2007年09期目次

  • Construction and Immunogenicity of Recombinant Plasmid Carrying Multiepitope Gene of Hepatitis C Virus

    WEI San-hua,YIN Wen,LEI Ying-feng,et al(Department of Clinical Laboratory Medicine,Tangdu Hospital,Fourth Military Medical University,Xi'an 710038,China)

    Objective To construct a eukaryotic expression vector of hepatitis C virus(HCV) multiepitope gene and study its immunogencity.Methods Digest prokaryotic expression vector pQE30-CtEm carrying the mimic epitopes at C and E2 domains and the tandem epitope at NS3-NS5 domain of HCV with BamH I and Hind III,and insert the obtained HCV multiepitope gene CtEm into eukaryotic expression vector pcDNA3.1(-).Transiently transfect CHO cells with the constructed recombinant plasmid in mediation of liposome and determine the expression of HCV multiepitope antigen.Immunize BALB/c mice with 100 μg of the constructed recombinant plasmid and determine the humoral and cellular immune responses.Results The constructed recombinant plasmid pcDNA3.1(-)-CtEm was successfully expressed in CHO cells and induced high antibody levels in mice.The specific lymphocyte proliferation level,IFN-γ and CTL activity of mice immunized with the constructed recombinant plasmid were significantly higher than those with empty vector or physiological saline.Conclusion The eukaryotic expression vector pcDNA3.1(-)-CtEm of HCV multiepitope gene was successfully constructed and induced specific humoral and cellular immune responses in mice.

    2007 09 [Abstract][OnlineView][Download 429K]

  • Construction and Identification of Recombinant Adenovirus for Expression of hTRAIL

    YANG Fang,YI Shan,LI Hong-jun,et al(Institute of Medical Biology,Chinese Academy of Medical Sciences and Peking Union Medical College,Kunming 650118,China)

    Objective To construct a recombinant adenovirus for expression of human TNF-related apoptosis inducing ligand(hTRAIL) gene.Methods Amplify gene fragment by PCR using the TRAIL gene extracted from recombinant plasmid pThioHisA-TRAIL as a template,insert into shuttle plasmid pShuttle-CMV and transform to E.coli DH 5α.Screen recombinant plasmid pShuttle-CMV-TRAIL and transform to competent BJ5183 cells previously transformed with pAdEasy-1.Screen recombinant plasmid pAdEasy-TRAIL and transfect to 293 cells in mediation of Lipofectamine to prepare replication-deficient adenovirus Ad-TRAIL carrying full-length of hTRAIL gene.Purify the prepared Ad-TRAIL virus particles by density gradient centrifugation with cesium chloride and determine the titer.Determine the transcription of TRAIL gene in 293 cells by RT-PCR.Transfect YTMLC cells with Ad-TRAIL and determine the expression of TRAIL by IFA and flow cytometry.Results The virus particle count and titer of purified Ad-TRAIL were 2.77×1012 VP/ml and 109.5(3.2×109)CCID50/ml respectively.The gene fragment at a length of about 843 bp was amplified by RT-PCR from transfected 293 cells,which was consistent with that expected.Both flow cytometry and IFA proved the expression of TRAIL protein in YTMLC cells,and the expressed product mainly existed in cytoplasm.Conclusion Recombinant adenovirus Ad-TRAIL for expression of hTRAIL was successfully constructed,which provided a basis for gene therapy of tumors.

    2007 09 [Abstract][OnlineView][Download 752K]

  • Construction of Eukaryotic Vector for Expression of Gene Encoding Tandem Repeats of Human Mucin 1 in Mammalian Cells

    ZHANG Shu-zi,ZHANG Wa,ZHANG Hai-hong,et al (Vaccine Research Center,College of Life Science,Jilin University,Changchun 130012,China)

    Objective To construct the eukaryotic expression vector of gene encoding 17 tandem repeats of human mucin 1(17MUC1 VNTRs),and express the gene in COS-7,HeLa and Lewis cells.Methods Synthesize 1MUC1 VNTR by PCR using the designed primer and clone into pGEM-T easy vector.Digest the constructed recombinant plasmid with SalⅠ and XhoⅠ,and ligate the digested product in turn to construct 17MUC1 VNTRs which was subcloned to eukaryotic expression vector VR1012.Transfect COS-7,HeLa and Lewis cells with the constructed recombinant VR1012-17m,then test for the transcription of mRNA by RT-PCR and the expression of 17m antigen protein by Western blot.Results Both restriction analysis and sequencing proved that the constructed recombinant plasmid VR1012-17m contained 17m gene.RT-PCR showed transcription of 17m mRNA in transfected COS-7,HeLa and Lewis cells.17m protein was expressed in all the transfected cells and showed specific reaction with McAb against MUC1 VNTR,as proved by Western blot.Conclusion The eukaryotic expression vector for 17m tumor antigen was successfully constructed and expressed in COS-7,HeLa and Lewis cells,which laid a foundation of further study on MUC1 vaccine and biotherapy of tumors.

    2007 09 [Abstract][OnlineView][Download 351K]

  • Cloning and Sequencing of Genomic A Segment of Infectious Bursal Disease Virus Vaccine Strain B87

    LIU Kai,WANG Xing-long,WANG Xue-li,et al (College of Animal Science and Veterinary Science,Jilin University,Changchun 130062,China)

    Objective To clone and sequence the full-length cDNA of genomic segment A of infectious bursal disease virus(IBDV) vaccine strain B87.Methods Extract the genomic RNA of IBDV by protease K digestion and phenol/chloroform extraction,then purify the genomic dsRNA by LiCl fractional precipitation for amplification of full-length cDNA of genomic segment A of IBDV strain B87 by one-step RT-PCR.Insert the amplified cDNA into pGEM-T vector,then sequence and analyze the result by using DNAStar software.Results Nucleotide sequencing proved that the homologies of cloned genomic A segment of IBDV vaccine strain B87,at a full-length of 3260 bp,were 99.5% and 95.2%~99.4% respectively to those of Gt and other strains,but was only 84.0% to that of strain OH of IBDV type Ⅱ.Conclusion The analysis of deduced amino acid sequences of 20 IBDV strains indicated that the 7 characteristic amino acid sites of A segment might be related to virulence.

    2007 09 [Abstract][OnlineView][Download 593K]

  • Influence of E1A Gene of Adenovirus Type 5 on Sensitivity of Breast Cancer Cells to Chemotherapeutics

    YANG Xin-yu,LI Qun,LI Xiang-liang,et al(Department of Common Surgery,Second Hospital of Jilin University,Changchun 130033,China)

    Objective To explore the influence of E1A gene of adenovirus type 5 on the sensitivity of breast cancer cells to chemotherapeutics.Methods Digest previously constructed recombinant plasmid pUC119-E1A with EcoR I and BamH I,and insert the obtained E1A gene into plasmid pEGFP-C1.Transfect human breast cancer cell strain SK-BR-3 with the constructed eukaryotic expression vector pEGFP-E1A in mediation of liposome and screen the transfected cells for stable expression of E1A gene.Determine the transcription level of E1A gene by RT-PCR.Observe the growth of transfected cells on soft agar plate and their sensitivity to cisplatin.Results A gene fragment at a length of about 380 bp was amplified from the cells transfected with recombinant plasmid pEGFP-E1A.Compared with those of SK-BR-34 cells untransfected or transfected with empty vector,the colonies of E1A gene-transfected SK-BR-3 cells formed on soft agar plate were significantly small,the colony formation rate was significantly low,and the sensitivity to cisplatin increased significantly.Conclusion The recombinant plamsid for stable expression of E1A gene was successfully constructed.E1A gene inhibited the growth of breast cancer cells and increased their sensitivity to chemotherapeutics significantly,which provided theoretical and practical basis for its clinical application.

    2007 09 [Abstract][OnlineView][Download 230K]

  • Construction of Recombinant Plasmid for Expression of Rabies Virus Glycoprotein in COS-7 Cells

    YAO Wei-min,SHU Shi,ZHOU Hua,et al (Changchun Institute of Biological Products,Changchun 130062,China)

    Objective To construct a recombinant plasmid for expression of rabies virus glycoprotein in COS-7 cells.Methods Culture rabies virus and extract negative strand RNA for amplification of glycoprotein gene by RT-PCR.Insert the amplified gene into eukaryotic expression vector pcDNA3.1(A) and transfect COS-7 cells with the constructed recombinant plasmid pcDNA3.1(A)/G in mediation of liposome.Determine the transient expression of rabies virus glycoprotein by ELISA and IFA.Results Restriction analysis showed successful construction of eukaryotic expression vector pcDNA3.1(A)/G.No expression of rabies virus glycoprotein was detected in the culture supernatant of transfected COS-7 cells.IFA proved the expression of rabies virus glycoprotein on surface of transfected COS-7 cells.Conclusion The constructed eukaryotic expression vector pcDNA3.1(A)/G was suitable for the expression of rabies virus glycoprotein in COS-7 cells,which laid a foundation of developing recombinant rabies virus glycoprotein vaccine.

    2007 09 [Abstract][OnlineView][Download 293K]

  • Development of A Method for Determination of Immunogenicity of H5N1 Avian Influenza Vaccine for Human Use

    ZHU Zhi-yong,DING Xiao-hang,ZHU Han-ping,et al(Key Lab of Vaccine against Hemorrhagic Fever with Renal Syndrome,Zhejiang Center for Disease Control and Prevention,Hangzhou 310009,China)

    Objective To develop a method for determination of immunogenicity of avian influenza vaccine for human use.Methods Prepare vaccine with avian influenza virus strain R1203.Immunize rabbits with the prepared vaccine at various dosages and determine the HI and neutralizing antibodies in sera.Perform cross HI test,cross single immunodiffusion test and cross neutralization test on antisera against human and avian influenza viruses.Results Both neutralizing and HI antibody titers of rabbits were low 14 d after the 1st dose,but increased significantly 14 d after the 2nd dose.The increase of HI antibody titer was dose-independent,while that of neutralizing antibody was significantly dose-dependent.Cross HI test showed cross reaction of different types,and the HI titers of most of samples were not less than 1∶40.Cross single immunodiffusion test showed no cross reaction of antisera of different types.The antisera induced by avian influenza virus could not neutralize human influenza virus but could neutralize the virus of the same type(R1194),with the highest neutralizing titer of 1∶240.Conclusion Neutralizing antibody titer could reflect the immunogenicity of avian influenza vaccine correctly,thus determination of neutralizing antibody might be used as the potency test on avian influenza vaccine.

    2007 09 [Abstract][OnlineView][Download 95K]

  • Immunogenicities of HBV DNA and Recombinant Modified Vaccinia Ankara-based HB Vaccines

    CHEN Yu,YANG Xiao-song,YANG Ping,et al (Vaccine Research Center,College of Life Science,Jilin University,Changchun 130012,China)

    Objective To compare the immunogenicities of HBV DNA and recombinant modified vaccina Ankara(rMVA)-based HB vaccines.Methods Immunize BALB/c mice with rMVA-based HB vaccine and HBV DNA vaccine respectively,then determine the humoral immune response by ELISA,and cellular immune response by ELISPOT.Results The serum HB antibody levels of mice immunized with rMVA-based HB vaccine and HBV DNA vaccine were 135 and 104 mIU/ml,and the numbers of spot-forming cells(SFC) were 550 and 314 cells per 106 lymphocytes respectively.Conclusion rMVA-based HB vaccine was more immunogenic than HBV DNA vaccine.

    2007 09 [Abstract][OnlineView][Download 196K]

  • Antibody Response Induced by Inactivated SV40 Vaccine in Macaques

    ZHANG Guang-ming,XIE Tian-hong,HE Zhan-long,et al(Institute of Medical Biology,Chinese Academy of Medical Sciences and Peking Union Medical College,Kunming 650118,China)

    Objective To evaluate the antibody response induced by inactivated SV40 vaccine in macaques.Methods Immunize macaques aged 6~12 months by intramuscular injection with inactivated SV40 vaccine for 2 times,at an interval of 10 weeks.Collect the venous blood 2 months after the 1st and the 2nd immunization respectively for determination of serum neutralizing antibody titer,and the peripheral blood 18 months after the 2nd immunization for SV40 genomic component.Results The serum antibody positive conversion rate of all the 12 macaques 2 weeks after the 1st immunization was 100%,and the mean antibody titer was 1∶16.However,the mean antibody titer 2 weeks after the 2nd immunization reached 1∶64~1∶128.No SV40 genomic component was detected by PCR in the peripheral blood of macaques 18 months after the 2nd immunization.Conclusion Inactivated SV40 vaccine induced specific antibody response in macaques.

    2007 09 [Abstract][OnlineView][Download 272K]

  • Stability of Freeze-dried Groups A and C Meningococcal Polysaccharide Conjugate Vaccine

    WU Kai,CHEN Yan,FAN Hui-lan,et al(Wosen Biotechnology Co.Ltd,Kunming 650106,China)

    Objective To explore the stability of freeze-dried groups A and C meningococcal polysaccharide conjugate vaccine at 2-8℃.Methods Store freeze-dried groups A and C meningococcal polysaccharide conjugate vaccine at 2-8℃ for 30 months.Take samples periodically for inspection of final containers,identity test,abnormal toxicity test,immunogenicity test as well as determination of polysaccharide content,moisture content and molecular size to evaluate the stability of vaccine.Results All the quality control indexes of freeze-dried groups A and c meningococcal polysaccharide conjugate vaccine after storage at 2-8℃ for 30 months met the requirements for product license.Conclusion Freeze-dried groups A and C meningococcal polysaccharide conjugate vaccine showed good stability at 2-8℃.

    2007 09 [Abstract][OnlineView][Download 56K]

  • Extraction and Immunological Function of Pilose Antler Polypeptide

    PAN Feng-guang,SUN Wei,ZHOU Yu,et al( College of Quartermaster Technology,Jilin University,Changchun 130062,China)

    Objective To extract and purify active polypeptides from the pilose antlers treated by various procedures and study their immunological function.Methods Extract active polypeptides from the pilose antlers treated by various procedures by molecular exclusion chromatography and ion exchange chromatography and determine its immunological activity by lymphocyte proliferation test and cytokine production test.Results The protein contents of active polypeptides extracted from freeze-dried,frozen and heat-treated pilose antlers were 9.2,1.3 and 1.1 mg/ml respectively.The polypeptides promoted the proliferation of murine T and B lymphocytes,activated macrophages to secrete IL-12 and showed immunopotentiation effect.Conclusion Pilose antler polypeptide enhanced cellular immunity significantly.

    2007 09 [Abstract][OnlineView][Download 312K]

  • Curative Effect of Recombinant Human Interferon α2b Vaginal Effervescent Tablet on Vaginitis Caused by Herpes Simplex Virus Type 2(HSV-2) in Guinea Pigs

    LI Jiu-li,TIAN Jian-dan,HE Ling-bing,et al( College of Life Science and Biotechnology,Shanghai Jiao Tong University,Shanghai 200030,China)

    Objective To observe the curative effect of recombinant human interferon(rhIFN) α2b vaginal effervescent tablet on vaginitis caused by herpes simplex virus type 2(HSV-2) in guinea pig model.Methods Establish experimental guinea pig model of vaginitis by infection with HSV-2 and treat with rhIFN α2b vaginal effervescent tablet at dosages of 600,3000 and 15 000 IU respectively,using acyclovir(0.1 g) and rhIFN α2b suppository 15 000 IU as controls.Score the pathological changes of appearance and tissue sections of viginae before and after treatment.Results The scores for pathological changes of appearance and tissue sections of viginae after treatment with rhIFN α2b vaginal effervescent tablet were significantly lower than those after treatment with acyclovir or rhIFN α2b suppository.Compared with that at dosages of 600 and 3000 IU,the rhIFN α2b vaginal effervescent tablet at a dosage of 15 000 IU was more effective in decreasing the scores.At the same dosage,rhIFN α2b vaginal effervescent tablet showed good curative effect as compared with rhIFN α2b suppository.Conclusion rhIFN α2b vaginal effervescent tablet showed significant curative effect on experimental vaginitis caused by HSV-2.

    2007 09 [Abstract][OnlineView][Download 98K]

  • Influence of Newborn Bovine Liver Bioactive Peptide on ALT and AST Activities in Sera of Mice

    FU Ying,WANG Yin(Zhejiang Academy of Medical Sciences,Hangzhou 310013,China)

    Objective To explore the curative effect of newborn bovine liver bioactive peptide on chemical liver injury of mice caused by carbon tetrachloride.Methods Divide model mice with liver injury caused by carbon tetrachloride into 3 groups and treated with newborn bioactive peptide at high,moderate and low dosages by oral route respectively,using the untreated model mice and normal mice as control.Determine the activities of ALT and AST of various groups before and after treatment respectively.Results The ALT activity of mice treated with newborn bovine liver bioactive peptide at high dosage decreased significantly,however,the AST activity showed no significant decrease.Conclusion Newborn bovine liver bioactive peptide showed assistant curative effect on chemical liver injury.

    2007 09 [Abstract][OnlineView][Download 58K]

  • Clinical Evaluation of Rapid Diagnostic Kit for HIV Antibody

    LI Xiu-hua,XU Si-hong,LI Jing-yun,et al( National Institute for the Control of Pharmaceutical and Biological Products,Beijing 100050,China)

    Objective To evaluate the sensitivity and specificity of rapid diagnostic kit for HIV antibody in test for HIV antibody in whole blood,serum or plasma samples.Methods Collect 493 whole blood samples from different populations and regions and separate the serum.Test the whole blood and serum samples of the same subject by diagnostic kit to be evaluated.Meanwhile,a total of 1175 HIV antibody positive and negative serum/plasma samples from different populations and regions were tested by the kit to be evaluated,using the reference kit as control.Results Of the 493 subjects,99 were diagnosed as HIV antibody positive by the kit to be evaluated,and the test results of whole blood and serum samples of the same subject were completely consistent.Compared with those of reference kit,the sensitivity and specificity of kit to be evaluated were 100% and 99.92% respectively.Conclusion The quality of rapid diagnostic kit for HIV antibody to be evaluated was equal to that of reference kit.

    2007 09 [Abstract][OnlineView][Download 61K]

  • Immune Activation of Immunocytokine Hu14.18-IL-2 in Patients with Melanoma

    SONG Ji-ping,Jacquelyn A.Hank,Paul M.Sondel( Beijing Tiantan Biological Products Co.Ltd,Beijing 100024,China)

    Objective To evaluate the in vivo immune activation of immunocytokine Hu14.18-IL-2 in the patients with melanoma.Methods Thirty-three patients were treated with Hu14.18-IL2,a humanized anti-GD-2 McAb linked to IL-2,by intravenous drip for 3 d as the 1st course at dosages of 0.8,1.6,3.2,4.8,6.0 and 7.5 mg/m2 respectively.The patients received the 2nd course of treatment at the 5th week after stabilization or remission of disease.The peripheral blood mononuclear cell(PBMC) antibody-dependent cytotoxicity,NK cell activity as well as in vitro proliferation and soluble receptor of IL-2 of patients before and after treatment were observed.Results Hu14.18-IL-2 induced the phagocytosis of lymphocytes,enhanced the killing activity of NK cells in peripheral blood and increased the level of soluble alpha chain of IL-2 receptor complex in sera.Conclusion Hu14.18-IL-2 showed immune activation in patients with melanoma.

    2007 09 [Abstract][OnlineView][Download 401K]

  • Application of Domestic Live Attenuated Varicella Vaccine in Immunocompromised Patients

    ZHOU Hua,CHEN Lie-sheng,SHEN Zhi-xiang,et al( Shanghai Institute of Biological Products,Shanghai 200052,China)

    Objective To observe the reactogenicity and antibody response of domestic live attenuated varicella vaccine prepared with Oka strain in the patients with acute leukemia or solid tumors or receiving immunosuppressants.Methods Immunize the above-mentioned immunocompromised patients with domestic live attenuated varicella vaccine prepared with Oka strain,determine the antibody levels before and after immunization and observe the adverse reactions.Results No abnormal reactions were observed in all the patients.Moderate or severe fever were observed in a few patients,most of whom were those with solid tumors.Most of the patients were positive for varicella antibody before immunization,and their antibody titers increased at various degrees after immunization.However,the antibody titers of patients treated with immunosuppressants showed no significant increase.Conclusion Domestic live attenuated varicella vaccine was safe and effective in immunocompromised patients.

    2007 09 [Abstract][OnlineView][Download 107K]

  • Culture of Vero Cells in Serum-free and Protein-free Medium

    GAO Zheng-lun,ZHANG Xian-ping,SHENG Jun,et al(College of Life Science,Jilin University,Changchun 130012,China)

    Objective To develop a method for culture of Vero cells in serum-free and protein-free medium.Methods Prepare serum-free and protein-free medium(PF) by adding yeast extract and soy hydrolysate into DMEM/F12 medium.Culture Vero cells in the prepared medium in common glass,polystyrene(PS-TC)-and polyvinyl butyral(PVB)-coated glass flasks respectively,using serum-containing medium(BM) as control.Observe the growth of Vero cells and plot a growth curve.Results Compared with BM,in common glass flask,the prepared PF medium could not support the growth and division of Vero cells;in PVB-coated glass flask,the medium supported the growth but could not support the the division of Vero cells.However,in PS-TC flask,the prepared PF medium supported both growth and division of Vero cells. Conclusion The prepared serum-free and protein-free medium was suitable for the culture of Vero cells in PS-TC flask.

    2007 09 [Abstract][OnlineView][Download 289K]

  • Development of Methods for Preparation of Monoclonal Antibody against Oxidized Low Density Lipoprotein

    XU Yan-hua,LUO Zu-xiu(Chengdu Hoist Biotechnology Co.Ltd,Chengdu 610225,China)

    Objective To develop the methods for preparation of monoclonal antibody(McAb) against oxidized low density lipoprotein(OxLDL). Methods Prepare McAb against OxLDL by in vivo culture in mice and purify by SuperoseTM 6 HR10/30 gel column chromatography.Meanwhile,prepare the McAb by in vitro cell culture,observe the growth of hybridoma cells and explore the condition for expression of McAb,then purify the McAb in cell culture supernatant by Streamline SPXL cation expanded bed chromatography and QXL anion exchange chromatography.Results The purity and recovery of McAb prepared by in vivo culture in mice were 95.7% and 65.4%,while those by in vitro cell culture were 94.7% and 47.8% respectively.Conclusion Two methods for preparation of McAb against OxLDL were developed,which laid a foundation of development of ELISA kit for OxLDL.

    2007 09 [Abstract][OnlineView][Download 320K]


@2012 Editorial Department of "Chinese Journal of Biologicals"