2013年06期目次

  • Immunogenicity of recombinant hepatitis B core antigen and its immunopotentiation to hepatitis B surface antigen

    LI Ji-lai,XU Jing,WANG Juan,WU Gang,ZHAO Li,XU Li-feng National Vaccine and Serum Institute,Bejing 100024,China

    Objective To evaluate the immunogenicity of hepatitis B core antigen(HBcAg)as well as its immunopotentiation to hepatitis B surface antigen(HBsAg).Methods The morphologies of HBcAg,HBsAg and virus-like particles(VLPs)of HBcAg+HBsAg were observed by electron microscopy.Mice were randomly divided into negative control(normal saline,NS),HBcAg(C),HBsAg(S),HBsAg+aluminum adjuvant(S+A)l,HBsAg+HBcAg(S+C)and HBsAg+HBcAg+aluminum adjuvant(S+C+A)l groups,and immunized i.m.with the corresponding antigens on days 0 and 14 respectively,of which the sera were collected and determined for anit-HBs,anti-HBc and antibody subclass against HBsAg.Meanwhile,the splenocytes of mice were collected and determined for IFNγ levels against HBsAg and HBcAg as well as IL-4 level against HBsAg.Results Elec-tron microscopy showed that both HBsAg and HBcAg formed VLPs with uniform structures,while the VLPs of mixed antigen were aggregated.The anti-HBs levels after the first immunization showed no significant difference in S+C and S groups(P = 0.074),while those after the second immunization were significantly higher in S+C group than in S group(P = 0.002).High anti-HBc levels were induced in both C and S+C groups.The antibody subclasses against HBsAg in S group were mainly IgG1,while those in S+C group were mainly IgG2a.The number of spots formed by IFNγ against HBsAg in S+C group was significantly larger than those in S,S+Al and S+C+Al groups(P < 0.05).Both the numbers of spots formed by IFNγ against HBcAg were large in C and S+C groups.However,the numbers of spots formed by IL-4 against HBsAg were higher in S+C+Al group than in S+Al group(P = 0.026).Conclusion HBcAg showed good immunogenicity,which enhanced the immune response of HBsAg and promoted the Th1 polarization of the response.It laid a foundation of development of therapeutic HB vaccine.

    2013 06 v.26 [Abstract][OnlineView][Download 374K]

  • Comparative study on different immune schemes of inactivated poliovirus vaccines prepared with Sabin and wild strains in rhesus monkeys

    YANG Hui-juan,CHEN Jun-ying,HE Zhan-long,LI Hua,YANG Yao-yun,YE Jun,SUN Qiang-ming,YUE Jun,SHI Hai-jing,MA Shao-hui Yunnan Key Laboratory of Vaccine Research & Development on Severe Infectious Diseases,Institute of Medial Biology,Chinese Academy of Medical Sciences and Peking Union Medical College,Kunming 650118,Yunnan Province,China

    Objective To evaluate the immunogenicity of different immune schemes of inactivated poliovirus vaccine prepared with Sabin strain(sIPV) and wild strain(wIPV) in rhesus monkeys.Methods Twenty-five rhesus monkeys were divided into five groups randomly,five for each.The monkeys in sIPV group were immunized with 3 doses of sIPV,while those in sIPV/OPV group with 2 doses of sIPV and 2 dose of OPV,those in wIPV group with 3 doses of wIPV,those in wIPF/OPV group with 2 doses of wIPV and 2 doses of OPV,and those in control group with 3 doses of M199 as diluent.OPV was given by oral route,while IPV and M199 by i.m.injection in deltoid of upper arm.The time interval between 2 doses was one month.Serum samples were collection before the first dose and one month after each dose re spectively,and determined for neutralizing antibody titers against poliovirus types Ⅰ,Ⅱ and Ⅲ by micro-neutralization test.Results High neutralizing antibody titers were detected in the monkeys except those in control group.All the posi tive conversion rates of antibodies against poliovirus types Ⅰ,Ⅱ and Ⅲ in sera of monkeys in four test groups were 100%.The neutralizing antibody levels against poliovirus types Ⅰand Ⅲ in sIPV and sIPV/OPV groups were significantly higher,while that against type Ⅱ was significantly lower,than those in wIPV and wIPV/OPV groups(each P <0.05).The immune response levels against poliovirus typesⅠ,Ⅱ and Ⅲ in wIPF/OPV group were comparable to those in wIPV group.However,compared with those in sIPV group,the immune response level against poliovirus type Ⅰ in sIPV/OPV group decreased significantly,and that against type Ⅱ showed no significant difference(P > 0.05),while that against type Ⅲ increased after the forth dose was given.Conclusion Both sIPV and wIPV induced effective immune responses in rhesus monkeys,which preliminarily proved the feasibility of IPV/OPV sequential immune scheme and provided a certain experimental basis for further development of immune strategy.

    2013 06 v.26 [Abstract][OnlineView][Download 241K]

  • Prokaryotic expression and immune protective effect of DnaJ protein of Streptococcus pneumoniae

    YOU Wen-xian,CAO Ju Department of Gastroenterology,The First Affiliated Hospital of Chongqing Medical University,Chongqing 400016,China

    Objective To express the heat shock protein 40(HSP40,DnaJ)of Streptococcus pneumonia and investigate its protective effect.Methods DnaJ gene was amplified from S.pneumonia of types 2,3,6B,14,19F and R6,and inserted into prokaryotic expression vector pET-32a.The constructed recombinant plasmid pET-32a-DnaJ was transformed to E.coli BL21(DE3)for expression under induction of IPTG.The expressed recombinant DnaJ protein was purified NiNTA chromatography,with which mice were immunized by i.p.and i.n.routes and determined for specific IgG and IgA titers in sera and saliva by ELISA.Murine splenocytes were stimulated with recombinant DnaJ protein and determined for cytokine level by ELISA.The binding ability of specific antiserum against DnaJ onto the surface of S.pneumonia was determined by flow cytometry.The ability of antiserum against DnaJ in inhibiting the adherence of S.pneumonia of type R6 to A549 cells was determined.Results DnaJ gene fragments each at a length of 1 119 bp was amplified from the six S.pneumoniae strains,of which the sequencing results were consistent with those reported in GenBank.Restriction analysis proved that recombinant expression plasmid was constructed correctly.Purified recombinant DnaJ protein,with a relative molecular mass of about 38 000,reached a purity of more than 90% and showed specific reaction with mouse anitserum against DnaJ.Immunization with DnaJ by i.p.route induced high titer specific antibody against DnaJ,which that by i.n.route increased the IgG level in sera and IgA level in mucosa(P < 0.01).However,immunization with DnaJ by i.n.route promoted the secretion of IL-10,IFNγ and IL-17A by murine splenocyte.Antiserum against DnaJ showed increased binding ab ility to S.pneumonia and inhibited the adherence of S.pneumonia to A549 cells.Conclusion DnaJ induced protective immune response in mice,indicating its potential as an antigen of S.pneumoniae protein vaccine.

    2013 06 v.26 [Abstract][OnlineView][Download 550K]

  • Genetic stability and bioinformatics of glycoprotein of rabies virus aG strain

    LI Jia,CAO Shou-chun,SHI Lei-tai,WANG Yun-peng,WU Xiao-hong,LIU Jing-hua,TANG Jian-rong,YU Yong-xin,DONG Guan-mu National Institutes for Food and Drug Control,Beijing 100050,China

    Objective To investigate the genetic stability of glycoprotein of rabies virus(RV)aG strain,and analyze the sequence and bioinformatics of GP genome.Methods GP genome sequences of aG strain of six passages,i.e.4aGV4,4aGV5,4aGV7,4aGV11,4aGV15 and 4aGV18,were amplified by RT-PCR and cloned into vector pGEM-T respectively,then sequenced and spliced.The aG strains of six passages were determined for titer,fluorescent titer and virulence,of which the GP genome sequences were analyzed by DNAStar and Mega4.0 software for homology to those of RV street virus strains isolated in China recently.Results The GP gene of aG strain of six passages were highly stable,in which no nucleotide mutation sites were observed.The RV titers of six passages were different,while the virulence indexes were basically in agreement.The GP antigenic domain of aG strain showed high homology to those of street virus strains in China,while the homology in extramembrane domain was significantly higher than those in transmembrane and intramembrane domains.The amino acid at key site 147 of RV aG strain was different from those of street virus strains,while the other key sites of various strains were highly homologous.No amino acid mutations were observed in key virulence sites of RV aG strain of six passages,indicating high genetic stability.The key virulence sites of aG strains were highly homologous to those of various street virus strains.The signal peptide structure of aG strain was highly homologous to those of various epidemic strains in China,while the GP was highly homologous to those of PV2061 strain isolated in France and HEP-Flury strain isolated in USA,and was homologous to those of street virus strains isolated in China.Conclusion The GP of RV aG strain showed high genetic stability,and was high homologous to those of street virus strains isolated in China recently,which provided an experimental basis for perfecting the quality control and complete evaluation of suitability of aG strain in China.

    2013 06 v.26 [Abstract][OnlineView][Download 538K]

  • Genetic characters of coxsackievirus B5 strains isolated in Kunming Region,Yunan Province,China in 2009

    SHAO Cong-wen,PAN Yue,DENG Xin-qiang,YE Jun,MA Zhong-fei,SUN Qiang-ming,MA Shao-hui Institute of Medical Biology,Chinese Academy of Medical Sciences and Peking Union Medical College,Yunnan Key Laboratory of Vaccine Research & Development on Severe Infectious Diseases,Kunming 650118,Yunnan Province,China

    Objective To analyze the sequences of partial VP1 genes of seven coxsackievirus B5(CVB5)strains isolated in 2009 in Kunming Region,Yunnan Province,China so as to investigate the genetic characters of CVB5.Methods A total of 200 fecal specimens were collected from the children diagnosed as patients with aseptic meningitis in Kunming Region in 2009,from which partial VP1 genes were amplified by semi-nested RT-PCR using designed general primers.The PCR products were sequenced directly,of which the nucleotide and deduced amino acid sequences were analyzed by Omiga and Mega 5.05 software,and a phylogenetic tree was plotted.Results A total of seven CVB5 strains were isolated,of which the homologies of nucleotide and amino acid sequences of partial VP1 genes were 92.7% ～ 98.5% and 93.6% ～ 100%,respectively.The homologies of nucleotide sequences of partial VP1 genes of the seven strains were 80.1% ～ 99.1% to those of other reference strains isolated in China,of which that to strain 98388-SD-CHN-1998 isolated in Shandong Province was low.The homologies of amino acid sequences of partial VP1 genes of the seven strains were 84.4% ～ 100.0% to those of other reference strains isolated in China,of which that to strain CoxB5-Henan-2010 isolated in Henan Province and strain 98388-SD-CHN-1998 isolated in Shandong Province were low.The homologies of nucleotide and amino acid sequences to were 77.5% ～ 89.1% and 92.1% ～ 96.3% to those of reference strains isolated in other countries,and 79.6% ～ 80.9% and 92.1% ～ 95.4% to those of prototype strain Faulkner,respectively.Phylogenetic tree showed that the seven Kunming isolates belonged to a separated branch,while the epidemic strains in China,except 98388-SD-CHN-1998,formed one branch.Conclusion The CVB5 strains isolated in Kunming in 2009 were of the same genetic group.

    2013 06 v.26 [Abstract][OnlineView][Download 699K]

  • Effect of hepatitis B virus X protein on apoptosis of mouse embryonic liver stem cells and expression of associated proteins

    SHEN Li-hong,LU Yong-liang,LI Hong-li,FENG Tao,HUANG Jia-yi Research Center of Molecular Medicine and Tumor,Chongqing Medical University,Chongqing 400016,China

    Objective To investigate the effect of hepatitis B virus X protein(HBx)on apoptosis of mouse embryonic liver stem cells(ELSCs)and expression of associated proteins.Methods ELSC14.5 cells were infected with recombinant adenovirus Ad-GFP-HBx,and determined for mRNA transcription and protein expression of HBx by RT-PCR and Western blot respectively,for change of nuclei by Hoechst33342 staining,and for apoptosis by TUNEL method and flow cytometry.The mRNA transcription levels of anti-apoptotic factors Bcl2 and Mcl1 as well as apoptosis-promoting factor Bax were determined by Real-time PCR,while the protein expression levels by Western blot.Results ELSC14.5 cells were effectively infected with Ad-GFP-HBx,and HBx was expressed specifically at both mRNA and protein levels.Nuclear condensation and chromatin marginization were observed in infected ELSC14.5 cells,while the apoptotic rate of cells decreased.The mRNA transcription and protein expression levels of Mcl1 and Bcl2 in infected cells increased,while those of Bax decreased.Conclusion HBx inhibited the apoptosis and encouraged the survival of mouse ELSCs by regulating the unbalanced proportion of anti-apoptotic and apoptosis-promoting factors in Bcl2 family.

    2013 06 v.26 [Abstract][OnlineView][Download 538K]

  • Cloning and prokaryotic expression of E2 gene of Western equine encephalomyelitis virus

    MA Jin-zhu*,WANG Hua-lei,ZHENG Xue-xing,ZHAO Yong-kun,GAO Yu-wei,WANG Tie-cheng,HUANG Geng,YANG Song-tao,XIA Xian-zhu *College of Life Science and Biotechnology,Heilongjiang Bayi Agricultural University,Daqing 163319,Heilongjiang Province,China

    Objective To construct the E2 gene of Western equine encephalomyelitis virus(WEEV) and express in prokaryotic cells.Methods E2 gene was amplified from recombinant plasmid pVL1393-C-E by PCR and inserted into prokaryotic expression vector pET-30a(+).The constructed recombinant plasmid pET-30a(+)-E2 was transformed to E.coli BL21(DE3) for expression under induction of IPTG.The expressed product was identified by SDS-PAGE and Western blot.Results PCR restriction analysis and sequencing proved that recombinant plasmid pET-30a(+)-E2 was constructed correctly.The relative molecular mass of expressed product was about 52 900.The expression level reached a peak value of 23.5% of total somatic protein 6 h after induction.The recombinant E2 protein mainly existed in a form of inclusion body and showed specific binding to mouse monoclonal antibody against His.Conclusion The E2 gene of WEEV was cloned and expressed in prokaryotic cells,which laid a foundation of study on immunogenicity of E2 protein and development of WEEV subunit vaccine.

    2013 06 v.26 [Abstract][OnlineView][Download 368K]

  • Construction of siRNA plasmid for human telomerase reverse transcriptase gene and its effect on growth and apoptosis of ovarian cancer cells in presence or absence of p53

    LUO Yi*,YAO Zhen-wei,YANG Ya-ying,YI Yong-fen *Department of Gynaecology and Obstetrics,The First Affiliated Hospital of Chongqing Medical University,Chongqing 400016,China

    Objective To construct the siRNA plasmid for human telomerase reverse transcriptase(hTERT)gene and investigate its effect on the growth and apoptosis of ovarian cancer cells in presence or absence of p53.Methods The siRNA targeting to hTERT gene was designed and synthesized,and inserted into expression vector pGenesil-1.Ovarian cancer SKOV3(p53 null)and OVCAR3(with p53)cells were transfected with the constructed recombinant plasmids pG1,pG2 and pGCR(negative control)separately,using the untransfected cells and empty vector-transfected cells as controls,and observed by fluorescent microscopy 48 h later,based on which the transfection efficiencies were calculated.The expression of hTERT at mRNA level in transfected cells was determined by PCR,while those of hTERT,p53 and p2a at protein level by Western blot.The telomerase activity in transfected cells was determined by TRAP method.The cell growth was observed by CCK-8 method.The cell cycle and cell apoptosis were determined by flow cytometry and Annexin Ⅴ-PE/7-AAD staining respectively.Results The siRNA plasmids pG1 and pG2 targeting to hTERT as well as negative control plasmid pGCR were constructed successfully,which showed high transfection efficiencies.As compared with those transfected with pGCR,the expression of hTERT at mRNA and protein levels(both P < 0.05) as well as telomerase activity in the cells transfected with pG1 and pG2 decreased significantly.Both pG1 and pG2 inhibited the growth of SKOV3 and OVCAR3 cells,while the inhibiting effect on the latter was superior to that on the former.The percentages of cells at G 0-G 1 phases in pG1 and pG2 transfection groups decreased significantly,while those at S phase increased significantly(P < 0.05);obvious apoptosis was observed in OVCAR3 cells after the expression of hTERT was inhibited;however,no obvious apoptosis was observed in the SKOV3 cells with the increased expressions of p53 and p21 proteins,while the expression level of p21 protein was significantly lower than those in pGCR transfection group(P < 0.05).Conclusion The constructed siRNA plasmid targeting to hTERT effectively and specifically silenced the expression of hTERT gene in ovarian cancer cells in vitro,decreased the telomerase activity,inhibited the growth and induced the apoptosis of tumor cells,of which the mechanism might be associated with the up-regulation of tumor suppression genes p53 and p21.

    2013 06 v.26 [Abstract][OnlineView][Download 873K]

  • Expression of mouse soluble B cell activating factor belonging to the TNF family in Pichia pastoris and purification and biological activity of expressed product

    OU Zi-qiang,TANG Yong,XIANG Jun-jian,LV Wei-dong,LIN Ting-ting Guangdong Province Key Laboratory of Molecular Immunology and Antibody Engineering,Jinan University,Guangzhou 510632,Guangdong Province,China

    Objective To express mouse soluble B lymphocyte activating factor(msBAFF)in Pichia pastoris,purify the expressed product and determine its biological activity.Methods Peripheral blood mononuclear cells of BALB/c were separated,from which msBAFF gene was amplified by RT-PCR and inserted into expression vector pPICZαA.The constructed recombinant plasmid pPICZαA-msBAFF was transformed to P.pastoris GS115 and induced with methanol.The expressed recombinant msBAFF protein was purified by ammonium sulfate precipitation and Q Sepharose XL anion exchange column chromatography.The splenic B lymphocytes of BALB/c mice were treated by purified msBAFF alone or combined with anti-IgM,and determined for activity.Results Restriction analysis proved that recombinant plasmid pPICZαA-msBAFF was constructed correctly.The relative molecular mass of expressed recombinant msBAFF was about 20 000,and the expression level reached the peak value 60 h after induction.Purified msBAFF was obtained by ammonium sulfate precipitation,dialysis and Q Sepharose XL anion exchange column chromatography,of which the yield was 20 mg/L.Recombinant BAFF enhanced the activity of mouse B lymphocytes.Conclusion Recombinant msBAFF with high biological activity was successfully expressed in P.pastoris and purified,which laid a foundation of further study on function of mouse BAFF gene and preparation of BAFF-based adjuvant and monoclonal antibody.

    2013 06 v.26 [Abstract][OnlineView][Download 475K]

  • Prokaryotic expression of ATP-dependent caseinolytic protease proteolytic subunit 1 gene of Mycobacterium tuberculosis

    ZHANG Chun-yan*,YANG Chun,LI Dai-rong,MU Liu-qing,YANG Jing,FENG Xin *Department of Pathobiology,Chongqing Medical Univercity,Chongqing 400016,China

    Objective To construct a prokaryotic expression vector for ATP-dependent caseinolytic protease proteolytic subunit 1(ClpP1) gene of Mycobacterium tuberculosis(MTB) and express recombinant protein in E.coli.Methods ClpP1gene was amplified by PCR from the genome of MTB H37Rv strain and inserted into prokaryotic expression vector pET-32a(+).The constructed recombinant plasmid pET-32 a(+)-ClpP1 was transformed to E.coli B21(DE3)and induced with IPTG.The expressed product was identified by SDS-PAGE and Western blot.Results Restriction analysis and sequencing proved that recombinant plasmid pET-32a(+)-ClpP1 was constructed correctly.The expressed recombinant protein,with a relative molecular mass of about 35 000,showed specific binding to mouse monoclonal antibody against His tag.Conclusion Recombinant plasmid pET-32a(+)-ClpP1 was constructed correctly,and recombinant protein was expressed in E.coli,which laid a foundation of biological characters of ClpP1 protein in MTB.

    2013 06 v.26 [Abstract][OnlineView][Download 429K]

  • Construction of eukaryotic expression vector for chimeric gene of hepatitis C virus neutralizing epitopes with hepatitis B virus S antigen and its expression in 293T cells

    SHU Fang*,LEI Ying-feng,LIN Fang,WANG Xi,LI Bin,ZHANG Li-jun,DONG Ke,ZHANG Hui-zhong,WEI San-hua * Department of Clinical Laboratory Medicine,Tangdu Hospital,Fourth Military Medical University,Xi’an 710038,Shaanxi Province,China

    Objective To construct a eukaryotic expression vector for chimeric gene of hepatitis C virus(HCV) neutralizing epitopes with hepatitis B virus(HBV)S antigen and express in 293T cells.Methods HBV S antigen gene was amplified from plasmid pHBV containing full-length HBV gene,into which the restriction site of Age I was introduced between amino acids 127 and 128.Conservative HCV linear neutralizing epitope genes in E1 and E2 regions and mimotope of hypervariable region 1 were inserted to the restriction site of Age I,and the obtained chimeric gene of HCV neutralizing epitopes with HBV S antigen was cloned into eukaryotic expression vector pCI-neo.293T cells were transfected with the constructed recombinant plasmids pCI-HBSE1 ～ 4 and determined for expression of chimeric gene by indirect IFA and Western blot.Results Restriction analysis proved that recombinant plasmids pCI-HBSE1 ～ 4 were constructed correctly.Strong green fluorescence was observed in the cytoplasm of 293T cells transfected with the recombinant plasmids.Western blot showed a protein band with a relative molecular mass of about 27 000.Conclusion A eukaryotic expression vector for chimeric gene of HCV neutralizing epitopes with HBV S antigen was successfully constructed and effectively expressed in 293T cells,which laid an experimental foundation of further preparation of chimeric HBV S antigen VLPs with HCV neutralizing epitope and study on inhibitory effect of neutralizing antibody on infections with HCV pseudovirus particles(HCVpp)and JFH-1 HCV in vitro culture system(HCVcc).

    2013 06 v.26 [Abstract][OnlineView][Download 507K]

  • Effect of interference of Apg-2 gene expression by RNA on apoptosis of BaF3-MIGR1 and BaF3-p210 cells

    CHEN Xi*,ZHONG Liang,XIAO Qing,WANG Xin,LI Chun-li,FENG Wen-li *Department of Clinical Hematology,Key Laboratory of Laboratory Medical of Education Ministry,Chongqing Medical University,Chongqing 400016,China

    Objective To investigate the effect of interference of Apg-2 gene expression by RNA on the apoptosis of BaF3-MIGR1 and BaF3-p210(Bp210) cells.Methods BaF3-MIGR1 and Bp210 cells,BaF3-MIGR1-Hspa42 and Bp210-Hspa42 cells in which Apg-2 expression was inhibited stably,as well as Bp210-HspaHK and BaF3-MIGR1HspaHK cells as negative control were observed for morphologies by Wright staining,for apoptosis by flow cytometry,and for expression of apoptosis-related Bax and Bcl-2 proteins by Western blot.Results A series of morphological changes including swelling,cytoplasmic vacuoles,chromatin aggregation,nuclear fragmentation were observed in the cells after interference of Apg-2 gene expression,and apoptotic body formation in partial cells.However,the cells at metaphase decreased significantly in quantity(P < 0.05).As compared with those of BaF3-MIGR1 and Bp210 cells,the apoptotic rates of BaF3-MiGR1-Hspa42 and Bp210-Hspa42 cells increased significantly(P < 0.05),while the Bcl-2 expression level decreased,and Bax expression level showed no significant change.Conclusion Interference of Apg-2 gene expression promoted the apoptosis of BaF3-MIGR1 and Bp210 cells through a possible mechanism of down-regulating the expression of anti-apoptotic protein Bcl-2.

    2013 06 v.26 [Abstract][OnlineView][Download 459K]

  • Prokaryotic expression and purification of staphylococcal enterotoxin B

    ZHANG Liang*,ZHANG Guo-li,CHEN Ping,ZHU Ping,YUE Yu-huan,WU Guang-mou,TIAN Yuan,LIU Xue,FENG Yue *College of Food Science and Engineering,Jilin Agriculture University,Changchun 130118,Jilin Province,China

    Objective To express staphylococcal enterotoxin B(SEB)in prokaryotic cells and purify the expressed product.Methods SEB gene was amplified by PCR using synthetic whole SEB gene sequence as a template,and inserted into prokaryotic expression vector pET-28a.The constructed recombinant plasmid pET-28a-SEB was transformed in to E.coli BL21(DE3)for expression under induction of IPTG.The expressed product was purified by salting out with ammonium sulphate,SP cation exchange chromatography and copper ion chelating chromatography then,after His Tag was digested with thrombin,further purified by copper ion chelating chromatography and DEAE anion exchange chromatography.Results Restriction analysis and sequencing proved that recombinant plasmid pET-28a-SEB was constructed correctly.The expressed recombinant protein,with a relative molecular mass of about 31 000,mainly existed in a soluble form,contained about 30% of total somatic protein and reached a purity of more than 90% after purification.Conclusion Recombinant prokaryotic expression vector for SEB gene was successfully constructed,and recombinant SEB was expressed in E.coli and purified,which laid a foundation of further study on the pathogenic mechanism and methods for control of SEB.

    2013 06 v.26 [Abstract][OnlineView][Download 401K]

  • Construction of lenfiviral expression vector for miR-34a and its effect on proliferation of human colon cancer SW480 cells

    SHAO Xin-hong,ZHANG Cai-quan Department of Gastrointestinal Surgery,The First Affiliated Hospital of Chongqing Medical University,Chongqing 400016,China

    Objective To construct a lentiviral expression vector for miR-34a and investigate its effect on proliferation of human colon cancer SW480 cells.Methods Partially complementary forward and reverse primers were designed based on has-pre-miR-34a sequence,of which dimmers were formed by annealing and amplified by PCR.The obtained double stranded DNA was used as a template for further amplification by PCR.The PCR product was digested with restriction endonuclease and insered into linearized lentiviral vector pGIPZ.The constructed recombinant plasmid pGIPZ-miR-34a was co-transfected to 293T cells with packaging plasmids Helper 1.0 and Helper 2.0 for packaging.The obtained recombinant lentivirus was determined by gradient dilution.SW480 cells were infected with recombinant lentivirus and determined for transcription level of miR-34a mRNA by qPCR,for proliferative activity by MTT and plate cloning method,and for distribution of cell cycle by flow cytometry.Results Restriction analysis and sequencing proved that recombinant lentiviral expression vector was constructed correctly.The titer of obtained recombinant lentivirus was 6.52 × 108 TU/ml.The recombinant lentivirus up-regulated the transcription level of miR-34a mRNA while inhibited the proliferative activity and increased the percentage at G0-G1 phases of infected SW480 cells(P < 0.01).Conclusion The lentiviral expression vector for miR-34a was successfully constructed,which increased the endogenous expression of miR-34a and inhibited the proliferation of SW480 cells.It laid a foundation of further study on function and action mechanism of miR-34a.

    2013 06 v.26 [Abstract][OnlineView][Download 594K]

  • Construction of anti-canine distemper virus phage display antibody library

    MA Yun-qing*,REN Wen-zhi,GAO Juan,CHEN Jian,YUE Yuan,GAO Hong-bo,GAO Wei *College of Animal Science and Veterinary Medicine,Jilin University,Changchun 130062,Jilin Province,China

    Objective To construct the T7 phage single-chain antibody fragment(scFv)library of anti-canine distemper virus(CDV).Methods Dogs were immunized s.c.with inactivated CDV for 3 times each at an interval of 2 weeks,of which the peripheral blood samples were collected 2 weeks after the 3rd immunization.Lymphocytes were isolated,from which total RNA was extracted for amplification of heavy chain(VH) and light chain(VL) of IgG by RT-PCR and spliced by SOE-PCR using a linker.The obtained scFv gene was digested with EcoRⅠ and Hind Ⅲ,inserted into expression vector T7 Select 10-3b,packaged by packaging protein,and transformed to E.coli BLT5403 for proliferation.Fifty plaques were randomly selected and identified by PCR,based on which the library was tested for titer.Results Both the lengths of amplified VH and VL genes were consistent with those expected,of which the coincidence rate of sequence to those of canine IgG were more than 90% as proved by blast software.The titer of constructed primary library was 3.2 × 107 pfu/ml,which reached 5.0 × 108 pfu/ml after proliferation.The recombination rate of fifty plaques was 94% as proved by PCR.Conclusion The T7 phage single-chain antibody fragment library of anti-CDV was successfully constructed,which provided a reference for prevention and treatment of canine distemper.

    2013 06 v.26 [Abstract][OnlineView][Download 457K]

  • Regulatory effect of Netrin-1 on expression of epithelial sodium channel α-subunit in alveolar epithelia cells and potential mechanism

    HE Jing,WANG Dao-xin Department of Respiratory Disease,The Second Affiliated Hospital,Chongqing Medical University,Chongqing 400010,China

    Objective To investigate the regulatory effect of Netrin-1 on expression of epithelial sodium channel α-subunit(α-ENaC)in alveolar epithelia A549 cells and the potential mechanism,as well as role of Netrin-1 in acute lung injury(ALI)and acute respiratory distress syndrome(ARDS).Methods A549 cells at logarithmic growth phase were incubated with 500 μg/L Netrin-1 for 0,3,6,12,24 and 48 h respectively,or Netrin-1 at various concentrations(0,0.5,5,50 and 500 μg/L)for 12 h,then divided into control,drug and inhibition groups,and added with RPMI1640 medium,500 μg/L Netrin-1 and 1 μmol/L PSB1115+500 μg/L Netrin-1 respectively,and further incubated for 12 h.The transcription levels of α-ENaC mRNAs in various groups were determined by RT-PCR,while the expression level of αENaC protein by Western blot.Results Both the expressions of α-ENaC at mRNA and protein levels in A549 cells after incubation with 500 μg/L Netrin-1 for 6 h were significantly higher than those in control group(P < 0.05)and reached peak values 12 h after incubation.However,both the expression levels in A549 cells after incubation with Netrin-1 at concentrations of more than 5 μg/L for 12 h were significantly higher than those in control group(P < 0.05),which were dose-dependent and reached the peak values after incubation with 500 μg/L Netrin-1.Both the expression levels were significantly higher in drug group than in control and inhibition groups(P < 0.01),indicating that PSB1115 inhibited the mRNA transcription and protein expression of α-ENaC significantly.Conclusion Netrin-1 up-regulated the expression of α-ENaC at gene level via adenosine A2b receptor pathway,which was beneficial to the prognosis of ALI and ARDS.

    2013 06 v.26 [Abstract][OnlineView][Download 539K]

  • Expression and identification of human coagulation factor Ⅶ in CHO-K1 cells

    XIAO Wei,ZHAO Rui,LI Chang-qing,BIAN Guo-hui,XIAO Xiao-pu,LIN Fang-zhao Institute of Blood Transfusion,Chinese Academy of Medical Sciences,Chengdu 610052,Sichuan Province,China

    Objective To express recombinant human coagulation factor Ⅶ(rhFⅦ)in CHO-K1 cells.Methods CHOK1 cells were transfected with plasmid pcDNA3.1-FⅦ and empty vector pcDNA3.1 respectively,from which G418-resistant cell strains were screened with 900 μg/ml G418.The cell culture supernatant were harvested and determined for rhFⅦ level by ELISA,for coagulation time by prothrombintime(PT) method,and for expression of rhFⅦ by Western blot.Results Three G418-resistant cell strains were obtained and named as CHO-FⅦ01 ～ 03 respectively,while that transfected with empty vector as CHO-CK.The rhFⅦs were detected in all the culture supernatants of CHO-FⅦ01 ～ 03 cells,of which the contents were lower than that in positive control group.The coagulation times of culture supernatant of CHO-CK cells were 2.4,2.1 and 3.1 times of those of CHO-FⅦ01 ～ 03 cells respectively.The culture supernatants of CHO-FⅦ01 ～ 03 cells contained rhFⅦ with a relative molecular mass of about 50 000.Conclusion Recombinant hFⅦ was successfully expressed in CHO-K1 cells,which laid a foundation of further study on hFⅦ.

    2013 06 v.26 [Abstract][OnlineView][Download 382K]

  • Expression of c-Jun N-terminal kinase in hypospadiac penis of fetal rats induced by maternal exposure to di(- 2-ethylhexyl)phthalate

    LI Ming-yong,QIU Lin,He Da-wei,LIN Tao,TU Sheng-fen,HUA Yi,ZhANG De-ying,LONG Chun-lan,WEI Guang-hui Department of Pediatric Urology Surgery,Children's Hospital of Chongqing Medical University,Chongqing 400014,China

    Objective To investigate the expression of c-Jun N-terminal kinase(JNK)in the hypospadiac penis of fetal rats induced by maternal exposure to di(-2-ethylhexyl)phthalate(DEHP)in order to explore the etiology of hypospadias.Methods Pregnant rats of 12 gestation days(GD12) were randomly divided into test and control groups,20 for each.The rats in test group were treated with DEHP dissolved in 1.5 ml soybean oil by lavage at a dosage of 750 mg /(kg.d),while those in control group with 1.5 ml soybean oil,once a day from GD12 to GD19.Half of rats in each group were raised to postnatal day(PND) 30,and live male pups were counted and weighed,of which anogenital distance(AGD) were measured,and the hypospadias was observed.Fetal rats of the rest pregnant rats were obtained on GD20 by caesare an section,and determined for expression levels of JNK 1/2 mRNA and protein by real-time quantitative PCR(qPCR) and immunochemical assay respectively.Results On PND30,the number,body weight and AGD of live male pups in test group decreased significantly as compared with those in control group(each P < 0.001),while the incidence rate of hypospadias was about 30.6%.However,compared with those in control group,the expression levels of JNK1 and JNK2 mRNAs in penis of fetal rats in test groups increased significantly(P < 0.05),while the expression levels of JNK1 and JNK2 proteins showed increasing tendencies(P < 0.05).Conclusion DEHP-induced congenital hypospadias might be associated with the abnormal expression of JNK signal pathway in hypospadiac penis of fetal rats.

    2013 06 v.26 [Abstract][OnlineView][Download 478K]

  • Construction of bacteriophage as a carrier of drug specific for hepatocellular carcinoma cells

    LI Qin,ZHAO Chen-yang,HUI Lin-ping,HE Lin,YU Tao Central Laboratory,The Forth Hospital Affiliated to Chinese Medical University,Shenyang 110032,Liaoning Province,China

    Objective To construct the bacteriophage as a carrier of drug specific for hepatocellular carcinoma(HCC) cells,and preliminarily evaluate its selective cytotoxicity.Methods Tumor specific peptide(TSP) were screened from phage-displayed random peptide library,recombined to bactriophage small capsid protein Soc by molecular cloning and an in vitro assembly strategies,and displayed on the surface of T4 bacteriophage.Epidoxorubicin(Epi)was loaded onto the bacteriophage capsid by using 1-ethyl-3(-3-dimethylaminopropyl)carbodiimide hydrochloride(EDC).In vitro tumor selective cytotoxicity of the constructed drug-carrying bacteriophage particles to HCC Bel-7402 cells and normal liver HL7702 cells was evaluated by MTT method at cell level.Results Soc-TSP fusion protein was displayed on the surface of T4 bacteriophage at a density of 846 copies/particle.The Epi-loading rate was about 2 000 molecules/particle.The killing rate of T4-carried Epi to Bel-7402 cells was 4 - 6 times higher than that of free Epi.However,T4-carried Epi showed no significant effect on HL-7702 cells.Conclusion Tumor-selective bacteriophage particle exerted their tumorkilling property via promoting the targeted drug delivery as well as the drug uptake by the tumor cells.

    2013 06 v.26 [Abstract][OnlineView][Download 559K]

  • Purification and physico-chemical property of recombinant human interferon β1a

    LI Liu-ping*,YANG Xu-wei,HUANG Zhi-bin,LA Wen-jun,WANG Yan *College of Chemistry,Jilin University,Changchun 130000,Jilin Province,China

    Objective To purify recombinant human interferon β1a(rhIFNβ1a)and analyze its physico-chemical property.Methods Recombinant CHO cells for expression of rhIFNβ1a were cultured on suspended micro-carriers in a 15 L bioreactor,of which the harvest liquid was concentrated by ultrafiltration and purified by Blue Sepharose 6 Fast Flow,Sephadex G-25 and CM ion exchange chromatography,then determined for rhIFNβ1a titer by vesicular stomatitis virus(VSV)inhibition test,for protein content by Lowry method,for isoelectric point by isoelectric focusing electrophoresis,for purity by SDS-PAGE and HPLC,for immunological activity by Western blot,for relative molecular mass by SDSPAGE and mass spectrometry,and for secondary structure by circular dichroism spectrum.Results Three batches of rhIFN-β1a samples were purified,of which the mean protein content was 0.284 0 mg/ml,the mean specific activity was 1.06 × 108 IU/mg,the purity was more than 95%,the mean recovery of protein was 58.36%,the relative molecular mass was 20 445.0,the isoelectric point was about 6.6,the immunological activity was high,and the secondary structure was basically consistent with that of national standard.Conclusion The rhIFNβ1a was successfully purified,and its physic-chemical property was determined,which laid a foundation of development of requirement for large-scale preparation,control test and industrialization of rhIFNβ1a.

    2013 06 v.26 [Abstract][OnlineView][Download 742K]

  • Inhibitory effect of nerve growth factor on infection of BHK-21 cells with Japanese encephalitis virus

    YANG Dong*,GU Peng-yi,LIU Hai-jun,LI Ning-he,LIU Chang,YAO Yu,LI He,SUN Ying-jie,SUN Jin-min *The Center of Laboratory Technology and Experimental Medicine,China Medical University,State Administration of Traditional Chinese Medicine Biological Engineering Research Laboratory Grade III,Shenyang 110001,Liaoning Province,China

    Objective To observe the inhibitory effect of nerve growth factor(NGF)on infection of BHK-21 cells infected with Japanese encephalitis virus(JEV).Methods BHK-21 cells were treated with NGF at various concentrations(100,50,25,12.5,6.3,3.1,1.6 and 0.8 μg/L)respectively.The toxic effect of NGF on BHK-21 cells was determined by MTT method.BHK-21 cells were infected with JEV at various dilutions(10-1,10-2,10-3,10-4,10-5,10-6 and 10-7),and determined for titer by plaque counting.BHK-21 cells infected with JEV were treated with NGF at various concentrations and observed for CPE,based on which the inhibitory effect of NGF on JEV was analyzed by plaque reduction rate.Results NGF at concentrations of less than 100 μg/L showed no cytotoxic effect on BHK-21 cells.The titer of JEV was Lg8.70 pfu/ml.The CPE of BHK-21 cells in NGF(12.5 μg/L)+ JEV group was relieved significantly.The plaque reduction rate showed an increasing tendency with the increased NGF concentration(r = 0.929,P < 0.05).The plaque reduction rate of BHK-21 cells treated with NGF at concentrations of more than 3.1 mg/L was above 10%,indicating significantly inhibitory effect on JEV.Conclusion The NGF at concentrations of 0.8 - 100 μg/L showed significantly inhibitory effect on JEV,which laid an experimental foundation of further study on mechanism of antiviral effect of NGF.

    2013 06 v.26 [Abstract][OnlineView][Download 579K]

  • Effect of polysaccharopeptide on Toll-like receptor 4 in peripheral blood mononuclear cells of patients with breast cancer

    WANG Jing,YU Shuang,CHEN Chu,DONG Bing,BAO Yi-xi Department of Clinical Laboratory,The Second Affiliated Hospital of Chongqing Medical University,Chongqing 400010,China

    Objective To investigate the effect of polysaccharopeptide(PSP)on Toll-like receptor 4(TLR4)in peripheral blood mononuclear cells(PBMCs)of patients with breast cancer.Methods PBMCs were isolated from the patients with breast and healthy individuals,and divided into PSP and control groups separately.The PBMCs in PSP group were treated with PSP and PHA at final concentrations of 25 and 100 μg/ml respectively,while those in control group with PHA at a final concentration of 100 μg/ml.Cellular immunofluorescence staining was applied to PBMCs in two groups with anti-TLR4 monoclonal antibody.The PBMCs from patients with breast cancer were divided into blank control,TLR4 antibody,PSP and PSP+TLR4 antibody groups,and determined for expression levels of IL-12,IL-6 and TNF-α by realtime quantitative PCR(Q-PCR).Results Enhanced fluorescence intensity was observed in PBMCs from healthy individuals in control group,while that in PSP group was weak.However,the fluorescence intensity of PBMCs from patients with breast cancer in PSP group was weaker than that in control group.As compared with those in blank control group,the ex pression levels of IL-12 and TNF-α in PBMCs of patients in TLR4 antibody group decreasd significantly(P < 0.05),while those of IL-12,IL-6 and TNF-α in PSP group increasd significantly(P < 0.05 or P < 0.01).However,compared with those in PSP and TLR4 antibody groups,the expression levels of IL-12,IL-6 and TNF-α in PSP+TLR4 antibody groups increased significantly(P < 0.05 or P < 0.01).Conclusion TLR4 may be one of the immune receptors of PSP.

    2013 06 v.26 [Abstract][OnlineView][Download 706K]

  • Immunosuppressive effect of kaempferol on delayed type hypersensitivity in mice

    TONG Chun-yu,CAO Ning,LIU Zhen-hua,CUI Yu-dong College of Life Science and Technology,Heilongjiang Bayi Agricultural University,Daqing 163319,Heilongjiang Province,China

    Objective To investigate the immunosuppressive effect of kaempferol(KA)on delayed type hypersensitivity(DTH)induced by dinitrofluorobenzene(DNFB)in mice.Methods BALB/c mice were randomly divided into physiological saline control,DTH model,cytoxan(CTX)(20 mg/kg)as well as low(2 mg/kg),moderate(4 mg/kg)and high(8 mg/kg)dosage KA groups according to the bodyweight,ten for each,then sensitized with DNFB and treated with the corresponding drugs for 7 d.DTH was induced on day 5 after sensitization by smearing 5 μl of 1% DNFB onto the back of right ears of mice,while the left ears were smeared with acetone/olive oil solvent at an equal volume as control.The bodyweight as well as wet weights of spleen and thymus of the mice were measured 7 d after treatment,based on which the spleen and thymus indexes were calculated.The two ears of mice were weighed 48 h after induction with DNFB to calculate the swelling level,the prepared into frozen sections and observed for pathological change by optical microscopy.Spleens of mice were collected aseptically,from which splenocytes were prepared and determined for proliferation activity after stimulation with ConA by MTT method.Results KA at a high dosage decreased the spleen and thymus indexes of mice with DTH significantly(P < 0.01 or P < 0.05),while that at moderate and high dosages inhibited the swelling of ears induced by DNFB significantly(P < 0.05 or P < 0.01).Meanwhile,KA decreased the lymphocyte infiltration in local ear tissues of mice with DTH and inhibited the proliferation of splenic lymphocytes(P < 0.05 or P < 0.01).Conclusion KA showed significantly immunosuppressive effect on DTH in mice by a possible mechanism of inhibiting the proliferation of lymphocytes.

    2013 06 v.26 [Abstract][OnlineView][Download 559K]

  • Preparation and preliminary application of fusion protein Rv0057-Rv1352 of Mycobacterium tuberculosis

    YANG You-rong*,FENG Jin-dong,ZHANG Jun-xian,ZHAO Wei-guo,LIU Yu,LIANG Yan,BAI Xue-juan,WANG Lan,WU Xue-qiong *Institute of Tuberculosis Research,The 309th Hospital of Chinese PLA,Beijing 100091,China

    Objective To prepare the fusion protein Rv0057-Rv1352 of Mycobacterium tuberculosis(M.tb),analyze its antigenicity by chemiluminescent EIA and evaluate its application.Methods Rv0057 and Rv1352 genes were synthesized,amplified and inserted into vector pET30aSETB to construct recombinant plasmid Rv0057/pET30aSETB and Rv1352/pET30aSETB respectively.Recombinant plasmid Rv1352/pET30aSETB was digested with NheⅠ and XhoⅠ,while Rv0057/pET30aSETB with SpeⅠ and XhoⅠ,and the recovered Rv1352 gene fragment was inserted into plasmid Rv0057/pET30aSETB.The constructed recombinant plasmid Rv0057-Rv1352/pET30aSETB was transformed to E.coli BL21(DE3)for expression under induction of IPTG.The expressed protein,in a form of inclusion body,was purified by His.Bind protein purification kit and used as an antigen for development of chemiluminescent EIA.The antibodies against M.tb in sera of patients were determined by the developed method,and the results were compared with those by commercial M.tb antibody diagnostic kit and by sputum smearing examination.Results The nucleotide sequence of constructed recombinant plasmid Rv0057-Rv1352/pET30aSETB was completely consistent with that designed.The expressed recombinant fusion protein Rv0057-Rv1352,with a relative molecular mass of about 30 500,mainly existed in a form of inclusion body and contained about 30% of total somatic protein.The concentration and purity of purified recombinant fusion protein were 1.6 mg / ml and 95% respectively.The sensitivity and specificity of developed chemiluminescent EIA using the fusion protein as an antigen were 66.70% and 90.00% for the antibody against M.tb in sera,while those of commercial M.tb antibody diagnostic kit were 80.00% and 63.33 % respectively.The sensitivity of sputum smearing examination for 30 cases of active pulmonary tuberculosis was 20.0%,which showed significant difference with that of chemiluminescent EIA(66.7%)(P < 0.01).Conclusion M.tb Rv0057-Rv1352 fusion protein was successfully prepared.Chemiluminescent EIA developed using the fusion protein as an antigen showed high sensitivity and specificity for antibody against M.tb and was of a broad prospect in application to rapid serological diagnosis of tuberculosis.

    2013 06 v.26 [Abstract][OnlineView][Download 589K]

  • Expression and correlation of T-cadherin and basic fibroblast growth factor in human uterine fibroids

    WANG Li-fang,TANG Liang-dan,JIA Li,CAO Yi Department of Obstetris and Gynecology,The First Affiliated Hospital of Chongqing Medical University,Chongqing 400016,China

    Objective To investigate the expression and correlation of T-cadherin and basic fibroblast growth factor(bFGF)in human uterine fibroids(UF).Methods Fifty patients with UF undergoing surgery in Department of Obstetris and Gynecology,The First Affiliated Hospital of Chongqing Medical University from September to December 2011 were selected,of whom UF(group A)and adjarcent normal myometrium(group B)were collected,using the myometrium of 10 non-UF patients with benign diseases as control(group C).The patients in groups A and B were further divided into subgroups based on the Color Doppler Flow Imaging(CDFI)grades and sex hormones levels before surgery.The locations of T-cadherin and bFGF in uterus were determined by SP immunohistochemical assay.The protein expression and mRNA transcription levels of bFGF and T-cadherin were determined by Western blot and RT-PCR respectively,of which the corrections to CDFI grade and sex hormone level were analyzed.Results T-cadherin in group A was mainly located on the leiomyoma cell membrane,while bFGF in the leiomyoma endochylema.The T-cadherin and bFGF mRNA transcription levels in group A were positively related to the protein expression levels(P < 0.01),both of which were significantly higher than those in groups B and C(P < 0.05).Both the mRNA transcription and protein expression levels of T-cadherin and bFGF increased with the increasing CDFI grade(P < 0.05 or P < 0.01).Conclusion T-cadherin and bFGF were highly expressed in uterin fibroids,indicating their promoting effect on onset and progress of the disease.

    2013 06 v.26 [Abstract][OnlineView][Download 654K]

  • One-step real-time fluorescent quantitative PCR assay for mumps virus in clinical saliva specimens

    SHI Hai-jing,WANG Jing-jing,CHEN Jun-ying,CHEN Wei,PAN Yue,ZHAO Gang,SUN Qiang-ming Institute of Medical Biology,Chinese Academy of Medical Sciences and Peking Union Medical College,Yunnan Key Laboratory of Vaccine Research & Development on Severe Infectious Diseases,Kunming 650118,Yunnan Province,China

    Objective To develop an one-step real-time fluorescent quantitative PCR assay for mumps virus(MuV)in clinical saliva specimens and provide a basis for clinical diagnosis,prevention and control of mumps.Methods A total of 101 clinical saliva specimens collected from suspected patients with mumps in Yunnan and Sichuan Province as well as Guangxi Zhuang Autonomous Region,China were determined by cell culture-MuV specific nested PCR and real-time PCR with TaqMan probe respectively,based on which the sensitivities of the two methods were compared.Results Thirty-one MuV-positive specimens were found by cell culture-MuV specific nested PCR,all of which belonged to genotype F as proved by phylogenetic tree.However,41 MuV-positive specimens were found by real-time PCR with TaqMan probe,including the 31 positive specimens proved by cell culture-MuV specific nested PCR,indicating that the positive rate of Muv by real-time RT-PCR with TaqMan probe(40.59%)was higher than that by traditional cell culture-nested PCR(30.69%).Conclusion The sensitivity,specificity and simplicity of real-time PCR with TaqMan probe were higher than those of traditional cell culture-MuV specific nested PCR,which might be used for the clinical diagnosis,prevention and control of mumps.

    2013 06 v.26 [Abstract][OnlineView][Download 673K]

  • Induction of neutralizing antibody in mice by plasmid DNA encoding envelope glycoprotein of Ebola virus

    HE Li-fang*,WU Wen-jun,WANG Li-li,CHEN Min,WANG Xin,YANG Ning,HUANG Zhen *Yunnan Walvax Biotechnology Co.Ltd.,Kunming 650106,Yunnan Province,China

    Objective To investigate the induction of neutralizing antibody in mice by plasmid DNA encoding envelope glycoprotein(GP)of Ebola virus.Methods BALB/c mice were injected i.m.with plasmids pcDNA3.1,pcDNA3.1GP(full length GP),pcDNA3.1-GPΔTM(GP with transmembrane domain deleted)and pcDNA3.1-GPΔMet(GP with original codon deleted)respectively,and determined for serum antibody titer against GP by ELISA,for specificity of antibody by Western blot,and for neutralizing efficiency by Ebola pseudovirus mock infection in 293E cells.Results Both full-length GP and the GP with transmembrane domain deleted induced effective immune response,and the induced GP antibody showed high specificity.GP antiserum inhibited the entry of Ebola pseudovirus into 293E cells.Conclusion Plasmid DNA encoding GP of Ebola virus induced high titer neutralizing antibody which neutralized Ebola pseudovirus effectively.

    2013 06 v.26 [Abstract][OnlineView][Download 489K]

  • A novel method for isolation and culture of mesenchymal stem cells from Wharton's jelly of human umbilical cord

    GAO Yong,JIANG Ming-de,WANG Zhao,WU Xiao-ling,HE Yong,WANG Qun-ru Department of Gastroenterology,General Hospital,Chengdu Military Command,Chengdu 610083,Sichuan Province,China

    Objective To develop a novel method for isolation and culture of mesenchymal stem cells(MSCs) from Wharton′s jelly of human umbilical cord.Methods Human umbilical cord tissues were collected,from which arteries and veins were removed,and minced into pieces each at a sized of 2 - 5 mm3.The pieces were treated with the mixture of 4 g/L collagenase Ⅰ and 1 g/L hyaluronidase at 37℃ for 1 h,then digested with 0.25% trypsin under the same condition for 30 min.The digested suspension was filtered with a 70 μm nylon mesh and prepared into a single-cell suspension,then cultured and subcultured.The growth curve of MSCs of passages 1,3 and 7(P1,P3 and P7)was plotted,while those of P3 were determined for surface marker by flow cytometry then subjected to osteogenic and adipogenic differentiations,and the results were observed by Alizarin red and oil red O staining.Results MSCs of P1 and P3 showed strong proliferation ability,while the proliferation ability of P1 was stronger than that of P3.However,the proliferation ability of P7 was weakened as compared with that of P3.The expression rates CD90(99.8%),CD105(100%)and CD166(100%)were high in MSCs of P3,while those of CD45(0.3%),CD14(0.1%),CD34(0.2%) and CD79a(0.3%)were low,and no HLA-DR was expressed.Red calcium node was observed by Alizarin red staining in MSCs of P3 after osteogenic differentiation.However,after adipogenic differentiation,liquid vacuoles were observed by oil red O staining.Conclusion The MSCs obtained from Wharton′s jelly of human umbilical cord showed high activity and strong proliferation ability,which provided an ideal cell seeds for further laboratory study and clinical application.

    2013 06 v.26 [Abstract][OnlineView][Download 609K]

  • Development of alkaline phosphatase cell ELISA method for single-chain phage antibody

    QIAO Yuan-yuan,ZHANG Da-jin,ZHAO Xiao-hang Center for Basic Medical Sciences,Navy General Hospital of Chinese PLA,Beijing 100048,China

    Objective To develop an alkaline phosphatase(ALP)cell ELISA method for single-chain phage antibody so as to eliminate the interference of leukocytes in peripheral blood and use the screened phage antibody for determination of clinical samples.Methods Single-chain phage antibodies binding to oesophageal carcinoma cells were screened from large-capacity phage antibody library,based on which the identification plates for oesophageal carcinoma KYSE-170 and EC109 cells as well as leukocytes were prepared.The binding activities of screened phage antibodies to KYSE-170 and EC109 cells as well as leukocytes were analyzed by ALP cell ELISA,using those by horse radish peroxidase(HRP)cell ELISA as parallel control,and the determination result of antibody to ovalbumin(OA) as negative control.Results ALP cell ELISA showed that the screened phage antibodies 1,2 and 3 bound to KYSE-170 and EC109 cells,of which the A405 values were within the detection range.However,the binding activities of three kinds of antibodies to two cell lines showed significant difference(P < 0.001).The binding activities of antibody against OA to the two cell lines were significantly lower than those of three kinds of phage antibodies(P < 0.001).The A405 values of three kinds of phage antibodies to leukocytes were within the detection range,which showed significant difference with those to KYSE-170 and EC109 cell lines(P < 0.01).Conclusion ALP cell ELISA may be used for single-chain phage antibody,by which the interference of leukocytes may be eliminated.The developed method is hopeful to be used for determination of clinical samples.

    2013 06 v.26 [Abstract][OnlineView][Download 453K]

  • Determination of benzalkonium chloride content in recombinant human interferon α1b eye drops by high performance liquid chromatography

    NIU Xiao-xia,CAO Yan,WU Mei-ying Beijing Tri-prime Genetic Engineering Co.,Ltd.,Beijing 102600,China

    Objective To determine the benzalkonium chloride content in recombinant human interferon α1b(rhIFNα1b)eye drops by high performance liquid chromatography(HPLC).Methods The benzalkonium chloride content in rhIFNα1b eye drops was determined by HPLC.A Waters Symmetry C18 column was used with the mobile phase of V(A)∶ V(B)= 65 ∶ 35[A: acetonitrile;B: 5 mmol/L ammonium acetate containing 1% triethylamine,adjusted with acetic acid to pH(5.0 ± 0.2)]at a flow rate of 1.0 ml/min.The detection wavelength,sample load and column temperature were 262 nm,20 μl and room temperature,respectively.The method was verified,and used for determination of benzalkonium chloride contents in three batches of rhIFNα1b eye drops.Results The method showed good specificity and suitability in determination of benzalkonium chloride content,of which the minimum detection limit and minimum quantitation limit were 0.53 and 1.48 μg/ml respectively.The standard curve showed good linearity within a benzlkonium chloride concentration range of 10 - 50 μg/ml(R2 = 0.999 1).The RSD of peak areas of six samples loaded were 1.78%.The mean recovery rate of samples at theoretical benzalkonium chloride concentrations of 22,24 and 26 μg/ml was 97.90%,with a RSD of 0.96%.The relative benzalkonium chloride contents in three batches of rIFNα1b eye drops were 19.27,19.89 and 19.76 μg/ml,which were 96.34%,94.46% and 98.80% of stated amounts,respectively.Conclusion The developed HPLC method was simple,sensitive and accurate,which might be used for determinatioin of benzalkonium chloride content in recombinant human interferon α1b eye drops.

    2013 06 v.26 [Abstract][OnlineView][Download 482K]

  • Determination of irregular antibody of erythrocytes by microtubes gel test

    AN Qin-fa,HU Zhi-hong,CUI Hui-qin Daming People’s Hospital,Daming 056900,Hebei Province,China

    Objective To evaluate the efficacy of microtubes gel test(MGT)in determination of irregular antibody of erythrocytes.Methods Serum specimens were collected from 1 147 patients receiving blood transfusion in Daming People’s Hospital,Hebei Province,China from September 2011 to December 2012,and screened for irregular antibody by MGT and test tube Coombs test(TT-Coombs’T)respectively,based on which MGT was evaluated for sensitivity and specificity severing the determination result by TT-Coomb’s T as a gold standard.The antibodies in positive specimens were further identified by MGT.Results Of the 1 147 specimens,10 were judged as positive for irregular antibody by both MGT and TT-Coombs’T,while 1 137 were negative,indicating a positive rate of 0.87%.Both the sensitivity and specificity of MGT were 100%.Further identification proved that,of the 10 positive specimens,9 were positive for Rh antibodies,including 5 for anti-E,2 for anti-c,1 for anti-D and 1 for anti-C,while 1 was positive for MNS antibody(anti-M).Conclusion MGT showed high sensitivity and specificity,of which the procedure was simple and rapid as compared with TTCoombs’T,and the judgment of result was easy to be standardized.The method was more suitable for screening of irregular antibody before blood transfusion in clinic.

    2013 06 v.26 [Abstract][OnlineView][Download 432K]

  • Application and prospect of systems biology in vaccinology

    WU Xing,LIANG Zheng-lun National Institutes for Food and Drug Control,Beijing 100050,China

    The development of novel"omics"technologies are fueling the development of a new science of systems biology.In this framework,the advancement of high-throughput technologies,together with the extensive identification of genomics,proteomics,etc.,facilitate large-scale biological measurements.These advances have led to the development of new investigative tools in the field of biology and medicine,especially in the study of prevention and treatment of intractable diseases such as diabetics and neoplastic diseases.This paper reviews the application and prospect of systems biology in vaccinology.

    2013 06 v.26 [Abstract][OnlineView][Download 464K]

  • Progress in clinical study on anti-infectious monoclonal antibodies

    WANG Cha,WEI Jing-shuang NCPC New Drug Research and Development Co.Ltd.,State Key Laboratory of Antibody Drug Development,Shijiazhuang 050015,Hebei Province,China

    The emergence of multidrug-resistent bacteria and viruses of new type is growing challenge for antibiotics therapy in the treatment of infectious diseases.Based on a body of preclinical work,monoclonal antibodies(mAbs)are considered to have great potential to treat the bacterial and viral infections,particularly nosocomial infections.Only one mAb is approved for use against an infectious disease,though there are many in various st ages of development.This review briefly summarizes the current progress in clinical research on anti-infectious mAbs.

    2013 06 v.26 [Abstract][OnlineView][Download 564K]

  • Establishment and verification of scale-down model of lifetime of chromatography medium

    YANG Hong-yan,SUI Li-li Fast Trak China Center,General Electric(China)Research and Development Center Co.Ltd.,Shanghai 201203,China

    Chromatography is one of the most popular techniques in modern biopharmaceutical manufacturing.To improve the safety and efficacy of the biopharmaceuticals,the usage of chromatography medium shall be inspected by regu latory authority.The lifetime of medium shall be determined by study and checked and approved by the regulatory authority.Prospective study on scale-down model has been widely accepted to lifetime of chromatography medium.This paper reviews the establishment and verification of scale-down model of life time of chromatography.

    2013 06 v.26 [Abstract][OnlineView][Download 425K]
    • 下载本期数据

@2012 Editorial Department of "Chinese Journal of Biologicals"