2007年06期目次

  • Expression of Chimeric Gene of Human Papillomavirus Structural Protein L1 and CTL Epitope of Candida albicans Mannoprotein in Insect Cells

    ZHU Shan-li,HOU Xiao-hong,OU Qin,et al(Department of Microbiology & Immunology,Wenzhou Medical College,Wenzhou 325000,China)

    Objective To construct recombinant plasmid carrying the genes of human papillomavirus(HPV)type 6b structural protein L1 and CTL epitope of Candida albicans(Ca)mannoprotein(Mp)and express in baculovirus-insect cell system.Methods Chimeric gene HPV6b L1/CaMp65 CTL145-153 was amplified by PCR with designed primers and inserted into baculovirus transfer vector pFastBacTM1,then transformed to E.coli DH10Bac.The constructed recombinant baculovirus rBacmid/HPV6b L1/CaMp65 CTL145-153 was tranfected to insect cell strain sf-9,and the expressed product was identified by SDS-PAGE and Western blot.Results HPV6b L1/CaMp65 CTL145-153 chimeric protein with a relative molecular mass of 56 000 was expressed in sf-9 cells and showed specific reaction with McAb against HPV6b L1.Conclusion HPV6b L1/CaMp65 CTL145-153 chimeric protein was successfully expressed in baculovirus-insect cell system,which laid a foundation of developing recombinant vaccine for prevention and treatment of HPV and Candida albicans infections.

    2007 06 [Abstract][OnlineView][Download 510K]

  • Function of Pre-stored mRNA in Conidia of Trichophyton rubrum

    ZHANG Qian,LIU Tao,SHENG Jun,et al(College of Life Science,Jilin University,Changchun 130061,China)

    Objective To analyze the function of pre-stored mRNA in conida of Trichophyton rubrum.Methods The homologies of 560 genes pre-stored in conidia of Trichomphyton rubrum to those of other filamentous fungi in NR database were analyzed.On the basis of this,the physiological processes in which the genes might be involved were classified by GO database.Results Of the 560 genes,177 were homologous to the Aspergillus fumigatus,Aspergillus nidulans,Neurospora crassa and Trichophyton rubrum in NR database.Some important genes and signaling pathways,which might be related to the induction of condial germination,were found.Conclusion It provided a basis for further study on the role of pre-stored genes in conidia of Trichomphyton rubrum as well as the mechanism of condial germination.

    2007 06 [Abstract][OnlineView][Download 165K]

  • Construction of Eukaryotic Vector for Expression of Tumor Suppress Gene WWOX in A549 Cells

    YANG Wei,YUAN Hong-yan,ZHU Ming-guang,et al(Department of Immunology,Basic Medicine College,Jilin University,Changchun 130021,China)

    Objective To construct eukaryotic expression vector for tumor suppress gene WWOX and observe the expression of the gene in A549 cells.Methods Extract WWOX gene from normal human fetal kidney HEK-293 cells by RT-PCR and clone into vector pMD18-T.After identification of the constructed recombinant plasmid pMD18-T-WWOX by restriction analysis,the WWOX gene was inserted into eukaryotic expression vector pEGFP-C1.Transfect A549 cells with the constructed recombinant plasmid pEGFP-C1-WWOX in mediation of liposome,and observe the expression of GFP-WWOX fusion protein by confocal microscopy.Results The sequence of amplified WWOX gene was identical to that reported in GenBank.Green fluorescence was observed in A549 cells 48 h after transfection with recombinant plasmid pEGFP-C1-WWOX.RT-PCR proved high expression of WWOX mRNA.Conclusion A eukaryotic expression vector pEGFP-C1-WWOX was successfully constructed,and GFP-WWOX fusion protein was expressed in A549 cells.

    2007 06 [Abstract][OnlineView][Download 449K]

  • Significance of Irregular Red Blood Cell IgG of Pregnant Women in Early Diagnosis of Non-ABO-HDN

    WU Yuan-jun,LIU Xing-ling,LIU Yan-hui,et al(Affiliated TungWah Hospital,Sun Yat-sen University,Dongguan 523110,China)

    Objective To explore the significance of irregular red blood cell(RBC)IgG of pregnant women in the early diagnosis of non-ABO-HDN.Methods Screen irregular RBC antibody in 4 200 pregnant women.The women positive for irregular RBC antibody were tested for specificity of antibody as well as type and titer of Ig.The umbilical cord blood or the blood of newborns from pregnant women positive for irregular RBC IgG were collected after parturition for diagnosis of HDN by test for free antibody in sera as well as direct RBC antiglobulin test and RBC antibody release test.Results Of the 4 200 pregnant women,44(1.05%)were positive for irregular RBC antibody,of which 20 were positive for IgG or IgG and IgM mixed antibody.The fetus of one pregnant woman positive for irregular RBC IgG died in uterus at the 20th weeks after pregnancy,and of the 19 newborns delivered by the other 19 pregnant women,10 were diagnosed as non-ABO-HDN.The distribution of specificity of pathogenic antibody in the dead fetus and the newborns with non-ABO-HDN was as follows:2 cases of anti-D,1 case of anti-c,2 cases of anti-E,1 case of anti-cE and 4 cases of anti-M(including 1 case complicated with anti-A).Conclusion The IgG and anti-IgM of Rh blood group system in pregnant women may cause HDN in newborn.The detection of irregular RBC antibody is of important significance in early diagnosis of non-ABO-HDN,differentiation of antibody type causing HDN,evaluation of severity of HDN and selection of therapeutic protocol.

    2007 06 [Abstract][OnlineView][Download 105K]

  • Bioinformatics of Four Pairs of PCR Primers for Simian Vacuolating Virus 40 DNA

    GE Chang-yong,SUN Mao-sheng(Institute of Medical Biology,Chinese Academy of Medical Sciences,Peking Union Medical College,Kunming 650118,China)

    Objective To compare the reasonability of design,conservation of amplified gene fragments and sensitivity in detection of target gene fragment of four pairs of PCR primers for simian vacuolating virus 40(SV40)DNA and provide theoretical basis for stable and effective method for SV40 DNA sequencing.Methods Four pairs of PCR primers and the conservation of amplified gene fragments were analyzed by DNAMAN 6.0.40 software based on known genomes of 23 different SV40 strains.Multiple sequence alignment was carried out by clustalX1.83 software.Phylogenetic tree was constructed by TreeView1.6.6 software.The characteristics of the four pairs of primers were analyzed by Oligo 6.71 software.Results All the sequences of four pairs of primers were not conservative for the SV40 strain with an accession number of DQ218418.The GCVp1 and GCT sequences of primers were conservative for four SV40 strains with accession numbers of J02400,NC_001669,AF316139 and AF316141.The VP1 sequences of primers were conservative for the SV40 strains except DQ218418.The T sequences of primers were conservative for the SV40 strains except DQ218418 and AF180737.Conclusion The synthetic primers were specific to 21 SV40 strains.Compared with common primers,the synthetic primers were of good conservation,wide adaptivity and ideal parameters.Specific primers shall be designed for highly variant SV40 strains.

    2007 06 [Abstract][OnlineView][Download 1404K]

  • Expression of Fas Protein in Hippocampus CA1 Region of Diabetic Rats with Focal Cerebral Ischemia-Reperfusion Injury

    ZHANG Ji-sheng,DING Xin,WU Jun,et al(Department of Neurology,Peking University Shenzhen Hospital,Shenzhen 518036,China)

    Objective To explore the pathological change and expression of Fas protein in hippocampus CA1 region of diabetic rats with focal cerebral ischemia-reperfusion injury(CIRI).Methods Establish diabetic rat model of middle cerebral artery occlusion(MCAO)by induction with streptozocin(STZ)and line-lock method.Observe the lesion of neuron and expression of Fas protein in hippocampus CA1 region of diabetic rats with CIRI by HE staining and immunohistochemical method,and compare the results with those of normal rats with CIRI.Results Compared with those of normal rats with CIRI,the quantity of neuron in hippocampus CA1 region of diabetic rats with CIRI decreased significantly,and the expression of Fas protein was up-regulated significantly.Conclusion Severe decrease of neuron in quantity was observed in hippocampus CA1 region of diabetic rats with CIRI,which might be due to the cell apoptosis mediated by Fas protein.

    2007 06 [Abstract][OnlineView][Download 463K]

  • Expression and Subcellular Location of Fusion Domain MM Protein Using Baculovirus Vector

    CHEN Jin-pin,ZHANG You-li,ZHU Yan,et al(The Affiliated Hospital of Jiangsu University,Zhenjiang 212001,China)

    Objective To express and locate a fusion gene(MM)consisting of c-Myb DNA-binding domain and Myeloid elf-like factor(MEF)AML1-binding region by using baculovirus vector.Methods Insert MM gene with green fluorescence protein(GFP)fused at N-terminus into baculovirus transfer vector BacPak8.Co-transfect insect cell Tn5 with the obtained recombinant plasmid and its parental virus AcPak6.Observe the subcellular location of GFP-MM fusion protein by fluorescent microscopy and determine the expressed product by Western blot.Results A large quantity of congregate expressed proteins were observed in Tn5 cells 48 h after transfection.Western blot showed specific reaction band with a relative molecular mass of about 60 000,which was consistent with that expected.Conclusion The MM protein expressed by baculovirus expression system was stable and located in nuclei correctly.In this way,a large quantity of expressed product might be obtained.

    2007 06 [Abstract][OnlineView][Download 212K]

  • Biological and Molecular Characteristics of Live Attenuated Japanese Encephalitis Vaccine Virus Strain SA14-14-2 Inoculated Intrathoracically to Culex tritaeniorhynchus

    LIU Zhi-wen,YU Yong-xin,ZHANG Hai-lin,et al(National Institute for the Control of Pharmaceutical and Biological Products,Beijing 100050,China)

    Objective To further evaluate the stability and safety of live attenuated Japanese encephalitis(JE)vaccine virus strain SA14-14-2 by studying the biological and molecular characteristics of the virus after intrathoracical inoculation to Culex tritaeniorhynchus.Methods Grind the Culex tritaeniorhynchus inoculated intrathoracically with strain SA14-14-2 then centrifuge.Collect the supernatant and inoculate to BHK21 cells for virus propagation.The obtained SA14-14-2 M1C1 virus liquid was determined for titer by PFU method,then subjected to neurovirulence test in mice and virulence reversion test in suckling mice.Extract viral RNA for amplification of E gene fragment by RT-PCR.Sequence the amplified gene fragment and compare with that of E gene of original SA14-14-2 strain.Results The titer of SA14-14-2 M1C1 virus liquid reached 1.4×107 PFU/ml.In neurovirulence test,no onset or death of mice was observed.In virulence reversion test,the virus titer after intracerebral passage in suckling mice was 1.9 LgLD50/0.03ml,which was less than 3.0 LgLD50 described in Chinese Pharmacopeia.No onset or death of suckling mice inoculated s.c.with the strain was observed.Compared with that of original SA14-14-2 strain,the nucleotide at site 1 340 of E region of amplified E gene changed from A to G,resulting the change of amino acid at E447 from D(Asp)to G(Gly).However,E447 was not one of the eight sites of gene mutation of SA14-14-2 strain during attenuation.Conclusion After intrathoracical inoculation to Culex tritaeniorhynchus,neither biological nor molecular characteristic of SA14-14-2 strain showed significant change,which further proved the safety of the JE vaccine virus strain.

    2007 06 [Abstract][OnlineView][Download 162K]

  • Biosafety of Recombinant Fowlpox Virus to Guinea Pigs

    JIN Ming-lan,JIN Ning-yi,LU Hui-jun,et al(College of Animal Science and Veterinary Medicine,Jilin University,Changchun 130062,China)

    Objective To study the biosafety of recombinant fowlpox to pregnant guinea pigs and their offspring.MethodsImmunize pregnant guinea pigs and their offspring with recombinant fowlpox virus at normal and large dosages separately.Analyze the virulence of recombinant fowlpox virus to pregnant guinea pigs as well as the influence on growth of offspring by clinical observation and histopathological method,and determine the residual viral DNA in offspring by PCR.Results During clinical observation,the pregnant guinea pigs showed no clinical symptoms or adverse reactions,and their mental status,water drinking and food taking were normal.The immuniztion with recombinant fowlpox virus caused no premature delivery,abortion,fetal death or ambly-fetus.The offspring showed no any clinical symptoms or physiological change in organs,and no viral DNA was detected.Conclusion The constructed recombinant fowlpox virus showed good biosafety to pregnant guinea pigs and their offspring.

    2007 06 [Abstract][OnlineView][Download 238K]

  • Immune Response Induced by DNA Vaccine Encoding SARS-CoV S1 and S2 Proteins in Mice

    WANG Rui-lin,JIN Ning-yi,JIN Hong-tao,et al(Genetic Engineering Laboratory,Academy of Military Medical Sciences,Changchun 130062,China)

    Objective To construct DNA vaccine encoding SARS-CoV S1 and S2 proteins and induce immune response in mice.Methods Synthesize the genes encoding S1 and S2 subunits of SARS-CoV according to the SARS-CoV genome sequence reported in GenBank and recombine with CTL epitope gene,then clone into expression vector pIRESlneo.Transfect BHK-21 cells with the constructed recombinant plasmids pIRCTL-S1 and pIRCTL-S2 respectively and determine the expressed product by IFA.Immunize mice with pIRCTL-S1 and pIRCTL-S2 respectively,and determine the CD4+ and CD8+T cell subgroups by flow cytometry,and serum antibody against SARS-CoV by ELISA.Results Both pIRCTL-S1 and pIRCTL-S2 were expressed in BHK-21 cells.The expressed proteins stimulated the proliferation of lymphocytes and induced specific antibody against SARS-CoV in mice.Conclusion The constructed DNA vaccines pIRCTL-S1 and pIRCTL-S2 induced both humoral and cellular immune responses in mice,which laid a foundation of development of SARS DNA vaccine.

    2007 06 [Abstract][OnlineView][Download 345K]

  • Humoral Immune Response Induced by Polytope DNA Vaccine in Chitosan-based Microspheres

    CHENG Yu-xing,ZHU Gao-hong,QU Su,et al(Chinese Academy of Medical Sciences and Peking Union Medical College,Kunming 650118,China)

    Objective To study the humoral immune response induced by polytope DNA vaccine in chitosan-based microsphere.Methods Prepare the constructed DNA vaccines pcDNA3.1-HME and pcDNA3.1-HME-3C3d into chitosan-based microspheres and immunize mice intranasally.Determine the specific antibody level induced in mice by micropore kit for protein detection.Results The specific IgGs to various epitopes were induced in immunized mice.However,the IgG level induced by chitosan-based DNA microsphere vaccine was significantly higher than that by DNA vaccine.Conclusion The chitosan-based microsphere delivery system enhanced the humoral immune response induced by polytope DNA vaccine.

    2007 06 [Abstract][OnlineView][Download 227K]

  • Activity and Specificity of AoGDW Peptide in Inhibiting Platelet Aggregation

    WANG Xiao-xia,NIU Bo,XIE Jun,et al(Department of Biochemistry and Molecular Biology,Shanxi Medical University,Taiyuan 030001,China)

    Objective To explore the activity and specificity of ω-aminooctanoic acid-glycine-aspartic acid-tryptophan(AoGDW)in inhibiting platelet aggregation.Methods Determine the activity of AoGDW in inhibiting human platelet aggregation by turbidimetry.Test for the detachment of AoGDW to human umbilical venous endothelial cell(HUVEC)by cell detachment test in vitro and MTT method.Results The IC50 of AoGDW and RGDS(arginine-glycine-aspartic acid-tryptophan-serine)in inhibiting the aggregation of human platelet were(4.71±2.51)μmol/L and(61.82±8.08)μmol/L respectively.The IC50 of AoGDW causing detachment to HUVEC was(16.50±0.68)mmol/L.Conclusion Rational design may enhance inhibitory potency of AoGDW peptide while retaining its specificity toward antiplatelet aggression,which may be an important consideration for the successful development of drugs as anti-thrombotic drugs.

    2007 06 [Abstract][OnlineView][Download 350K]

  • Optimization of Condition for Expression of HBV PreS2-MBP Fusion Protein in E. coli

    LIU Zhao-hui,SHAO Li-jun,LI Meng,et al(Changchun Institute of Biological Products,Changchun 130062,China)

    Objective To optimize the condition for expression of HBV PreS2-MBP fusion protein in E.coli.Methods Analyze the influence of host bacterium,culture medium,temperature and time for induction as well as inducer and its concentration on expression level of HBV PreS2-MBP fusion protein in E.coli by SDS-PAGE and BandScan software.Subculture recombinant E.coli strain for 100 passages and test the expression stability of HBV PreS2-MBP fusion protein.Results The expression level of HBV PreS2-MBP fusion protein reached 43.5% of total somatic protein in E.coli BL21 strain cultured in GS medium under induction of 0.5 mmol/L IPTG or 1.5 mmol/L lactose at 28℃ for 4 h.The expression level of fusion protein in E.coli BL21 strain subcultured for 100 passages showed no significant difference with that in original strain.Conclusion The condition for expression of HBV PreS2-MBP fusion protein in E.coli was optimized.

    2007 06 [Abstract][OnlineView][Download 252K]

  • Determination of Residual Host Genomic DNA in DNA Vaccine by Real-time PCR

    LI Liang-zhu,LIU Yong,WANG Yi-jie,et al(Institute of Medical Biology,Chinese Academy of Medical Science & Peking Union Medical College,Kunming 650118,China)

    Objective To develop a real-time PCR for determination of residual host genomic DNA in DNA vaccine.MethodsAmplify residual host genomic DNA from DNA vaccine by PCR using designed primers target to E.coli 23S ribosome RNA gene and determine the real-time fluorescence intensity of amplified product.Determine the intermediate product during purification of DNA vaccine by the developed real-time PCR.Results The whole determination was completed within 30 min.The developed real-time PCR showed good specificity and a sensitivity of 10 fg/μl.The correlation coefficient of standard curve of the method was-0.99.Conclusion The developed real-time PCR may be used for the determination of residual host genomic DNA in DNA vaccine.

    2007 06 [Abstract][OnlineView][Download 261K]

  • Optimization of Condition for Refolding of Recombinant Human Osteogenic Protein-1

    LI Jun-ling,WANG Shi-li,HAN Jin-xiang,et al(Shandong Medicinal Biotechnology Centre,Key Laboratory for Biotech-Drugs,Ministry of Health,Jinan 250062,China)

    Objective To optimize the condition for refolding of recombinant human osteogenic protein-1(rhOP-1)in a form of inclusion body.Methods Split the recombinant E.coli for expression of rhOP-1 by ultrasomication in ice bath and extract the inclusion body.Dissolve the inclusion body with 8 mol/L urea,purify by SP-Sepharose chromatography,refold by gradient dialysis.Analyze the content of target protein dimer by TotalLab software and the biological activity by in vitro and in vivo tests.Results The purity of target protein after purification reached more than 97%.The optimal condition for refolding of target protein was as follows:4℃,pH 9.0,protein concentration 0.4-0.8 mg/ml,urea concentration 2 mol/L,L-Arg concentration 0.4 mol/L.The refolding rate of target protein reached more than 75%,and the refolded protein showed high biological activity.Conclusion The condition for refolding of rhOP-1 inclusion body by gradient dialysis was optimized.

    2007 06 [Abstract][OnlineView][Download 680K]

  • Isolation and Identification of Swine Streptococus suis Serotype 2

    GAO Wei,HAN Wen-yu,LEI Lian-cheng,et al(College of Animal Science and Veterinary Medicine,Jilin University,Changchun 130062,China)

    Objective To identify the 5 strains of swine Streptococus suis isolated from a pig farm in Sichuan Province,China.Methods Observe the morphology,culture characteristics,pathogenicity in animals and biochemical reactions of the 5 strains of swine Streptococus suis,and identify their serotypes and virulence factors by PCR.Results All the 5 strains were identified as swine Streptococus suis serotype 2 and contained virulence factors MRP and EF,of which strains S1,S2 and S5 showed pathogenicity in BALB/c mice while the other 2 strains showed no pathogenicity.Conclusion The pestilence in the pig farm was caused by swine Streptococus suis serotype 2,which provided a basis for further prevention and control of infection with swine Streptococus suis serotype 2.

    2007 06 [Abstract][OnlineView][Download 359K]

  • Development of A Procedure for Preparation of Plasmid DNA for Gene Therapy

    LI Xue-mei,TIAN Ming-rao,JIN Ning-yi,et al(Department of Agriculture,Jilin University,Changchun 130062,China)

    Objective To explore a procedure for preparation of plasmid DNA for gene therapy.Methods Remove macromolecular RNA from cultured recombinant E.coli with plasmid pIRVP3HNIL18 by calcium chloride precipitation,and other impurities such chromosomal DNA,micromolecular RNA and protein by Q-Sepharose and SOURCE ion exchange chromatography.Results The yield,purity,relative recovery and protein content of final product of plasmid pIRVP3HNIL18 were 1.638 mg plasmid DNA/g bacteria,1.839,12.55% and 0.002 8 mg/ml respectively.No RNA or chromosomal DNA was detected.Conclusion The developed procedure was effective for removal of impurities and suitable for preparation of therapeutic-grade plasmid DNA.

    2007 06 [Abstract][OnlineView][Download 384K]

  • Purification and Identification of Recombinant Schistosoma japonicum 14-3-3 Protein

    ZHONG Zheng-rong,SHEN Ji-long,HU Yuan-sheng,et al(Department of Laboratory,The First Affiliated Hospital of Bengbu Medical College,Bengbu 233004,China)

    Objective To purify and identify recombinant Schistosoma japonicum(Sj)14-3-3 protein.Methods Purify recombinant Sj 14-3-3 protein by 2 times of gel cutting and 2 times of electroelution.Test the purified protein for concentration by Coomassie brilliant blue staining,purity by SDS-PAGE and specificity by Western blot,and compared with those purified by column chromatography.Results The purified protein showed a single band with relative molecular mass of about 32 500 on SDS-PAGE profile,and reached a concentration of 3 mg/ml,which was significantly higher than that of protein purified by column chromatography(1 mg/ml).Western blot showed good specificity of the purified protein.Conclusion Highly purified and specific Sj 14-3-3 protein was obtained by developing an economic and effective purification procedure consisting of 2 times of gel cutting and 2 times of electroelution.

    2007 06 [Abstract][OnlineView][Download 298K]

  • Curative Effect of Acupuncture Separation Combined with Injection of Recombinant Human Interferon α2b around Bulbar Conjunctiva on Encapsulated Filtering Bled

    WANG Ying,XU Shao-lin,CHENG Zhuo(Department of Ophthalmoloy,Second Hospital of Jilin University,Changchun 130041,China)

    Objective To observe the curative effect of acupuncture separation combined with injection of recombinant human interferon α2b(rhIFN α2b)around bulbar conjunctiva on encapsulated filtering bled after trabeculectomy.Methods Twelve patients with encapsulated filtering bled after trabeculectomy were subjected to acupuncture separation,then injected with rhIFN α2b around bulbar conjunctiva and followed-up for intraocular pressure(IOP)and the form of filtering bled.Results The mean IOPs before and after treatment were(27.67±2.96)mmHg and(15.25±3.02)mmHg respectively,which showed significant difference.The filter bled type Ⅰ,Ⅱ and Ⅲ were observed in 3,8 and 1 eyes respectively.Conclusion Acupuncture separation combined with injection of rhIFN α2b around bulbar conjunctiva showed good curative effect on encapsulated filtering bled at early stage and increased the success rate of filtering surgery.It was value of popularization in clinic.

    2007 06 [Abstract][OnlineView][Download 56K]


@2012 Editorial Department of "Chinese Journal of Biologicals"