2008年04期目次

  • Prokaryotic Expression,Purification and Immune Effect of HPV16 MuE6/E7 Chimeric Protein

    YU Li,AN Jing,HAN Ping,et al (Lanzhou Institute of Biological Products,Lanzhou 730046,China)

    Objective To express HPV16 MuE6/E7 chimeric protein and study its immunogenicity and antitumor activity.MethodsTransform the constructed recombinant expression vector for MuE6/E7 protein to E.coli BL21(λDE3) for expression under induction of IPTG.The expressed product,in a form of inclusion body,was separated,purified,de-naturalized and re-naturalized,then further purified by ion exchange and molecular sieve chromatography,and identified by SDS-PAGE,Western blot,HPLC and mass spectrography.Immunize C57BL/6 mice with the purified protein to evaluate the immunogenicity and antitumor activity.Results The expressed product,with a relative molecular mass of about 21 000,contained about 23.06% of total somatic protein and reached a purity of 97.17% after purification.High titer specific antibody against HPV was induced in the mice immunized with 3 doses of purified MuE6/E7 protein.The antibody titer was positively related to the dosage of purified protein.The protein protected the immunized mice against challenge with TC-1 tumor cells and prolonged the survival time of mice with tumor caused by TC-1 cells.Conclusion Recombinant MuE6/E7 protein was expressed and purified and showed a certain curative effect on the mice with tumor caused by TC-1 cells,which laid a foundation of further development of therapeutic HPV vaccine.

    2008 04 [Abstract][OnlineView][Download 411K]

  • Construction and Expression of Recombinant Adenovirus Carrying Human 6Ckine/IFNγ Fusion Gene

    XUE Gang,CHENG Ying,CAO Yong-kuan,et al (Department of General Surgery,Chengdu Army General Hospital,Chengdu 610083,China)

    Objective To construct the recombinant adenovirus carrying human secondary lymphoid organ chemokine(6Ckine)/IFNγ fusion gene and express in eukaryotic cells.Methods Amplify human 6Ckine cDNA and IFNγ cDNA from human lymph node and peripheral blood mononuclear cells by RT-PCR respectively and insert into adenovirus shuttle plasmid pShuttle sequentially.Amplify 6Ckine/IFNγ fusion gene from the constructed recombinant plasmid pShuttle-6Ckine/IFNγ by PCR and clone to plasmid pDC316.Co-transfect HEK293 cells with large adenoverus helper plasmid pBHGlox△E1 and the constructed shuttle plasmid pDC316-6Ckine/IFNγ to prepare recombinant adenovirus Ad-6Ckine/IFNγ.The obtained recombinant adenovirus Ad-6Ckine/IFNγ was amplified,purified and used for infection of HepG2 cells,and the expressed fusion protein was identified by immunofluorescence method and Western blot.Results The constructed recombinant adenovirus Ad-6Ckine/IFNγ reached a titer of 1.26×1010 IFU/ml after amplification and purification and showed no contamination with wild type adenovirus as proved by PCR.The 6Ckine/IFNγ was expressed in a secretory form in HepG2 cells.Conclusion Recombinant adenovirus Ad-6Ckine/IFNγ was successfully constructed and expressed in eukaryotic cells,which laid a foundation of further study on gene therapy and immunotherapy of tumors.

    2008 04 [Abstract][OnlineView][Download 272K]

  • Cloning of Neurturin Gene and Its Expression in Vero Cells

    MA Kai-li,SUN Mao-sheng,SHI Rui,et al(Institute of Medical Biology,Chinese Academy of Medical Science,Peking Union Medical College,Kunming 650118,China)

    Objective To clone Neurturin(NTN) gene and express in Vero cells.Methods Amplify human NTN cDNA by RT-PCR and clone to eurkaryotic expression vector pcDNA3.Transfect Vero cells with the constructed recombinant plasmid pcDNA3/hNTN in mediation of Lipofectamine 2000.Identify the expressed product by RT-PCR and IFA.Observe the morphology and growth of transfected Vero cells.Results The positive clones for stable expression of NTN was obtained after transfection of Vero cells with recombinant plasmid pcDNA3/hNTN,and the target protein was expressed.The morphology and growth of transfected Vero cells showed significant change.Conclusion The Vero cell strain for stable expression of NTN was established,which laid a foundation of gene therapy of Parkinson disease in rhesus.

    2008 04 [Abstract][OnlineView][Download 321K]

  • Expression of Antimicrobial Peptide Dermaseptin S4 Gene and Its Mutant in E. coli

    ZHANG Qing-hua,HAN Yue-wu,XIE Jun-qiu,et al(Institute of Biochemistry and Molecular Biology,College of Basic Medical Sciences,Lanzhou University,Lanzhou 730000,China)

    Objective To construct a recombinant E.coli strain for expression of antimicrobial peptide Dermaseptin S4(DS4) gene and its mutant K4-S4 and express the target proteins.Methods Design and amplify DS4 and K4-S4 genes by overlap extension PCR and clone into vector pUC-18 respectively.Identify the constructed recombinant plasmids pUC18-DS4 and pUC18-K4-S4 by restriction analysis and sequencing,and insert the obtained target genes into expression vector pGEX-4T-1respectively.Transform the constructed recombinant plasmids pGEX-4T-1-DS4 and pGEX-4T-1-K4-S4 to E.coli BL21(DE3) plyS for expression under induction of IPTG.Identify the expressed product by 12%SDS-PAGE.Results DS4 and K4-S4 proteins,both with relative molecular masses of 34 000,were expressed.The expressed product mainly existed in a form of inclusion body.The expression level reached a peak value 5 h after induction.Conclusion A recombinant E.coli strain for expression of D4 and K4-S4 genes was successfully constructed,and the target protein was expressed,which laid a foundation of further study on function and application of D4 and K4-S4 proteins.

    2008 04 [Abstract][OnlineView][Download 263K]

  • Cloning and Prokaryotic Expression of HE Gene of Swine Hemagglutinating Encephalomyelitis Virus

    CHANG Ling-zhu,SONG De-guang,HE Wen-qi,et al(College of Animal Science and Veterinary Medicine,Jilin University,Changchun 130062,China)

    Objective To construct the prokaryotic expression system for hemagglutinin esterase(HE) protein of swine hemagglutinating encephalomyelitis virus(HEV) 67N strain and provide a candidate gene for the development of specific immunodiagnostic method.MethodsClone the gene encoding epitope enrich regions of HE protein of swine HEV 67N strain to construct recombinant plasmid pET-HE and express in E.coli.The expressed product,in a form of inclusion body,was washed,lyzed and further purified by metal chelate chromatography,then identified by SDS-PAGE and Western blot.Results The expressed HE protein contained 42.2% of total somatic protein.It reached a protein content of 0.6 mg/ml and a purity of 85.4% after purification and was recognized by rabbit serum against swine HEV.ConclusionThe prokaryotic expression vector for HE gene of swine HEV was successfully constructed.The expressed recombinant HE protein showed good specificity and might be used as a candidate antigen for determination of serum antibody against swine HEV.

    2008 04 [Abstract][OnlineView][Download 263K]

  • Construction and Peokaryotic Expression of Recombinant Plasmid Containing Colorectal Cancer-associated 15-Hydroxyproglandin Dehydrogenase Gene

    LI Mei-ning,FENG Yan,CHANG Bing-mei,et al(Department of Biochemistry and Molecular Biology,Shanxi Medical University,Taiyuan 030001,China)

    Objective To construct a recombinant plasmid for prokaryotic expression of colorectal cancer-associated 15-hydroxyproglandin dehydrogenase(PGDH)gene and express in E.coli.Methods The encoding sequence of PGDH gene was amplified by RT-PCR using the total RNA of normal human colorectal mucous tissue as template and inserted into prokaryotic expression vector pBV220.The constructed recombinant plasmid was transformed to E.coli DH5α for expression under induction of temperature.The expressed product was purified by Ni2+-NTA affinity chromatography and identified by SDS-PAGE and Western blot.Results Both PCR and restriction analysis proved that recombinant plasmid pBV220-PGDH-His6 was correctly constructed.The PGDH protein,with a relative molecular mass of about 29 000,was expressed in a form of inclusion body.The expression level reached a peak value(about 30% of total somatic protoin)3 h after induction.The expressed protein reached a purity of more than 95% after purification and showed good specificity as proved by Western blot.Conclusion The recombinant plasmid for prokaryotic expression of PGDH gene was successfully constructed and PGDH protein was expressed.

    2008 04 [Abstract][OnlineView][Download 154K]

  • Cloning and Expression of Gene Fragment Encoding TpN47 Lipoprotein of Treponema pallidum

    WANG Xian-ling,ZHANG Wei-jun,ZOU Quan-ming,et al(Department of Clinical Microbiology and Immunology,The Third Military Medical University,Chongqing 400038,China)

    Objective To express the TpN47(68～410 aa)lipoprotein of Treponema pallidum(Tp)in E.coli.Methods The gene fragment TpN47(202～1 230 bp) was amplified from the genome of Tp by PCR and,after T-A cloning,inserted into expression vector pET-22 b(+).The constructed recombinant plasmid pET-22 b(+)-TpN47 was identified by restriction analysis and transformed to E.coli BL21(DE3) for.expression under induction of IPTG.The expressed product was purified by nickel ion affinity chromatography and identified by Western blot.Results The target gene fragment,at a length of about 1 000 bp,was amplified by PCR.Restriction analysis proved that prokryotic expression vector pET-22 b(+)-TpN47 was correctly constructed.The expressed product,with a relative molecular mass of about 39 000,existed in a form of inclusion body and contained about 43% of total somatic protein,and reached a purity of more than 95% after purification.Western blot showed specific reaction of the protein with the sera of patients with syphilis.Conclusion The expressed TpN47 protein showed good immunoreactivity,which provided an experimental basis for the development of more effective diagnostic kit for syphilis.

    2008 04 [Abstract][OnlineView][Download 219K]

  • Effect of Trichinella on Growth of Human Colorectal Caricnoma HCT-8 Cells in BALB/c Mice

    LI Xia,ZHANG Guo-cai,ZHANG Xi-chen,et al(College of Animal Science and Veterinary Medicine,Jilin University,Changchun 130062,China)

    Objective To observe the effect of infection with trichinella on the growth of human colorectal carcinoma HCT-8 cells in BALB/c mice.Methods BALB/c mice were infected with trichinella at various dosages and inoculated with HCT-8 cells on different days before or after infection.Dissect the mice 20 d after inoculation,measure the size and weight of tumors to calculate the tumor-inhibiting rate,and determine the T lymphocyte subgroup.Results Both the size and weight of tumors of mice in test group were significantly lower than those in control group.However,the numbers of CD3+,CD4+ and CD8+ cells,the ratio of CD4+ cells to CD8+ cells,the percentage of tumor-free mice as well as the death rate and tumor-inhibiting rate in test group were significantly higher than those in control group.The tumor-inhibiting effect of mice infected with trichinella before inoculation with HCT-8 cells was better than those infected after inoculation.Conclusion Trichinella showed a certain inhibiting effect on the growth of HCT-8 cells in BALB/c mice.

    2008 04 [Abstract][OnlineView][Download 33K]

  • Biochemical Property of Deoxyribonuclease EWD3 from Eisenia foetida

    KANG Hui-fang,FAN Hui-jie,WANG Xiao-yuan,et al(Department of Biological and Chemistry,Shanxi Medical University,Taiyuan 030001,China)

    Objective To study the main biochemical property of a deoxyribonuclease(EWD3) from Eisenia foetida.Methods Determine the EWD3 for protein content by Lowry method,for relative molecular mass by SDS-PAGE and MALDL-TOF,for isoelectric point by capillary isoelectric focusing electrophoresis.The Km value of enzymatic reaction was also determined.Meanwhile,the acid and alkaline resistances,thermal stability,as well as effect of activator and inhibitor on enzyme activity of EWD3,were studied.Results The protein content,relative molecular mass,isoelectric point and Km value of EWD3 were 2.1 mg/ml,63460,6.20 and 1.52 mg/ml respectively.EWD3 was heat-labile,and its optimal pH value was 4.4～5.2.Magnesium,calcium and manganese ions and EDTA inhibited the enzyme activity of EWD3 significantly.All the parameters were significantly different from those of various DNases reported previously.Conclusion The deoxyribonuclease EWD3 from Eisenia foetida showed special enzymatic characters.

    2008 04 [Abstract][OnlineView][Download 241K]

  • Expression of MMP-2 and MMP-9 in Malignancies of Systema Lymphaticum

    WEI Su-hua,XU Wen,JIANG Yu-zhen,et al(Department of Hemopathy,The Second Hospital,Jilin University,Changchun 130041,China)

    Objective To explore the relationship between metal matrixprotease(MMP) and the malignancies of systema lymphaticum.Methods Determine the expression of ProMMP-2,MMP-2,ProMMP-9 and MMP-9 in sera of 62 patients with malignancies of systema lymphaticum(35 at primary onsetstage,12 at recurrence stage and 15 at remission stage) by SDSPAGE zymograph and computer protein quantitative determination system,using those in 20 healthy persons as control.Results The expression levels of MMP-2,MMP-2 and MMP-9 in the sera of patients at primary onset and recurrence stages were significantly higher than those in control group.The expressions of ProMMP-2,MMP-2,ProMMP-9 and MMP-9 in sera of patients at remission stage showed no significant difference with those in control group.However,the expression levels of ProMMP-9 in the sera of patients at primary onset and recurrence stages showed no significant difference with those in the patients at remission stage and in control group.The expression levels of ProMMP-2,MMP-2,ProMMP-9 and MMP-9 in sera of patients at primary onset stage showed no significant difference with those at recurrence stage.Conclusion The expression levels of MMP-2 and MMP-9 were positively related to the onset and progress of malignancies of systema lymphaticum.ProMMP-2,MMP-2 and MMP-9 mignt be used as the indexes for the judgment of disease course and evaluation of curative effect.It laid a foundation of further development of relevant MMP inhibitor for the treatment of malignancies of systema lymphaticum.

    2008 04 [Abstract][OnlineView][Download 36K]

  • Detection of Toxic Shock Syndrome Toxin 1 Gene of Staphylococcus aureus

    WANG Min,FU Jiong,LI Xian-ping(Department of Clinical Laboratory,The Second Xiangya Hospital of Central South University,Changsha 410011,China)

    Objective To detect the tst gene encoding toxic shock syndrome toxin 1(TSST-1) of Staphylococcus aureus and explore the carrier condition of the gene.Methods The tst gene encoding TSST-1 of S.aureus was amplified in vitro by PCR.Meanwhile,the tst gene in 84 S.aureus strains isolated in clinic were detected by PCR and sequenced.Results The tst gene was successfully detected by PCR and sequenced.Of the 84 clinical isolates of S.aureus,16(19.05%) were positive for tst gene.The sequencing result showed that tst gene was highly homologous to the TSST-1 gene reported in GenBank.Conclusion PCR was of good reproducibility,specificity and sensitivity in detection of tst gene.The tst gene positive strains were in a high proportion in the clinical isolates of S.aureus.

    2008 04 [Abstract][OnlineView][Download 75K]

  • Purification and Immune Effect of C3-Binding Protein(C3-BP)of Streptococcus pneumoniae

    SHAN Pu,WANG Jian-hua(National Vaccine and Serum Institute,Beijing 100024,China)

    Objective To purify C3-binding protein(C3-BP)from Streptococcus pneumoniae type 9V and study its immune effect.Methods Purify C3-BP from the culture supernatant of S.pneumoniae type 9V by trichloroacetic acid precipitation and ion exchange,gel filtration and affinity chromatography,then determine for purity and relative molecular mass by SDS-PAGE and for C3-binding activity by Western blot.Immunize NIH mice with the purified C3-BP and determine the serum IgG by ELISA and the spleen lymphocyte proliferation level by MTT method.Challenge the mice with S.pneumoniae and evaluate the protective effect of C3-BP.Results The purified C3-BP was the single chain protein with a relative molecular mass of about 90 000,and reached a concentration of 0.21 mg/ml and punity of 90.7%.The IgG titer of immunized mice reached 1∶38 802.The stimulating indexes of lymphocytes in both spleen and thymus of immunized mice were significantly higher than those of control.The C3-BP protected the immunized mice against the challenge not only with S.pneumonia type 9V,but also with types 2,6B and 14.Conclusion A method for purification of C3-BP from S.pneumoniae was developed.The purified C3-BP showed serotype-independent cross protective effect as well as good humoral and cellular immune effects.

    2008 04 [Abstract][OnlineView][Download 169K]

  • Residual Lysing Agent Content and Safety of Influenza Virus Subunit Vaccine

    ZHANG Yan,QI Feng-chun,ZHANG Xue-mei,et al(School of Public Health,Jilin University,Changchun 130021,China)

    Objective To determine the residual lysing agent content and safety of influenza virus subunit vaccine.Methods Prepare 3 batches of influenza virus subunit vaccine and determine the residual contents of lysing agents Tween-80 and CTAB in the vaccine by colorinetric method.Observe the safety of vaccine by animal test.Results The mean residual contents of Tween-80 and CTAB in the 3 batches of vaccine were 68.3～72.0 and 36.3～38.7 μg/ml respecively.No anaphylactic reaction was observed in guinea pigs,and no abnormal toxicity in mice or guinea pigs. Conclusion The residual lysing agent content in the prepared influenza virus subunit vaccine met the relevant requirements,and the vaccine showed good safety.

    2008 04 [Abstract][OnlineView][Download 19K]

  • Adaptability of Influenza Virus in Vero Cells

    LUO Zhen-wu,QIU Feng,WEI Xiao-lu,et al (Institute of Medical Biology,Chinese Academy of Medical Sciences,Peking Union Medical College,Kunming 650118,China)

    Objective To study the adaptability of influenza virus strain recommended by WHO for production of vaccine in Vero cells and lay a foundation of preparation of influenza vaccine with Vero cells.Methods The condition for culture of influenza virus,such as medium,trypsin concentration,pH value and time for virus harvest,were optimized.The influenza virus strain for production of vaccine was subcultured continuously in Vero cells for determination of HI titer and for PT-PCR analysis.Results The optimal medium,trypsin concentration,pH value and time for virus harvest for culture of influenza virus were F12+DMEM,1.5 μg/ml,7.5 and 3 d respectively.The virus cultured under the optimized condition reached a high HI titer.However,after subculture in Vero cells for 4 passages,the HI titer of virus decreased to 0,and the result of RT-PCR was negative.Conclusion The HI titer of influenza virus strain recommended by WHO for vaccine production decreased gradually after subculture in Vero cells.

    2008 04 [Abstract][OnlineView][Download 76K]

  • Preparation and Activity of Monoclonal Antibody against Intercellular Adhesion Molecule-1

    SUN Hong,WAN Zhong-hai,ZHANG Guo-li,et al(Institute of Veterinary Medicine,Academy of Military Medicine,Changchun 130062,China)

    Objective To prepare the monoclonal antibody(McAb) against human intercellular adhesion molecule-1(ICAM-1) with biological activity.Methods Immunize BALB/c mice with human ICAM-1,then screen the hybridoma cell strain stably secreting McAb against ICAM-1 by selective culture with HAT and clone by limiting dilution.The secreted McAb was purified by caprylic acid-ammonium sulfate precipitation and affinity chromatography,then determined for subclass and titer by indirect ELISA,for specificity by Western blot and for neutralizing activity by cell adhesion test.Results A hybridoma cell strain 3F2 stably secreting McAb against ICAM-1,with a chromosome modal number of 98～104,was screened.The secreted McAb was IgG1 and reached a protein content of 1.253 mg/ml,a purity of 83.6% and a titer of 1:2.56×105 after purification.The purified McAb bound to ICAM-1 specifically,inhibited the adhesion of ICAM-1 to leucoma cells and showed significant neutralizing activity to ICAM-1.Conclusion The McAb against ICAM-1 was successfully prepared,which laid a foundation of further study on its application.

    2008 04 [Abstract][OnlineView][Download 157K]

  • Inhibitory Effect of Murine Bone Marrow-derived Mesenchymal Stem Cells on Lymphocyte Proliferation of Rats with Xenogenic Liver Transplantation

    LI Xiu-dong,LIU Ya-juan,CHEN Qiang,et al(Department of Toxicology,School of Public Health,Jilin University,Changchun 130021,China)

    Objective To observe the migration and colonization of murine bone marrow-derived mesenchymal stem cells(MSCs) in the thymus,spleen and bone marrow of model rats with xenogenic liver transplant as well as the inhibitory effect of MSCs on lymphocyte proliferation,and explore the possibility of induction of immunotolerance to xenogenic liver transplant by MSCs.Methods Establish rat model of xenogenic liver transplant by imbedding the hepatic tissue pieces of KM mice into the cut of liver of Wister rats.The MSCs of suckling KM mice were isolated,purified and cultured in vitro,then labeled with DAPI and injected into the caudal vein of model rats.Observe the migration and colonization of MSCs in thymus,spleen and bone marrow by laser scanning confocal microscopy 24 h and 5 d after injection.Determine the inhibitory effect of MSCs on the proliferation of lymphocytes of model rats by lymphocyte proliferation test in vitro.Results MSCs were observed in thymus,spleen and bone marrow 24 h and distributed around blood vessels 5 d after injection.Both the proliferations of lymphocytes in thymus and spleen of model rats treated with MSCs were significantly inhibited as compared with those untreated with MSCs.Conclusion Murine MSCs were migrated to the immune organs of rats with xenogenic liver transplantation and showed inhibitory effect on lymphocyte proliferation.

    2008 04 [Abstract][OnlineView][Download 128K]

  • Repair of Skin Photoaging of Guinea Pigs by Bone Marrow-derived Mesenchymal Stem Cells

    YAO Chun-li,XIA Jian-xin,CHEN Yu-bing(Department of Plastic Surgery,The Second Hospital of Jilin University,Changchun 130041,China)

    Objective To explore the repair of skin photoaging by bone marrow-derived mesenchymal stem cells(MSCs) and provide a novel route for the treatment of skin photoaging.Methods Establish guinea pig model of skin photoaging by simultaneous irradiation with UV-A and UV-B ultraviolet radiators for 50 d,2 h per day.The MSCs of suckling KM mice were isolated and cultured in vitro,then labeled with DAPI and injected i.d.into the irradiation region of model guinea pigs.Observe the migration and colonization of MSCs in irradiation region by laser scanning confocal microscopy 24 h,5 d and 10 d after injection,and the repair of skin photoaging by macroscopic examination and HE staining.Results MSCs were observed in epidermis 24 h,in both epidermis and hair follicle tissue 5 d and diffused to all cortical layers 10 d after injection.Both macroscopic examination and HE staining showed that,compared with those of control,the skin photoaging of guinea pigs treated with MSCs was significantly delayed,and improved significantly after stopping irradiation.Conclusion MSCs delayed and repaired skin photoaging significantly.

    2008 04 [Abstract][OnlineView][Download 210K]

  • Effect of Blood Fat Regulator Xuezhikang on Proliferation and Collagen Synthesis of Cardiac Fibroblast of Newborn Rats

    ZHAO Shu-jian,JIN Yu-huai,YE Ping,et al (Department of Cardiology,The Third Hospital of Hebei Medical University,Shijiazhuang 050051,China)

    Objective To observe the effect of Xuezhikang,an extract of cholestin,on the proliferation and collagen synthesis of cardiac fibroblasts(CFs) caused by angiotensin Ⅱ(AngⅡ) in newborn rats and explore the potential molecular mechanism of the effect.Methods Isolate the CFs of newborn Sprague-Dawley rats by trypsin digestion method,and induce the proliferation of CFs by AngⅡ.Observe the effect of Xuezhikang on the number and cell cycle of CFs by MTT method and flow cytometry.After treatment with AngⅡ and Xuezhikang at various concentrations for 48 h,the yield of collagen in culture supernatant was determined by Sirius staining,and the expression of TGF-β1 protein by ELISA.The expressions of collagen gene and TGF-β1 mRNA were determined by RT-PCR.Results AngⅡ promoted the proliferation of CFs significantly.However,Xuezhikang showed significant dose-dependent inhibitory effect on proliferation of CFs induced by AngⅡ.The drug increased the percentage of cells at G0/G1 phase,decreased those at S and G2/M phases,and decreased the expressions of collagen gene and TGF-β1 mRNA.Conclusion Xuezhikang inhibited the proliferation of CFs and production of collagen induced by AngⅡ.The mechanism for this might be the inhibition of TGF-β1 expression.

    2008 04 [Abstract][OnlineView][Download 66K]

  • Evaluation of Clinical Application of Rapid Diagnostic Kit for Human Cytomegalovirus pp65 IgG-AI

    LIU Qian,HUANG Wei,CUI Huan,et al(Department of Microbiology,Anhui Medical University,Hefei 230032,China)

    Objective To evaluate the clinical application of rapid diagnostic kit for human cytomegalovirus(HCMV) pp65 IgG-avidity index(AI).Methods Determine the specific IgG in confirmed positive and negative serum specimens preserved in our laboratory with prepared HCMV pp65 IgGAI kit by indirect ELISA to analyze the sensitivity,specificity and accuracy.Determine the pp65 IgG in 89 serum specimens collected in clinic,of which 5 were from the patients with active HCMV infection,by the prepared kit,and compare the results with those by imported kit.In addition,determine the AI values of pp65 IgG positive specimens by indirect ELISA using urea as protein denaturant and compare the results with those by imported IgM kit to evaluate the significance of AI in diagnosis of HCMV infection.Results All the determination results of confirmed positive and negative serum specimens were identical to those confirmed previously,indicating that the sensitivity,specificity and accuracy of prepared kit were 100%.The determination results of IgG by prepared and imported kits showed no significant difference.By determination of AI value with prepared kit,the primary(or suspected primary) HCMV infection rate of patients from whom the clinical specimens were collected was 46.07%,which was significantly higher than that by determination of IgM with reference kit(23.59%).Of the 5 specimens diagnosed as active HCMV infection,4 were proved as IgG positive by either prepared or reference kit,indicating that the coninicidence rate of the two kits was 100%.However,3 of the 5 specimens were proved as positive by determination of AI value with the prepared kit,while only one as positive by determination of IgM with reference kit.The specimens from patients with HCMV pulmonitis diagnosed in clinic were proved as positive by determination of AI value with the prepared kit,but as negative by determination of IgM with reference kit.Conclusion The developed rapid diagnostic kit for pp65 IgG-AI may be used for preliminary diagnosis of HCMV infection.It is simple,rapid and of good reproducibility and good prospect in clinical application.

    2008 04 [Abstract][OnlineView][Download 44K]

  • Adverse Reaction and Immune Effect of Domestic Adjuvant-free Rabies Vaccine Prepared with Vero Cells

    LIU Hua-zhang,HUANG Gui-hua,CAO Qing,et al(Guangzhou Center for Disease Control and Prevention,Guangzhou 510080,China)

    Objective To observe the adverse reaction and immune effect of domestic adjuvant-free rabies vaccine prepared with Vero cells.Methods Immunize 93 subjects with domestic adjuvant-free rabies vaccine prepared with Vero cells on days 0,3,7,14 and 28 after exposure respectively.Observe the immediate adverse reaction within 30 min as well as local and systemic adverse reactions with in 72 h after immunization.Collect the serum samples on days 3,7,14 and 45 respectively for determination of antibody.Results No immediate adverse reaction was observed.The incidences of local and systemic adverse reaction were 5.8% and 4.5% respectively.The serum antibody positive conversion rate was 22.58% on day 7 and reached 100% on day 14.The GMTs of serum antibody were 2.12 and 8.52,and 15.43 IU/ml on days 7,14 and 45 respectively,which showed significant difference.The GMTs on days 14 and 45 of female and male subjects showed no significant difference.The GMTs of various age groups showed significant difference on day 14 while no significant difference on day 45.Conclusion The domestic adjuvant-free rabies vaccine prepared with Vero cells was safe and showed good immune effect.

    2008 04 [Abstract][OnlineView][Download 23K]

  • Optimization of Condition for Modification of Recombinant Human Interferon-α1b with Polyethylene Glycol

    YANG Yu-feng,HUANG Jing,XU Fan-hong,et al(Shanghai Institute of Biological Products,Shanghai 200052,China)

    Objective To optimize the condition for modification of recombinant human interferon-α1b(rhIFN-α1b) with polyethylene glycol(PEG).Methods Modify rhIFN-α1b with 3 kinds of PEG derivatives of the second generation,determine the apparent relative molecular masses of PEG and the modified rhIFN-α1b,and optimize the condition for modification,such as pH value,temperature,time and dosage of PEG.The modified rhIFN-α1b was primarily separated,purified and determined for activity.Results The apparent relative molecular mass of PEG was higher than that in theory.The modification rate of rhIFN-α1b with PEG20000(92.1%) were significantly higher than that with PEG5000 or with PEG10000.The dosage of PEG and pH value showed significant influence,while time and temperature showed little influence on the modification effect.The optimal pH value for modification was 9.0,and the optimal molar ratio of rhIFN-α1b to PEG20000 was 1∶10.The mono-pegylated rhIFN-α1b reserved 14.4% of activity in vitro,while poly-pegylated rhIFN-α1b showed no activity.Conclusion The optimal PEG derivative for modification of rhIFN-α1b was screened,and the condition for modification was optimized,which laid a foundation of further study on modification of interferon with PEG.

    2008 04 [Abstract][OnlineView][Download 258K]

  • Rapid Detection of Hepatitis B Virus YMDD Gene Variant by Real-time Fluorescent PCR

    CHEN Jian,WANG Man,CHEN Hua,et al(Department of Biochemistry and Molecular Biology,East China University of Science and Technology,Shanghai 200237,China)

    Objective To develop a sensitive,specific and rapid real-time fluorescent PCR for detection of hepatitis B(HB) virus YMDD gene variant and provide a basis for clinical treatment of HB.Methods Develop a real-time fluorescent PCR by using designed primers and TaqMan-MGB probe.Thirty-six clinical specimens of HBV-YMDD variants and 20 specimens of wild type HBV were detected by the developed real-time fluorescent PCR,and the results were compared with those of DNA sequencing.Results The sensitivity,specificity and minimum detection limit of the developed real-time fluorescent PCR were 101 copies/μl,100% and 101 DNA copies/30 μl reaction system respectively The detection result of developed PCR was completely consistent with that of DNA sequencing.Conclusion The developed real-time fluorescent PCR was sensitive,specific and accurate for the detection of HB virus YMDD gene variant and of important significance in clinical monitoring of resistance to lamivudine.

    2008 04 [Abstract][OnlineView][Download 124K]

  • Large Scale Culture of Rotavirus by Vero Cell Microcarrier Technique

    ZHANG Yong-xin,LIU Xin,ZHANG Guang-ming,et al(Institute of Medical Biology,Chinese Academy of Medical Science,Peking Union Medical College,Kunming 650118,China)

    Objective To culture rotavirus in a large scale by using Vero cell microcarrier technique.Methods Culture Vero cells with microcarrier in a 5 L bioreactor,to which rotavirus strain P[2]G3 was inoculated at a MOI of 0.05 when the cell density reached 1.5×106 cells/ml.Incubate the mixture at 37℃ for 6 d and take samples for observation of CPE and determination of infectious titer of virus daily.Results The infectious titer of virus increased gradually at early stage of culture and reached a peak value 48 h after culture.The virus titer in culture supernatant and lysate of cells were 5.5 and 3.75 CCID50/ml respectively.Conclusion The titer of rotavirus after large scale culture by Vero cell microcarrier technique increased significantly.

    2008 04 [Abstract][OnlineView][Download 164K]


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