of Open Reading Frame 2 of Human Herpes Simplex Virus Type 2 Latency Associated Transcript and Enhanced Green Fluorescent Protein

    YANG Hui-lan,BAI Li-li,FAN Jian-yong,et al(Department of Dermatology,Guangzhou General Hospital of Guangzhou Military Command,Guangzhou 510010,China)

    Objective To establish a Vero cell strain for stable expression of fusion protein of open reading frame 2(ORF2) of human herpes simplex virus type 2(HSV-2)latency associated transcript(LAT)and enhanced green fluorescent protein(EGFP).Methods Recombinant plasmid pEGFP-ORF2 was constructed and identified by restriction analysis and sequencing,then transfected to Vero cells in vitro.The recombinants for stable expression of fusion protein were screened with G418 for scale-up culture,in which the expression of EGFP was observed by fluorescent microscopy,and the transcription of target gene by RT-PCR.Results Both restriction analysis and sequencing proved that recombinant plasmid pEGFP-ORF2 was constructed correctly.The expression of fusion protein was proved by fluorescent microscopy in the screened Vero cell strain.RT-PCR showed transcription of target gene in Vero cells transfected with recombinant plasmid pEGFP-ORF2.Conclusion A Vero cells strain for stable expression of fusion protein of HSV-2 LAT ORF2 and EGFP was successfully established,which laid a foundation of further study on function of HSV-2 LAT ORF2.

    2010 06 v.23 [Abstract][OnlineView][Download 282K]

  • Effect of Advanced Glycosylation End Products on Expression of Tissue Transglutaminase mRNA in Rat Proximal Tubular Epithelial Cells

    LIU Hui,YU Xiao-yan,SUN Bo,et al(Department of Laboratory Pharmacology and Toxicology,College of Pharmacy,Jilin University,Changchun 130021,China)

    Objective To investigate the effect of advanced glycosylation end products(AGEs)on the expression of tissue transglutaminase(tTG)mRNA in rat proximal tubular epithelial cells NRK-52E as well as on the degradation of extracellular matrix(ECM).Methods NRK-52E cells were cultured in vitro,then treated with various concentrations of AGE-BSA and BSA synthesized in vitro for 48 h respectively.The fibronectin(FN)and type Ⅳ collagen(Col Ⅳ)contents in supernatant of cell culture were determined by ELISA,while the expression of tTG mRNA by RT-PCR.Results Compared with those treated with BSA,the FN content in supernatant of cells treated with 50 ～ 200 mg /L AGE-BSA increased significantly(P < 0.01),and the Col Ⅳ content in those treated with 25～100 mg /L AGE-BSA increased significantly(P < 0.05).The increase of expressed tTG mRNA was positively related to the increase of secreted FN(r = 0.911)and Col Ⅳ(r = 0.872).Conclusion AGEs induced the expression of tTG mRNA in rat proximal tubular epithelial cells and increased the contents of FN and Col Ⅳ as major components of ECM,indicating that tTG might inhibit the degradation of ECM during cumulation of ECM caused by AGEs and be involved in the pathogenesis of diabetic nephropathies.

    2010 06 v.23 [Abstract][OnlineView][Download 301K]

  • Construction of Expression Vector of siRNA Targeting to VP1 gene of Enterovirus 71

    LV Yu-chun,CHEN Quan,YAN Sha,et al(Department of Immunology,College of Basic Medicine,Chongqing Medical University,Chongqing 410016,China)

    Objective To construct and identify the expression vector of siRNA targeting the VP1 gene of enterovirus 71(EV71).Methods The expression vectors pSVSi1,pSVSi2 and pSVSi3 of siRNA targeting EV71 VP1 gene were constructed and transfected to A549 cells in mediation of liposome.The positive clones were screened,in which the expression of EV71 VP1 gene at mRNA and protein levels were identified by fluorescent quantitative PCR and Western blot respectively.Results Sequencing proved that recombinant plasmids pSVSi1,pSVSi2 and pSVSi3 were constructed correctly.Of the three recombinant plasmids,pSVSi1 was more effective than the other two in inhibiting the expression of VP1 gene in A549 cells,with inhibitory rates at mRNA and protein levels of 87% and 89% respectively.However,the inhibitory rates of pSVSi2 and pSVSi3 to the expression of VP1 gene at mRNA level were 60% and 70% respectively.Conclusion The expression vector of siRNA targeting the VP1 gene of EV71 was successfully constructed,and a siRNA inhibiting VP1 gene significantly was screened,which laid a foundation of siRNA gene therapy of hand-footmouth disease.

    2010 06 v.23 [Abstract][OnlineView][Download 313K]

  • Expression of Human Basic Fibroblast Growth Factor in Plant Hairy Root and Pichia pastoris

    FAN Wei-quan,MU Xu-peng,WEI An-hui,et al(College of Traditional Chinese Pharmacy,Beijing University of Traditional Chinese Medicine,Beijing 100102,China)

    Objective To express human basic fibroblast growth factor(hbFGF)in plant hairy root and Pichia pastoris and optimize the condition for fermentation.Methods The hbFGF gene was cloned into expression vectors pCAMBIA1301 and pPICZα to construct recombinant plasmids pCAMBIA1301-hbFGF and pPICZα-hbFGF respectively.Recombinant plasmid pCAMBIA1301hbFGF was transformed to soybean hairy root in the mediation of Agrobacterium rhizogenes,while pPICZα-hbFGF to P.pastoris X-33 strain by electrotransformation.Positive transformants were screened by PCR for expression under induction.The strains expressing hbFGF highly and stably were screened and cultured by fermentation,based on which the condition for fermentation was optimized.The expressed products were analyzed by SDS-PAGE and Western blot then purified.Results Both restriction analysis and sequencing proved that recombinant plasmids pCAMBIA1301-hbFGF and pPICZα-hbFGF were constructed correctly.PCR showed that hbFGF gene was integrated to both the genomes of soybean hairy root and P.pastoris.The hbFGF expressed in soybean hairy root and P.pastoris contained 31% and 42% of total somatic proteins respectively,and both showed good reactogenicities.Either of the expressed hbFGF showed a single band with relative molecular mass of about 18 000 on SDS-PAGE profile.The conditions for fermentation in the two systems were optimized.Conclusion Human bFGF may be highly expressed in both soybean hairy root and P.pastoris,which lays an experimental basis of large-scale production of bFGF.

    2010 06 v.23 [Abstract][OnlineView][Download 346K]

  • Biological Function of Recombinant Protein Transduction Domain-SARA Fusion Protein

    JIA Hui-wei,HUANG Chen,LI Jing,et al(Department of Nephrology,Xijing Hospital,Fourth Military Medical University,Xi'an 710032,China)

    Objective To preliminarily study the biological function of recombinant protein transduction domain(PTD)SARA fusion protein in inhibiting renal fibrosis.Methods The transmembrane efficiency of recombinant PTD-SARA fusion protein was determined by immunocytochemical assay.The morphology of HK2 cells was observed by microscopy.The E-cadherin,α-smooth muscle actin(α-SMA)and phospho Smad3 protein levels were determined by Western blot to compare the inhibitory effects of PTDSARA and SARA on TGF-β1-induced renal tubular epithelial to mesenchymal transition.Results Recombinant PTD-SARA fusion protein could enter both cytoplasm and karyon of HK2 cells effectively.Compared with SARA,PTD-SARA up-regulated the expression of E-cadherin and down-regulated those of α-SMA and phosphoSmad3 protein significantly(P < 0.05).Conclusion Compared with SARA,PTD-SARA may enter the cytoplasm and karyon effectively and inhibit the TGF-β1-induced renal tubular epithelial to mesenchymal transition significantly,which lays a foundation of further study on prevention and treatment of renal fibrosis with recombinant PTD-SARA fusion protein.

    2010 06 v.23 [Abstract][OnlineView][Download 466K]

  • Effect of DNA Methylation on Expression of Insular Differentiation-related Genes

    FANG Ai-ping,ZHANG Yue,LIN Bao-hang,et al(Shenzhen People's Hospital,The Second College of Clinical Medicine,Jinan University,Shenzhen 518020,Guangdong Province,China)

    Objective To investigate the effect of DNA methylation on the expression of insular differentiation-related genes.Methods The DNA methylation levels of four insular differentiation-related genes,i.e.Pdx-1,MafA,Nkx6.1 and Oct4 in 129 /J mouse embryo stem(mES),NIT1 and NIH3T3 cells were determined by methylated DNA co-immunoprecipitation-real-time quantitative PCR(MeDIP-qPCR)technique,and the expression levels of mRNA of the four genes in three cell lines were determined by real-time quantitative RT-PCR,based on which the relationship between DNA methylation levels and expressions of the genes were analyzed.Results Pdx-1,MafA and Nkx6.1 genes were lowly methylated in mES and NIT1 cells,while highly methylated in NIH3T3 cells,which showed significantly difference(P < 0.05).No methylation of Oct4 gene was observed in mES cells,while low methylation in NIT1 and NIH3T3 cells.However,the methylation level of Oct4 gene in NIT1 cells was significantly lower than that in NIH3T3 cells(P < 0.05).Lowly methylated Pdx-1,MafA and Nkx6.1 genes were highly expressed in NIT1 cells,while showed no expression in NIH3T3 or mES cells(P < 0.05).However,Oct4 gene was highly expressed in mES cells,while showed no expression in NIT1 or NIH3T3 cells.Conclusion The DNA methylation level of transcription initiator may influence the expressions of Pdx-1,MafA,Nkx6.1 and Oct4 genes and involve in the differentiation of β cells.

    2010 06 v.23 [Abstract][OnlineView][Download 269K]

  • Construction and Self-activation of Bait Vector for Shigella flexneri Invasion Gene ipaC

    CHEN Zhi-bao,SONG Bo-cui(College of Life Science and Technology,Heilongjiang August First Land Reclamation University,Daqing 163319,Heilongjiang Province,China)

    Objective To construct a bait vector for Shigella flexneri invasion gene ipaC and determine its self-activation.Methods Shigella flexneri invasion gene ipaC was amplified by PCR and cloned into bait vector pSos,and the constructed recombinant plasmid pSos-IpaC was transformed to yeast cdc25H.The expressed fusion protein was identified by Western blot,and the constructed bait vector was determined for self-activation.Results Both restriction analysis and sequencing proved that recombinant plasmid pSos-IpaC for yeast double-hybrid system was constructed correctly.Western blot showed that,by using the bait vector,the fusion protein with a relative molecular mass of about 214 000 was expressed.The bait vector showed no self-activation effect on yeast cdc25H,and the expressed fusion protein was located correctly in host cytoplasm.Conclusion Bait vector pSos-IpaC was successfully constructed,which showed no self-activation effect on yeast cdc25H.

    2010 06 v.23 [Abstract][OnlineView][Download 207K]

  • Development of Procedure for Fermentation and Purification of Recombinant Human B7-H1IgV and Antitumor Activity of Purified Protein

    CHEN Lin,LI Jing,HOU Jian-wei,et al(Biotechnology Center,The Fourth Military Medical University,Xi'an 710032,China)

    Objective To develop the fermentation and purification procedures for large-scale production of recombinant human B7-H1IgV(rhB7-H1IgV)and determine the antitumor activity of purified protein.Methods Recombinant E.coli DH5α with B7-H1IgV gene was cultured by fermentation and induced with IPTG for expression,then cleaved by ultrasonication,from which inclusion bodies were separated for gradient re-naturalization,and the target protein was purified by nickel ion affinity chromatography for 2 times,then identified by SDS-PAGE and Western blot,and determined for antitumor activity.Results The expressed rhB7H1IgV in E.coli DH5α cultured by the developed fermentation procedure contained(10 ～ 11)% of total somatic protein.The expressed rhB7-H1IgV reached a HPLC purity of more than 95% after purification,and showed good reactogenicity as proved by Western blot.The purified rhB7-H1IgV induced high anti-B7-H1 titer in BALB /c mice,and showed significantly inhibitory effect on growth of tumors.Conclusion The developed procedures for fermentation and purification was simple and rapid,which laid a foundation of further development and application of purified rhB7-H1IgV.

    2010 06 v.23 [Abstract][OnlineView][Download 301K]

  • Cloning and Sequencing of Complete BoNT Gene of Clostridium botulinum Type A for Therapy

    ZHAO Ya-jing,WEI Ran,LIU Chen-ming,et al(Lanzhou Institute of Biological Products,Lanzhou 730046,China)

    Objective To clone and sequence the complete BoNT gene of Hall strain of Clostridium botulinum type A for production of type A botulinum toxin for therapy in China,investigate its genetic characters and provide a basis for quality control of type A botulinum toxin.Methods Complete BoNT gene was amplified by PCR from the master and working seeds for production of type A botulinum toxin for therapy,and cloned into vector pGEM-T to construct recombinant plasmid pGEM-T-BoNT for sequencing.Results The cloned complete BoNT gene sequence of Hall strain,at a length of 3 891 bp,encoded 1 297 amino acids.The proportions of nucleotides A,G,T and C were 40.58%,16.17%,33.13% and 10.13%,while the GC and AT contents were 26.29% and 73.71%,respectively.A nonsense mutation(G→A at site 3591) was observed between master and working seeds,resulting no change of deduced amino acids.The homologies of nucleotides and amino acids of both master and working seeds to those of standard Hall strain in GenBank were 99.99% and 100% respectively.Conclusion The BoNT gene of Hall strain of Clostridium botulinum type A was highly stable in genetic during long-term storage in laboratory and subculture for production.

    2010 06 v.23 [Abstract][OnlineView][Download 1992K]

  • Cloning and Identification of Promoter of Special Expression Protein SAG1 of Toxoplasma gondii Tachyzoite

    AN Na,ZHANG Rui-yan,ZHAO Zhi-yuan,et al(College of Life Science,Jilin Agriculture University,Changchun 130118,China)

    Objective To clone the promoter sequence of special expression protein SAG1 of Toxoplasma gondii tachyzoite and provide a tool for genetic manipulation of Toxoplasma gondii.Methods The non-encoding region at 5'-terminus of SAG1,the encoding region of 3'-terminus of dense grandule protein 2(GRA2)and red fluorescent protein(RFP)gene were amplified by PCR for construction of recombinant plasmid pMD/SAG-RFP-GRA which was transfected to Toxoplasma gondii tachyzoite by electroporation.The expression of RPF was observed under inverted fluorescent microscope.Results Both restriction analysis and sequencing proved that recombinant plasmid pMD /SAG-RFP-GRA was constructed correctly.Red fluorescence was observed in the Toxoplasma gondii tachyzoite 24 h after transfection with the recombinant plasmid,indicating transcription activity of the cloned promoter of SAG1.Conclusion The promoter sequence of SAG1 was successfully cloned,which could mediate the expression of exogenous gene in Toxoplasma gondii tachyzoite.

    2010 06 v.23 [Abstract][OnlineView][Download 216K]

  • Effect of Lysophosphatidic Acid on Expression of P120catenin in Human Ovarian Cancer Cells

    JIANG Xin,PENG Zhen-nian,XIONG Xue-hua,et al(Department of Pathology,Laboratory of Stem Cell and Tissue Engineering,Chongqing University of Medical Sciences,Chongqing 400016,China)

    Objective To observe the effect of lysophosphatidic acid(LPA)on expression of P120catenin(P120ctn)in human ovarian cancer SKOV3 cells and investigate the action mechanism of LPA and P120ctn in onset and development of ovarian cancer.Methods SKOV3 cells were treated with LPA at various concentrations,and determined for proliferation rate by MTT method.SKOV3 cells were treated with LPA at optimal concentration plus tyrosine protein kinase inhibitor Genistein at various concentrations respectively,and determined for proliferation-inhibiting rate by MTT method.SKOV3 cells were divided into control,LPA and LPA + Genistein groups,in which the expression of P120ctn were analyzed by immunofluorescent cytochemical method and Western blot,and transcription of P120ctn mRNA by RT-PCR 6,12 and 24 h after culture.Results The proliferation rate of SKOV3 cells reached a peak value 24 h after treatment with 20 μmol /L LPA.However,200 μmol /L Genistein significantly inhibited the promoting effect of LPA on SKOV3 cell proliferation.The expression level of P120ctn in membrane of SKOV3 cells treated with LPA decreased,while that in cytoplasm increased.However,the expression level of P120ctn in membrane of SKOV3 cells treated with LPA + Genistein increased,while that in cytoplasm decreased.Conclusion LPA may induce the change of P120ctn expression in SKOV3 cells by a potential mechanism associated with the phosphorylation of the relevant tyrosine protein kinase.

    2010 06 v.23 [Abstract][OnlineView][Download 398K]

  • Effect of Plasmid Dosage and Immunization Occasions on Immune Response Induced by Gene-gun Delivered DNA Vaccine

    XU Zhi-yong,ZHANG Zhou,CHEN Pei,et al(State Key Laboratory for Infectious Disease Prevention and Control,National Center for AIDS/STD Control and Prevention,Chinese Center for Disease Control and Prevention,Beijing 100050,China)

    Objective To investigate the effect of plasmid dosage and immunization occasions on the immune response induced by gene-gun delivered DNA vaccine.Methods DNA vaccine plasmid p1.0-Env carrying the Env gp145 gene of HIV-1 was constructed.BALB/c mice were inoculated with the constructed plasmid at various dosages by gene-gun for various times,using those inoculated by i.m.injection as control.The specific antibody level against Env of immunized mice was determined by ELISA.Results The antibody level induced with the DNA vaccine delivered by gene-gun was significantly higher than that by i.m.injection.At a dosage range of 0.1～2.25 μg,the specific antibody level against Env induced by gene-gun increased with the increasing dosage of DNA vaccine.However,no further enhancement of humoral immune response was observed after the dosage of DNA vaccine increased to 5 or 10 μg.The delivery of DNA vaccine by gene-gun for 1 and 2 times induced only low specific antibody responses,while that for 3 times induced high antibody response.Conclusion Delivery by gene-gun enhanced the antibody response induced with DNA vaccine.The optimal dosage of DNA vaccine for mice was about 2.25 μg.The immunization with 2.25 μg of DNA vaccine by gene-gun for 3 times induced high humoral immune response.

    2010 06 v.23 [Abstract][OnlineView][Download 229K]

  • Preparation of Inactivated Hemorrhagic Fever with Renal Syndrome Vaccine by Primary Kidney Cells of Gerbil Mice Cultured on Cell-Stack Apparatus

    YAO Wei,ZHONG Jian-qin,SU Bo,et al(Zhejiang Tianyuan Bio-Pharmaceutical Co.,Ltd.,Hangzhou 311100,China)

    Objective To prepare inactivated hemorrhagic feverwith renalsyndrome(HFRS)vaccine by primary kidney cells of gerbil mice cultured on Cell-Stack apparatus(also called cell factory)instead of typical roller culture so as to increase the yield and decrease the use of gerbil mice.Methods Primary kidney cells of gerbil mice were cultured on Cell-Stack apparatus,using those cultured in 15 L rolling bottle as control.The growths of cells,as well as titers and antigen contents of harvested virus cultured by the two methods were compared.The harvested virus liquid was concentrated by ultrafiltration,then purified by Sepharose-4FF chromatography and subjected to control tests.Results At the same stage of culture,the quantity of cells on Cell-Stack apparatus was equal to that in rolling bottle,while the quantity of primary kidney cells of gerbil mice inoculated required by the former was only 1 /3 of that by the latter.About(10 ～ 20)% of cells inoculated with virus in roller culture were exfoliated after harvest for 5 times,while those cultured on Cell-Stack apparatus showed little exfoliation,only 20% of which were exfoliated after harvest for 9 times.The titer and antigen content of each virus harvest of culture on Cell-Stack apparatus were higher than those of roller culture,which were stable within a certain range.The virus liquid after concentration and purification was qualified in control tests.Conclusion The yield of vaccine prepared by culture of primary kidney cells of gerbil mice on Cell-Stack apparatus increased,while the cultural space and contamination decreased,indicating that Cell-Stack apparatus might be used for large-scale production of inactivated HFRS vaccine instead of roller culture.

    2010 06 v.23 [Abstract][OnlineView][Download 191K]

  • Preparation and Properties of PEGylated Recombinant Human Interferon α1b

    YANG Yu-feng,LU Yu,SHI Wei,et al(Shanghai Institute of Biological Products,Shanghai 200052,China)

    Objective To prepare PEGylated recombinant human interferon α1b(PEG-rhIFNα1b)and analyze its properties.Methods Recombinant human IFNα1b was modified by PEG,and the modified mixture was separated and purified by ion exchange chromatography,based on which PEG-rhIFNα1b was concentrated by ultrafiltration,and determined for concentration by Lowry method,for purity by SDS-PAGE and RP-HPLC,for isoelectric point by isoelectric focusing electrophoresis,for relative molecular mass by mass spectrography,for in vitro activity by cytopathic inhibition assay,and for antigenicity by ELISA.Results The yield of PEGrhIFNα1b was 33%.The purity of PEG-rhIFNα1b was 96.1% by SDS-PAGE and 98.1% by RP-HPLC.The isoelectric point,relative molecular mass,specific activity and activity reservation rate of PEG-rhIFNα1b were 6.22,39 946,1.28 × 106 IU /mg and 14.4% respectively,while the antigenicity decreased by at least 625 folds.Conclusion The pharmacological properties of prepared PEGrhIFNα1b were improved.PEG-rhIFNα1b might be developed to a novel safe and long-acting interferon.

    2010 06 v.23 [Abstract][OnlineView][Download 377K]

  • Effect of Paridis Extract on Proliferation of Human Colon Cancer SW480 Cells and Mechanism of The Effect

    LI Xi,WANG Ji-hong,XIAO Ya-xiong(Laboratory of Biochemistry and Molecular Biology of Experimental Teaching Center,Chongqing Medical University,Chongqing 400016,China)

    Objective To observe the effect of Paridis extract on proliferation of human colon cancer SW480 cells and investigate the mechanism of the effect.Methods SW480 cells cultured in vitro were treated with various concentrations(10,20,40 and 80 μg/ml)of Paridis extract for 12,24,36 and 48 h separately.The inhibiting rate to SW480 cells was determined by MTT method.The SW480 cells cultured in vitro were treated with Paridis extract at median inhibitory concentration for 24 h,then observed for morphology and analyzed the effect on cell cycle by flow cytometry.The transcription of stem cell factor(SCF)mRNA in SW480 cells was determined by RT-PCR,and expression of SCF protein by Western blot.Results The Paridis extract at various concentrations showed inhibitory effect at different degrees on SW480 cells,in dose-and time-dependent modes.Morphological change of SW480 cells,such as denaturation and necrosis,were observed after treatment.The percentage of SW480 cells at S phage decreased 24 h after treatment with Paridis extract,while those at G0-G1 and G2 /M phases increased(P < 0.05).Meanwhile,the expression level of SCF increased(P < 0.05).Conclusion Paridis extract might inhibit the mitosis of tumor cells by inhibiting the synthesis of protein and DNA,thereby inhibit the proliferation of SW480 cells,and the inhibitory effect might be SCF-independent.

    2010 06 v.23 [Abstract][OnlineView][Download 432K]

  • Development of Purification Procedure for Tumor DNA Vaccine

    YANG Xiao-ling,LV Jing,ZHU Zhi-qiang,et al(Department of Biochemistry and Molecular Biology,Shanxi Medical University,Taiyuan,030001,China)

    Objective To develop a purification procedure and lay a foundation of industrial production of tumor DNA vaccine.Methods Plasmid DNA was crudely extracted by alkaline lysis,then purified by gel filtration,affinity and ion-exchange chromatography,and subject to overall control tests.Results The ratio of A260 to A280 of plasmid DNA after purification by three steps of chromatography was 1.85 ～ 1.92,in which the proportion of super-coiled plasmid DNA was not less than 94%,and the endotoxin content was far less than 10 EU /mg.Overall control tests proved that all the five batches of purified plasmid DNAs met the national quality standards.Conclusion The tumor DNA vaccine purified by the developed procedure showed high purity,in which the endotoxin was removed effectively.

    2010 06 v.23 [Abstract][OnlineView][Download 218K]

  • Effect of Prophylactic Administration with Angelica Polysaccharides on Expression of Adhesion Molecules and Cell Cycle of Bone Marrow Mononuclear Cells of Radiation Injured Mice

    ZHANG Yan,WU Hong,GUAN Xue-jing,et al(Laboratory of Stem Cell and Tissue Engineering,Department of Histology and Embryology,Chongqing Medical University,Chongqing 400016,China)

    Objective To investigate the effect of prophylactic administration with Angelica polysaccharides(APS)on the expression of adhesion molecules and cell cycle of bone marrow mononuclear cells(BMNCs)of radiation injured mice.Methods BALB /c mice were divided into one normal control,one normal saline(NS)control and two test groups.The mice in normal control group were untreated,while those in NS control and the two test groups were injected i.p.with NS as well as 2 and 8 mg /kg APS respectively,once a day for 7 d,then subjected to radiation and further injected for 13 d.The peripheral blood of mice in various groups were collected on days 7 and 14 after radiation and counted for WBC,RBC and PLT,while the bone marrow were collected and counted for BMNCs.The expressions of adhesion molecules CD44 and CD49d on surface as well as cell cycle of Sca-1+BMNCs were determined by flow cytometry.The transcription level of CyclinD2 mRNA in BMNCs was determined by semi-quantitative RTPCR,while the expression level of CyclinD2 by Western blot.Results Compared with those in normal control group,the counts of WBC,RBC,PLT and BMNCs,the expression levels of CD44 and CD49d on surface of Sca-1+BMNCs,as well as CyclinD2 mRNA transcription and protein expression levels of mice in NS control group decreased significantly(P < 0.05),while the percentage of cells at G0 /G1 phase increased significantly(P < 0.05).However,the counts of WBC,RBC,PLT and BMNCs as well as CyclinD2 mRNA transcription and protein expression levels of mice in the two test groups increased significantly(P < 0.05),while the expression levels of CD44 and CD49d on surface of Sca-1+BMNCs and the percentage of cells at G0 /G1 phase decreased significantly(P < 0.05).Conclusion Prophylactic administration with APS accelerated the cell cycle of BMNCs from G1 phase to S phase and promoted the recovery of hematopoietic function by decreasing the expression levels of adhesion molecule on surface of Sca-1+ BMNCs and up-regulating the expressions of CyclinD2 mRNA and protein of radiation injured mice.

    2010 06 v.23 [Abstract][OnlineView][Download 538K]

  • Preparation of Monoclonal Antibody against Poliovirus TypeⅠ

    LI Shu-xiang,SHEN Xin-liang,SHI Nan,et al(National Vaccine and Serum Institute,Beijing 100024,China)

    Objective To prepare the monoclonal antibody(McAb)against poliovirus and lay a foundation of developing ELISA for the virus.Methods The bulks of live attenuated and inactivated poliovirus typeⅠ were purified by ultrafiltration and ultracentrifugation.The purified antigen of live attenuated poliovirus was used for immunization of BALB /c mice,and the obtained splenic cells were fused with SP2 /0-A14 cells.An indirect ELISA was developed with the purified antigen of inactivated poliovirus and used for screening of positive cell strains secreting specific McAb.The secreted McAb was purified from the ascites of mice by ncaprylic acid-ammonium sulfate method and identified.Results Two cell strains stably secreting McAb against poliovirus,7G5 and 5E4,were obtained.The purified McAbs 7G5 and 5E4 were bound to the capsid proteins VP2 and VP3 of D antigen of poliovirus respectively,and showed high stability after storage at various temperatures.McAb 7G5 was not suitable for storage at pH4.The relative affinity of 5E4 was higher than that of 7G5.McAb 7G5 was identified as IgM,while 5E4 as IgG2a.McAbs 7G5 and 5E4 recognized similar epitopes,with neutralizing titers of 1:64 and 1:128 respectively.Conclusion The McAbs 7G5 and 5E4 against poliovirus were successfully prepared.

    2010 06 v.23 [Abstract][OnlineView][Download 219K]

  • Curative Effect of Component Blood Transfusion on Autoimmune Hemolytic Anemia

    LI Lan,XUE Jian-cheng(Department of Blood Transfusion,The Second People's Hospital of Shenzhen,Shenzhen 518035,Guangdong Province,China)

    Objective To investigate the curative effect of component blood transfusion on autoimmune hemolytic anemia(AHIA)in clinic.Methods The clinical data on 15 patients with AHIA transfused with washed red cells(group A)and 15 patients transfused with suspended red cells(group B)were reviewed and compared.Results The Hb,RBC and Hct of patients in both groups A and B increased 24 h after transfusion.All the indexes showed significant difference in the same group before and after transfusion,while showed no significant difference between the two groups.The clinical symptoms of 5 patients in group A improved significantly after treatment by transfusion of washed red cells combined with plasma exchange.Conclusion Transfusion with washed red cells and with suspended red cells showed no significant difference in clinical curative effect.However,the transfusion with washed red cells combined with plasma exchange showed significantly curative effect on AHIA.

    2010 06 v.23 [Abstract][OnlineView][Download 127K]

  • Optimization of Condition for Serum-free Culture of Vero Cells and Influenza A(H1N1)Virus on Microcarriers

    TIAN Shi-hua,SUN Ming-bo,ZHANG Xin-wen,et al(Institute of Medical Biology,Chinese Academy of Medical Sciences and Peking Union Medical College,Kunming 650118,China)

    Objective To optimize the conditions for serum-free culture of Vero cells and influenza A(H1N1)virus on microcarriers and lay a foundation of developing Vero cell-based influenza vaccine.Methods Vero cells were inoculated at various concentrations and cultured on microcarriers at various concentrations in stirring flask to prepare influenza virus.The effects of pH value,TPCK-trypsin content,MOI of virus inoculated and time for harvest on hemagglutinatin(HA)titer were evaluated.Results The condition for culture of Vero cells and influenza virus was optimized as follows:(1.0 ～ 5.0)× 105 Vero cells were inoculated onto serumfree microcarriers at a concentration of 3 mg /ml,and cultured in virus medium at a pH value of 7.2 ～ 7.4 and a TPCK-trypsin content of 1.0 μg /ml,then inoculated with influenza virus at a MOI of 1.0.TPCK-trypsin was supplied 48 h,and the virus in supernatant and cells were harvested 72 h after culture.The titer of influenza virus prepared under the optimal condition reached 1:512.Conclusion The condition for serum-free culture of Vero cells and influenza A(H1N1)virus on microcarriers were optimized,which laid a foundation of large-scale preparation of influenza virus by using bioreactor.

    2010 06 v.23 [Abstract][OnlineView][Download 364K]

  • Validation Test on Inactivation of Rabies Vaccine:Development of Cell Culture Method

    WANG Xia,XU Ge-lin,SUN Wen,et al(Wuhan Institute of Biological Products,Wuhan 430060,China)

    Objective To develop a cell culture method for validation of inactivation of rabies vaccine.Methods Nine batches of rabies vaccine samples and CTN virus suspension at various contents were cultured in Vero cells for 21 d then,after fixation of cells with acetone,determined by IFA,and the results were compared with those by mouse method.The reproducibility and sensitivity of the developed cell culture method were verified.Results All the determination results of nine batches of vaccine samples by the developed cell culture method were negative,while all those of CTN virus suspension were positive,which were consistent with the determination results by mouse method.Cell culture method reached a sensitivity of 3.3 FFU /ml and showed high reproducibility.Conclusion A cell culture method was developed,which showed high sensitivity and reproducibility and was suitable for the validation of inactivation of rabies vaccine.

    2010 06 v.23 [Abstract][OnlineView][Download 301K]

  • Development of A Method for Determination of Biologic Activity of Recombinant Human Ciliary Neurotrophic Factor

    LI Ping,JIANG Lin,MA Ya-ru,et al(Lanzhou Institute of Biological Products,Lanzhou 730046,China)

    Objective To develop a method for determination of biologic activity and for effective quality control during preparation and production of recombinant human ciliary neurotrophic factor(rhCNTF).Methods Qualitative and semi-quantitative determinations of rhCNTF were carried out by cultures of ciliary ganalia cells and dorsal root ganglia of chicken embryo respectively.The activity of rhCNTF was determined by TF-1.CN5a.1 cell proliferation method.The biologic activity of rhCNTF in obesity was determined by bodyweight decrease test in normal mice.The four methods were investigated for experimental conditions and evaluated for advantages and shortcomings.Results All the four methods were suitable for the determination of biologic activity of rhCNTF.However,the TF-1.CN5a.1 cell proliferation method combined with bodyweight decrease test in normal mice was more effective.Conclusion The quality of rhCNTF may be controlled by TF-1.CN5a.1 cell proliferation method combined with bodyweight decrease test in normal mice.

    2010 06 v.23 [Abstract][OnlineView][Download 394K]

  • Inactivation Effect of Binary Ethylenimine on Type Ⅱ Poliovirus Sabin Strain

    WANG Bai-yan,ZHANG Xin-wen,CAI Wei,et al(Institute of Medical Biology,Chinese Academy of Medical Sciences and Peking Union Medical College,Kunming 650118,China)

    Objective To observe the inactivation effect of binary ethylenimine(BEI)on type Ⅱ poliovirus Sabin strain.Methods Sabin strain was inactivated at various temperatures(37,33,28 and 25℃)with BEI at various concentrations(0.002,0.001,0.0005 and 0.00025 mol /L),and determined for infectious titer of virus by microtitration and for D antigen contents by ELISA,using those inactivated with formaldehyde as control,based on which the verification test on virus activation was performed.Results Sabin strain was completely inactivated with 0.002 mol /L BEI at 37℃ for 48 h.The recovery rate of D antigen content of the inactivated virus was 90.6%,which was significantly higher than that inactivated with formaldehyde(60.6%).No live virus was detected by verification test.Conclusion BEI showed less damage to D antigen of type Ⅱ poliovirus Sabin strain and good protection to active constituents of vaccine.

    2010 06 v.23 [Abstract][OnlineView][Download 285K]

  • Development of A Method for Removal of Endotoxin from DNA Vaccine against Liver Cancer

    LV Jing,YANG Xiao-ling,ZHU Zhi-qiang,et al(Department of Biochemistry and Molecular Biology,Shanxi Medical University,Taiyuan 030001,China)

    Objective To develop a method for removal of endotoxin from DNA vaccine against liver cancer.Methods The endotoxin was removed from DNA vaccine pVAX1-aLCGV against liver cancer by Triton X-114 liquid phase separation,and the result was verified by pyrogen test in rabbits.The endotoxin content in vaccine was determined by limulus amoebocyte lysate(LAL)test.The TNF-α and IL-8 contents in plasma of rabbits before and after injection as well as serum antibody titers of rats after injection with the DNA vaccine were determined by ELISA.SMMC-7721 and HepG2 cells were transfected with the DNA vaccine before and after treatment with Triton X-114 separately,and the transfection efficacies were compared.Results The endotoxin content in DNA vaccine after treatment with Triton X-114 met the relevant requirements,and the vaccine was qualified in pyrogen test.The TNF-α and IL-8 contents in plasma of rabbits after injection with the DNA vaccine before treatment with Triton X-114 were significantly higher than those after treatment.However,the immunogenicity of DNA vaccine after treatment with Triton X-114 showed no significant difference with that before treatment,while the transfection efficacy of the former was higher than that of the latter.Conclusion Triton X-114 removed the endotoxin in DNA vaccine pVAX1-aLCGV effectively,which laid a foundation of clinical application of the vaccine.

    2010 06 v.23 [Abstract][OnlineView][Download 136K]

  • Determination of Relative Binding Affinity of Vascular Endothelial Growth Factor Trap by Indirect ELISA

    BI Hua,DING You-xue,WANG Lan,et al(National Institute for the Control of Pharmaceutical and Biological Products,Beijing 100050,China)

    Objective To determine the relative binding affinity of vascular endothelial growth factor(VEGF)Trap by indirect ELISA.Methods The VEGF165 at a fixed amount was bound to VEGF Trap,and the free VEGF165 was determined by double antibody sandwich ELISA,based on which the median inhibition concentration(IC50)of VEGF Trap as well as its ratio to the IC50 of reference in reaction system,i.e.the relative binding affinity,were calculated.Results Each of the three VEGF Trap samples of the same batch was determined for 3 times,and the relative binding affinities were(100.86 ± 9.43)%,(88.46 ± 13.94)% and(82.66 ± 9.60)% respectively,at a mean of(90.66 ± 12.60)%,which met the requirement for quality control.The variation coefficients of three tests were 9.35%,15.76% and 11.61% respectively.Conclusion ELISA showed high precision,and its determination result was objective with few influencing factors,suggesting that the method might be used for routine determination of relative binding affinity of VEGF Trap.

    2010 06 v.23 [Abstract][OnlineView][Download 272K]

  • Progress in Research on Herpes Simplex Virus Type Ⅱ Vaccine

    ZHANG Hua-jie,ZHANG Shu-min(National Institute for the Control of Pharmaceutical and Biological Products,Beijing 100050,China)

    Herpes simplex virus typeⅡ(HSV-2)is a pathogen mainly causing human genital herpes,a widespread sexually transmitted disease in the world especially in underdeveloped countries and regions.HSV-2 infection not only seriously threats human health,but also causes great economic loss.Numerous studies on development of HSV-2 vaccines,including inactivated vaccine,live attenuated vaccine,recombinant subunit vaccine,peptide vaccine and DNA vaccine,have been taken for many years.Although progresses have been made,there is yet no licensed HSV-2 vaccine in the world so far.In this paper,the progresses in development of HSV-2 vaccine in recent years are reviewed.

    2010 06 v.23 [Abstract][OnlineView][Download 160K]

  • Association of TNF-α and IL-8 with Pathogenic Mechanism of Chronic Obstructive Pulmonary Disease

    WANG Dong,ZHANG Dong-yue(Department of Respiratory Medicine,The First People's Hospital of Lanzhou City,Lanzhou 730050,China)

    Chronic obstructive pulmonary disease(COPD)is one of the common diseases in respiratory system.The late researches showed close association of some cytokines,such as TNF-α and IL-8,with the onset of COPD.As major inflammatory mediators,TNF-α and IL-8 involve in the inflammatory response of airway,which accelerate the onset and progress of local inflammation by their interaction,result in the structural reconstruction of airway and the formation of airflow obstruction and thereby influence the onset and progress of COPD.This paper reviews the association of TNF-α and IL-8 with pathogenic mechanism of COPD.

    2010 06 v.23 [Abstract][OnlineView][Download 145K]

  • Progress in Research on Determination of Rabies Virus Antibody by ELISA

    WANG Wei-wei,ZHANG Guo-qiang,XU Li-feng(National Vaccine and Serum Institute,Beijing 100024,China)

    Effective prophylaxis of rabies virus is essential for avoiding large-scale epidemic of rabies.ELISA is specific and rapid,thus has a unique superiority in monitoring the antibody level in population.This article reviews the progress in research on determination of rabies virus antibody by ELISA.

    2010 06 v.23 [Abstract][OnlineView][Download 132K]


@2012 Editorial Department of "Chinese Journal of Biologicals"