2010年01期目次

  • Sequencing of Whole Genome of Varicella-Zoster Virus 84-7 Strain

    LI Xiu-ling,WANG Xiao-xiao,ZHANG Zhong-yang,et al (National Vaccine & Serum Institute,Beijing 100024,China)

    Objective To analyze the gene sequence and its character of varicella-zoster virus(VZV)84-7 strain isolated in China.Methods Purified virus particles of VZV were obtained by ultracentrifugation,from which viral genomic DNA was extracted with benzene-chloramine then ultrasonicated,and the gene fragments at lengths of 1 500 ～ 3 000 bp were recovered and cloned into vector pUC18 to construct viral DNA library.The genomic DNAs were sequenced,and the results were compared with those of wild Dumas strain,based on which a phylogenetic tree was generated to analyze the homologies of VZV84-7 strain to other strains.Results The whole genome of VZV84-7 strain consisted of 125 083 bp,with a G + C content of 46.1%.The lengths of regions UL,TRL,IRL,Us,TRS and IRS were 104 746,89,89,5 235,7 462 and 7 462 bp,while the G + C contents were 44.3%,69.7%,69.7%,42.8%,59.3% and 59.3%,respectively.The copy numbers of repeat regions R2,R4 and R5 in the genome were 7,9 and 1 respectively.A total of 71 ORFs were found in the genome of VZV84-7 strain,of which 3 genes were located within the repeat sequences,therefore the sequence of ORFs 69 ～ 71 in IRS region were completely identical to that of ORFs 62 ～ 64 in TRS region.A total of 248 nucleotide substitutions including a part of insertion and deletion mutations were observed as compared with Dumas strain,most of which were single nucleotide polymorphisms(SNPs).Transitions were more frequent than transversions(66% vs 34%).Phylogenetic tree showed that the homologies of VZV84-7 strain to other VZV strains were more than 95%.Conclusion Though VZV84-7 strain was unique genetically,it was highly homologous to other VZV strains.

    2010 01 v.23 [Abstract][OnlineView][Download 365K]

  • Cloning and Prokaryotic Expression of Bordetella pertussis Adenylate Cyclase Toxin Gene

    SHI Bi-zhu△,ZHANG Hua-jie,MENG Min-jie,et al (△School of Life Science and Biopharmacy,Guangdong Pharmaceutical University,Guangzhou 510006,China)

    Objective To clone Bordetella pertussis adenylate cyclase toxin(CyaA,ACT)gene,and express and purify recombinant CyaA protein.Methods The gene encoding CyaA was amplified from genomic DNA of B.pertussis CS strain by PCR and cloned into vector pET30a.The constructed recombinant plasmid pET30a /cyaA was transformed to competent E.coli BL21(DE3) for expression under induction of IPTG.The expressed recombinant protein was de-naturalized with 8 mol /L urea,re-naturalized by dialysis,purified by DEAE anion exchange chromatography and identified for reactogenicity by Western blot.Results PCR,restriction analysis and sequencing proved that recombinant plasmid pET30a /cyaA was constructed correctly.The expressed recombinant protein mainly existed in a form of inclusion body,contained about 20% of total somatic protein,reached a purity of about 90% after purification and showed specific reactions with sera of mice immunized with whole cell and acellular pertussis vaccines.Conclusion B.pertussis cyaA gene was successfully cloned,and recombinant CyaA protein was expressed in E.coli,which laid a foundation of study on application of CyaA.

    2010 01 v.23 [Abstract][OnlineView][Download 404K]

  • Infectivity of Full-length cDNA Clone of Attenuated Japanese Encephalitis Virus Strain SA14-14-2

    LI Jing△,YU Yong-xin,DONG Guan-mu,et al(△Wuhan Institute of Biological Products,Wuhan 430060,China)

    Objective To establish an infectious transcript on the basis of full-length cDNA clone of Japanese encephalitis virus(JEV),obtain the recovery virus and provide a method for study on pathogenic mechanism and molecular virology of JEV as well as development of JEV vaccine.Methods The full-length cDNA of JEV was cloned into modified vector pBluescript KS Ⅱ(+),followed by in vitro transcription using T7 promoter and Lipofectamine-mediated transfection to BHK-21 cells.The recovery virus was identified by RT-PCR,sequencing,indirect IFA and plaque formation test.Results Obvious CPE was observed in the BHK-21 cells transfected with transcript.The harvested recovery virus was identified as JEV.Conclusion A method for obtaining infectious JEV RNA by in vitro transcription of full-length cDNA clone was developed,which laid a foundation of study on molecular biology of JEV and vaccine development.

    2010 01 v.23 [Abstract][OnlineView][Download 323K]

  • Expression,Purification and Immunogenicity of Tetravalent Recombinant Group A Streptococcus Protein

    YU Gang,YANG Jing-sheng,QUAN Jia-wu(Wuhan Institute of Biological Products,Wuhan 430060,China)

    Objective To express tetravalent recombinant protein against group A streptococcus(GAS)of serotypes M1,3,6 and 18 in prokaryotic cells,purify the expressed product and determine its immunogenicity.Methods The target gene was amplified by PCR using cloning vector pUC18-Strep4 as a template and cloned into expression vector pQE30.The constructed recombinant plasmid was transformed to E.coli M15,and positive clones were screened for expression under induction of IPTG.The expressed recombinant protein was purified by Ni2+-NTA gel affinity chromatography.Rabbits were immunized with the expressed protein,and their serum antibody titers were determined by ELISA.The type-specific antibodies against M1,M3,M6 and M18 of GAS were determined by IFA,and serum bactericidal antibody activity by in vitro bactericidal test.Results Restriction analysis and sequencing proved that recombinant plasmid pQE30-Strep4 was constructed correctly.The expressed product in a soluble form,with a relative molecular mass consistent with that expected,contained about 30% of total somatic protein and reached a purity of more than 90%.Specific bactericidal antibody titers against serotypes M1,3 and 6 of GAS were induced in rabbits.Conclusion The tetravalent recombinant protein against GAS of serotypes M1,3,6 and 18 was successfully expressed.The purified recombinant protein induced specific bactericidal antibodies against serotypes M1,3 and 6 of GAS.

    2010 01 v.23 [Abstract][OnlineView][Download 730K]

  • Regulatory Effect of ALU Transcribed by RNA Polymerase Ⅱ on PKR Phosphorylation

    YANG Mei△,SHEN Wei,WU Jin-feng,et al (△Department of Gastroenterology,The Second Affiliated Hospital,Chongqing Medical University,Chongqing 400010,China)

    Objective To investigate the influence of RNA polymerase Ⅲ promoter of ALU on transcription of RNA polymerase Ⅱ(Pol Ⅱ)as well as the regulatory effect of ALU transcribed by Pol Ⅱ on double-stranded RNA-dependent protein kinase (protein kinase R,PKR)phosphorylation.Methods The full-length of ALU gene was inserted downstream to the CMV promoter of vector pcDNA3.1(-),and the constructed recombinant plasmid pcDNA3.1-ALU was transfected to HEK293 cells.Total RNA was extracted from the transfected cells,and the cells stably expressing ALU were screened by RT-PCR and treated with interferon.The PKR phosphorylation level was determined by Western blot.Results The full-length of ALU gene inserted downstream to the CMV promoter was effectively transcribed by RNA polymeraseⅡ.The PKR phosphorylation level in HEK293 cells stably expressing ALU,either treated or untreated with interferon,was significantly higher than that in HEK293 cells as control.Conclusion The RNA polymerase Ⅲ promoter of ALU showed no significant influence on transcription of Pol Ⅱ.However,unlike the Pol Ⅲ-driven ALU transcript,the ALU transcribed by Pol Ⅱ lost its antagonism to interferon and,on the contrary,activated PKR by forming doublestranded RNA.

    2010 01 v.23 [Abstract][OnlineView][Download 301K]

  • Inhibitory Effect of Terpene Derivative V-54 from Alisma orientalis Extract on Replication of Picornaviridae Viruses

    WANG Li-chun△,LIAO Yun,LONG Run-xiang,et al(△Institute of Medical Biology,Chinese Academy of Medical Sciences and Peking Union Medical College,Kunming 650118,China)

    Objective To investigate the inhibitory effect of terpene derivative V-54 from Alisma orientalis extract on the replication of picornaviridae viruses as well as mechanism of the effect.Methods KMB17 cells were infected with hepatitis A virus H strain(HAV-H),poliovirus Sabin Ⅰ(PV-Ⅰ)and coxsackievirus B2 strain(Cox-B2)respectively,then treated with V-54 and determined for virus titers,based on which the dynamics of inhibitory effects on replication of PV-Ⅰ and Cox-B2 as well as influence of V-54 on apoptosis of KMB17 cells were analyzed.Results V-54 inhibited the replication of HAV-H,PV-Ⅰ and Cox-B2 in KMB17 cells.The titers of PV-Ⅰ and Cox-B2 in KMB17 cells treated with V-54 decreased significantly as compared with those untreated.The inhibitory effects of V-54 on replications of PV-Ⅰ and Cox-B2 were dose-dependent.The apoptosis rates of KMB17 cells increased significantly within 24 h after treatment with V-54,then decreased to normal level gradually.Conclusion V-54 inhibited the infection and replication of picornaviridae viruses by a potential mechanism of binding to the relevant receptors on cell surface and resulting in the decrease of virus infection efficacy.

    2010 01 v.23 [Abstract][OnlineView][Download 359K]

  • Effect of Habenular Lesion on Expression of GABAA Receptor Subunit α1 in Hepothalamus

    MU Li△,TSANG Lai-ling,ZHANG Xiao-hu,et a(l△Department of Physiology,Norman Bethune College of Medical Sciences,Jilin University,Changchun 130021,China)

    Objective To observe the effect of habenular lesion on expression of GABAAreceptor subunit α1(GABAAα1)in the hypothalanus and investigate the potential molecular mechanism of functional relationship between habenular nucleus and hypothalamus.Methods A rat model of habenular lesion was established,using the rats receiving sham operation as control.The hypothalamus of rats were isolated,from which total RNA and total protein were extracted.The GABAAα1 mRNA transcription and protein expression levels in hypothalamus of rats were determined by RT-PCR and Western blot respectively.Results Both the GABAAα1 mRNA transcription and protein expression levels in hypothalamus of rats with habenular lesion were significantly higher than those in the rats receiving sham operation.Conclusion Habenular lesion increased the expression level of GABAAα1 in hypothalamus significantly,indicating the potential important role of GABAA receptor in physiological regulatory course involving habenular nucleus and hypothalamus.

    2010 01 v.23 [Abstract][OnlineView][Download 228K]

  • Separation and Purification of Heme Peptide-Iron and Its Capacity of Scavenging Free Radical

    ZHANG Bin△,MA Mei-hu (△College of Food Science & Technology,Hunan Agricultural University,Changsha 410128,China)

    Objective To separate and purify heme peptide-iron and analyze its capacity of scavenging free radical.Methods The lysate of porcine haematoglobin with trypsin under conditions of vacuum and low-voltage high frequency pulse was separated and purified by metal chelate affinity chromatography using polyacrylamide as a carrier.The obtained heme peptide-iron was analyzed for molecular structure by MALDI-TOFMS and bioinformatics software,for antioxidation capacity using vitamin C(Vc)as control,for capacity of scavenging superoxide anion by pyrogallol method,for capacity of scavenging hydroxyl radical by deoxyribose (DR)method,and for inhibitory effect on lipid peroidation by chloroform-methanol method.Results The purified heme peptide-iron consisted of 139 amino acids,with a relative molecular mass of 16 065.08,a ferri ion content of 11 013.5 μg /L,a theoretical pI value of 8.10,an extinction coefficient of 6 990,a fat coefficient of 92.73,an instability coefficient of 1.18 and a total mean hydrophobicity of 0.023,and showed high capacity of scavenging superoxide anion and antioxidant in vitro.Conclusion The purified heme peptideiron showed ability in clearance of free radical.

    2010 01 v.23 [Abstract][OnlineView][Download 805K]

  • Inhibitory Effect of Mesenchymal Stem Cells on Thymoma Induced by Ionizing Radiation in Mice

    CHEN Yu-bing△,WANG Hong-yan,LIU Li-ping,et al (△Department of Radiotherapy,Second Hospital,Jilin University,Changchun 130021,China)

    Objective To observe the inhibitory effect of mesenchymal stem cells(MSCs)on the thymoma induced by ionizing radiation in mice.Methods The mouse model of thymoma was replicated by ionizing radiation by typical Kaplan method.The MSCs of C57BL /6 mice were isolated and cultured by whole bone marrow adherent culture method,labeled with DAPI and injected into model mice through tail vein.Partial model mice were killed 1,5 and 10 d after injection respectively,and their thymus tissues were observed for location of MSCs in thymoma by laser confocal microscopy.The thymus tissues of partial model mice were collected 6 months after the first high dose systemic radiation,and observed for pathological change by HE staining,based on which the thymoma formation was judged.Results Laser confocal microscopy showed that the MSCs injected through tail vein were migrated to the thymus tissues of mice.Pathological observation showed that the structures of cortices and medulla of thymus tissues of mice treated with MSCs were clear,while lymphoid tumor cells were in a small number and uneven shape and size,and nuclear division was observed occasionally.The thymoma formation rate of mice treated with MSCs was 37.50% ± 7.55%,which decreased significantly as compared with that of model mice untreated(57.00% ± 9.78%).Conclusion The mouse model of thymoma induced by ionizing radiation was successfully established.The MSCs injected may be migrated to the thymus tissue and decrease the thymoma formation rate.

    2010 01 v.23 [Abstract][OnlineView][Download 367K]

  • In Vitro Effect of Piliated Pseudomonas aeruginosa and Its Flagellin Combined with rhIL-12 on Immunological Function of Patients with Chronic Hepatitis B

    FU Yong-hang△,YANG Xiang-yang,LI Ru-bing,et al (△Department of Laboratory Medicine,The 458th Hospital of PLA,Guangzhou 510602,China)

    Objective To investigate the in vitro effect of piliated Pseudomonas aeruginosa(PPA)and its flagellin combined with rhIL-12 on the immunological function of patients with chronic hepatitis B (HB).Methods The PBMCs of patients with chronic HB were co-incubated with PPA,the flagellin of PPA,rhIL-12,PPA + rhIL-12 and flagellin + rhIL-12 respectively,using those of healthy persons as control.The IFNγ content in culture supernatant of PBMCs was determined by ELISA.Results PPA and its flagellin induced only low IFNγ levels,while the IFNγ level induced by rhIL-12 was significantly higher than that of negative control.However,the IFNγ levels induced by PPA + rhIL-12 and flagellin + rhIL-12 were significantly higher than those by PPA,flagellin and rhIL-12,and the effect of rhIL-12 was dose-dependent.Conclusion PPA and its flagellin combined with rhIL-12 significantly increased the IFNγ levels in PBMCs of patients with HB and healthy persons in vitro,and enhanced their cellular immune response.

    2010 01 v.23 [Abstract][OnlineView][Download 415K]

  • Expression of hFGF-21 with Optimized Codon in P.pastoris

    MA Zhong-hui△,ZHAO Qiao-xiang,GAO Xin,et al (△Faculty of Animal Science and Veterinary Medicine,Heilongjiang August First Land Reclamation University,Daqing 163319,Heilongjiang Province,China)

    Objective To express human fibroblast growth factor-21(hFGF-21)protein with optimized codon in P.pastoris and lay a foundation of study on role of hFGF-21 in therapy of diabetes mellitus.Methods Human FGF-21 gene was synthesized by optimization of codon and inserted into vector pPICZαA,and the constructed recombinant plasmid pPICZαA-hFGF-21 was transformed to P.pastoris GS115 by electrotransformation.Recombinant P.pastoris strain was screened by dot immunoblot for expression under induction of methanol.The expressed product was identified by SDS-PAGE and Western blot,and the condition for induction was optimized.Results PCR,restriction analysis and sequencing proved that recombinant expression vector pPICZαA-hFGF-21 was constructed correctly.Five positive clones of recombinant P.pastoris strains were screened.SDS-PAGE showed that the target protein with a relative molecular mass of 25 000 was expressed in supernatant of recombinant P.pastoris after induction,and the expression level reached 6 μg /L.Western blot showed good reactogenicity of expressed protein.The expression level of target protein reached a peak value after induction of recombinant P.pastoris at pH 4.0 for 96 h.However,methanol concentration showed no significant effect on expression level.Conclusion A eukaryotic expression vector for hFGF-21 with optimized codon was constructed successfully,and hFGF-21 was expressed in a secretory form in P.pastoris GS115.

    2010 01 v.23 [Abstract][OnlineView][Download 643K]

  • Eukaryotic Expression Vectors for mp65 and sap2 Genes Protects Mice against Systemic Candida albicans Infection

    YIN Xiao-lin,LI Bo,ZHAI Xue-qiong,et al(Department of Immunology,Hebei Medical University,Shijiazhuang 050017,China)

    Objective To investigate the protective effect of eukaryotic expression vectors for mp65 and sap2 genes on systemic Candida albicans infection in mice.Methods BALB /c mice were immunized with plasmids pcDNA3.1-mp65 and pcDNA3.1sap2 separately or in combination.The specific IgGs against MP65 and Sap2 proteins in peripheral blood of immunized mice were determined by ELISA,and the percentages of CD4+ and CD8+ T lymphocytes in spleens by flow cytometry.The survival rate of immunized mice after challenge with Candida albicans was observed.Results The specific IgGs against MP65 and Sap2 proteins were detected in peripheral blood of immunized mice.Both the plasmids increased the percentage of CD4+ T lymphocytes in spleen and the survival rate of mice after systemic infection with Candida albicans.Conclusion Plasmids pcDNA3.1-mp65 and pcDNA3.1-sap2 induced humoral immune response and CD4+ T lymphocyte-mediated cellular immune response in mice,and showed a certain protective effect against systemic infection with Candida albicans.

    2010 01 v.23 [Abstract][OnlineView][Download 240K]

  • Induction of Immune Response and Protection against Toxoplasma gondii Infection by Recombinant T.gondii Peroxiredoxin Protein in Mice

    WU Kai,HU Jian-min,YIN Guo-rong,et al (Department of Parasitology,Shanxi Medical University,Taiyuan 030001,China)

    Objective To observe the humoral and cellular immune responses as well as protection against Toxoplasma gondii infection induced by recombinant T.gondii peroxiredoxin protein (rTgPrxz)at various dosages in mice.Methods Sixty BALB /c mice were divided into five groups randomly,and injected s.c.with 20,40,60,80 μg of rTgPrx(dissolved in 100 μl of PBS)and 100 μl of PBS respectively for 3 times at intervals of 2 weeks.Each mouse was challenged with 1 × 104 tachyzoites by intragastric route on day 14 after the last immunization and observed for health daily.All the mice were killed on day 30 after challenge and determined for IgG in sera and sIgA in intestine washes.The splenic lymphocytes and intestinal intraepithelial lymphocytes (IELs)of mice were isolated and counted.Meanwhile,the tachyzoites in livers and brains of mice were counted.Results The serum IgG levels of mice injected with 60 μg of rTgPrx were significantly higher than those with 20 and 40 μg of rTgPrx and with PBS.However,no significant differences were observed in the sIgA levels in intestine washes of mice in various groups.The counts of splenic lymphocytes were significantly higher in 80 μg of rTgPrx group than in PBS and 20 μg of rTgPrx groups,while those of IELs showed no significant difference in various groups.The counts of tachyzoites in livers and brains of mice in 60 and 80 μg of rTgPrx groups were significantly lower than those in PBS and 20 μg of rTgPrx groups.Conclusion Immunization with 60 and 80 μg of rTgPrx by subcutaneous injection induced effective humoral and cellular immune responses as well as protection against Toxoplasma gondii infection in mice.

    2010 01 v.23 [Abstract][OnlineView][Download 252K]

  • Effect of Celecoxib on Proliferation,Invasion,VEGF Expression and Radiosensitivity of Nasopharyngeal Carcinoma HNE-1 Cell Line

    CHEN Jiong-yu,HONG Chao-qun,WU Xiao,et al(Cancer Hospital,Medical College,Shantou University,Shantou 515041,Guangdong Province,China)

    Objective To investigate the effect of Celecoxib,a selective inhibitor of cyclooxygenase-2(COX-2),on the proliferation,invasion,vascular endothelial growth factor (VEGF)expression and radiosensitivity of nasopharyngeal carcinoma HNE-1 cell line.Methods HNE-1 cells were treated with Celecoxib at various concentrations and determined for proliferation by MTT method,for invasion and metastasis by cell invasion test,for VEGF mRNA transcription level by RT-PCR,for VEGF expression level by ELISA,and for radiosensitivity by clone formation test.Results The Celecoxib inhibited the proliferation and invasion of HNE-1 cells and down-regulated the VEGF mRNA transcription and VEGF protein expression significantly,both in dose-dependent modes.The invasion abilities and VEGF expression levels of HNE-1 cells treated with Celecoxib at various concentrations showed significant difference.Clone formation test showed significantly synergistic anti-tumor effect of Celecoxib at a dosage of 125 μmol /L and radiation therapy.Conclusion Celecoxib significantly inhibited the proliferation,invasion and VEGF expression,and increased the radiosensitivity of HNE-1 cells.

    2010 01 v.23 [Abstract][OnlineView][Download 833K]

  • Immune Response Induced by Human Anti-idiotypic Antibody G22 ScFv against Nasopharyngeal Cancer

    WANG Jing,LI Guan-cheng,TONG Yong-qing,et al (Cancer Research Institute of Central South University,Changsha 410078,China)

    Objective To observe the immune response induced by human anti-idiotypic antibody G22 ScFv against nasopharyngeal cancer in mice and investigate the feasibility of G22 ScFv as a nasopharyngeal cancer vaccine.Methods G22 ScFv was expressed in E.coli,then purified and used for immunization of BALB /c mice.The specific humoral immune responses of mice were determined by indirect ELISA and competitive inhibition ELISA,and the splenic T lymphocyte phenotypes by flow cytometry.Results The expressed fusion protein reached a purity of more than 95% after purification and showed good reactogenicity.ELISA proved that G22 ScFv induced Ab3 and increased the counts of CD4+ and CD8+ T lymphocytes in spleens of mice.Conclusion G22 ScFv induced both humoral and cellular immune responses,which laid a foundation of clinical application of human anti-idiotypic antibody as vaccine against nasopharyngeal cancer.

    2010 01 v.23 [Abstract][OnlineView][Download 511K]

  • Analysis of Antigenicity of Polypeptides at N-and C-terminus of ORF2 of Hepatitis E Virus

    MA Hong-xia△,SONG Xiao-guo,HUANG Wei-jin,et al (△College of Life Sciences,Jilin University,Changchun 130012,China)

    Objective To analyze the antigenicity of polypeptides at N-and C-terminus of ORF2 of hepatitis E virus(HEV).Methods Microtiter plate was coated with the polypeptide pS4-1 at N-terminus and the polypeptide pS4-6 at C-terminus of type 4 HEV ORF2 respectively and used for determination of anti-HEV IgG in a series of serum samples of monkeys infected with HEV by EIA,based on which the antigenicity of the two polypeptides were analyzed,and the determination results were compared with those by imported kit.Results In a series of serum samples of monkeys infected with HEV,the IgG against antigen at N-terminus of HEV ORF2 appeared early and last for a short time,and its peak value appeared almost at the same time with that of ALT.However,the IgG titer against antigen at C-terminus of HEV ORF2 was low at early stage of infection,while increased gradually and showed almost no decreasing tendency during the observation period of 14 weeks.Both the sensitivities of HEV IgG kits prepared with polypeptides pS4-1 and pS4-6 were higher than those of imported kit.Conclusion Both pS4-1 and pS4-6 showed good antigenicity,while showed different reactogenicity at various stages of HEV infection.

    2010 01 v.23 [Abstract][OnlineView][Download 705K]

  • Safety and Immunogenicity of Domestic Influenza A H1N1 Virus Cleavage Vaccine

    ZHUANG Mao-xin △,PAN Hong-xing,YAO Gen-hong,et al (△Changchun Institute of Biological Products,Changchun 130062,China)

    Objective To observe the safety and immunogenicity of domestic influenza A H1N1 virus cleavage vaccine.Methods A clinical trial was performed according to a random,control and blind principle.Each of the aged,adolescent and children groups consisted of 220 subjects of whom 110 were inoculated with 15 μg of influenza A H1N1 virus cleavage vaccine and the other 110 with 30 μg,for 2 times by a schedule of 0 and 21 d.Adult group consisted of 330 subjects of whom 110 were inoculated with 15 μg of influenza A H1N1 virus cleavage vaccine,110 with 30 μg,and 110 with placebo,for 2 times by a schedule of 0 and 21 d.Another 220 adults were inoculated with 15 and 30 μg of influenza A H1N1 virus cleavage vaccine,110 for each,by a schedule of 0 and 28 d.The total,systemic and local adverse rates,HI antibody positive conversion rates,protective rates as well as GMT level and increasing folds of HI antibody in various groups after inoculation were observed.Results The total adverse reaction rate of the 1210 subjects was 21.8%,most of which were of degree 1,no adverse reactions of degree 3 or above,other abnormal reactions or severe adverse events were observed.The HI antibody positive conversion rate and protective rate induced by 30 μg of vaccine showed no significant difference with those induced by 15 μg.No significant differences were observed in the HI antibody positive conversion rate,protective rate,GMT levels and increasing folds after inoculation with the first dose of vaccine at the same dosage by different schedule.Conclusion Domestic influenza A H1N1 virus cleavage vaccine showed good safety and immunogenicity.The inoculation with one dose of 15 μg vaccine by a schedule of 0 and 21 d induced satisfactory immune effect in the population aged 12 ～ 60 years.

    2010 01 v.23 [Abstract][OnlineView][Download 178K]

  • Relationship between Angiontensin Ⅱ Type 2 Receptor Gene Polymorphism and Essential Hypertension in Han Women in Jining Region,Shandong Province,China

    LI Chuan-fang△,GAO Dong-sheng(△School of Medicine,Shandong University,Jinan 250012,China)

    Objective To investigate the relationship between the polymorphism of angiontensin Ⅱ type 2 receptor(AT2R) gene A1675G and essential hypertension (EH)in Han women in Jining Region,Shandong Province,China.Methods The genomic DNA were extracted from the peripheral blood of 127 female patients with EH and 119 healthy women in Jining Region,from which A1675 genes were amplified by PCR and tested for polymorphism by high resolution melting(HRM)method.Results The frequencies of AA,AG and GG genotypes in female patients with EH were 26.7%,53.5% and 18.8%,while those in healthy women were 27.7%,42.0% and 30.3%,respectively,which showed no significant difference.However,the frequencies of allelic genes A and G were 53.5% and 46.5% in the patients with EH and 48.7% and 51.3% in healthy women,respectively,which showed no significant difference.Conclusion The polymorphism of AT2R gene A1675G showed no significant relationship to EH in Han women in Jining Region.

    2010 01 v.23 [Abstract][OnlineView][Download 295K]

  • Identification of Cross Contamination of Human Cell Lines by STR Profiling

    WU Yu,FENG Jian-ping,LIN Lin,et al(National Institute for the Control of Pharmaceutical and Biological Products,Beijing 100050,China)

    Objective To identify the cross contamination of human cell lines by STR profiling.Methods The 16 STR loci of human,monkey and mouse cell lines as well as cross contamination model of human cell line were determined by PCR-capillary electrophoresis to evaluate the feasibility of the method to identification of cell cross contamination.Twenty-seven human cell lines used for production of biologics and 5 cell lines used for production of tissue engineering products,from various manufactures,were analyzed by the developed STR profiling method.Results Human cell lines showed characteristic STR profiles,while monkey and mouse cell lines showed no such profiles.The developed STR profiling method could be used for authentication of cross contamination models of human cell lines with morphological difference or similarity.No cross contamination was found in 27 human cell lines for production of biologics.However,2 cell lines used for production of tissue engineering products were identified as mixed cells.Conclusion STP profiling showed high specificity and could be used for authentication of cross contamination between human cell lines or origin of cell individual.

    2010 01 v.23 [Abstract][OnlineView][Download 896K]

  • Modification of Neutral Protease with Monomethoxypolyethylene Glycol

    ZHANG Chen△,ZHANG Ying,GUO Feng,et al (△School of Life Sciences,Anhui Agriculture University,Hefei 230036,China)

    Objective To modify neutral protease with monomethoxypolyethylene glycol(mPEG)and study the zymological property of modified protease.Methods Neutral protease was modified chemically by mPEG5000 activated with cyanuric chloride,and the infrared spectrums and zymological properties of prepared mPEG-neutral protease before and after modification were compared.Results The modification rate of neutral protease was 41.7%.Significant changes were observed in several characteristic peaks of modified neutral protease on infrared spectrum.The optimal temperature and pH value of the modified protease showed no significant changes,while the thermal stability increased significantly.Conclusion The temperature and pH value for modification of neutral protease with mPEG were optimized,and the modified protease showed high thermal stability compared with natural protease.

    2010 01 v.23 [Abstract][OnlineView][Download 422K]

  • Analysis of Non-reduced SDS-PAGE Purity of Recombinant Monoclonal Antibody

    CHENG Li-jun,WEI Jing-shuang,ZHANG Shi-xiong,et al (New Drug R&D Center,North China Pharmaceutical Corporation,Shijiazhuang 050015,China)

    Objective To analyze the non-reduced SDS-PAGE purity of recombinant monoclonal antibody(McAb)and exclude the artifacts formed during preparation of test samples.Methods The condition for routine SDS-PAGE was modified based on the structural characters of recombinant McAb molecules.The recombinant McAb samples were treated with various loading buffers under various conditions for SDS-PAGE,and the results were compared.Results Almost all the minor bands appeared during determination of purity of McAb by non-reduced SDS-PAGE were eliminated by addition of alkylation reagent into test sample buffer to block the free sufhydryl groups.The minor bands with high relative molecular mass could be diminished effectively by decreasing the ambient temperature during non-reduced SDS-PAGE.Conclusion The artifacts formed during preparation of test samples due to the disulfide bonds in McAb may be eliminated by modification of condition for non-reduced SDS-PAGE.

    2010 01 v.23 [Abstract][OnlineView][Download 399K]

  • Development of A Quantitative ELISA Method for EV71 Antigen

    JIA Hui△,CAI Fang,GAO Qiang,et al(△College of Life Sciences,Kunming University of Science and Technology,Kunming 650224,China)

    Objective To develop a double antibody sandwich ELISA method for quantitative determination of EV71 antigen during production of inactivated EV71 vaccine.Methods Monoclonal and polyclonal antibodies against EV71 antigen were prepared by immunizing rabbits with complete EV71.A double antibody sandwich ELISA method was developed using polyclonal antibody as coating antibody and HRP-labeled monoclonal antibody as enzyme-labeled antibody and used for determination of EV71 antigen content in test samples,then verified and applied.Results The linear range and R2 value of the developed ELISA method were 5 ～ 80 U /ml and 0.994 respectively,indicating good linearity.The quantitation limit of the developed method was 5 U /ml.The recovery rate of internal reference of EV71 antigen at various concentrations was 88% ～ 119.0%,with a variation coefficient of less than 15%.No cross reaction with harvested hepatitis A virus liquid,inactivated Japanese encephalitis virus liquid,influenza H5N1 virus cleavage vaccine,calf serum,human albumin,Vero cells,MEM or harvested CoxA16 liquid was observeed.The determination result of EV71 antigen content in test samples of bulk of inactivated EV71 vaccine during purification reflected the tendency of antigen purification.The developed method showed high accuracy in determination of antigen content in dissociated final product of EV71 vaccine adsorbed onto aluminium salt.Conclusion A double antibody sandwich ELISA method for quantitative determination of EV71 antigen was developed,which showed good linearity and high specificity,sensitivity,accuracy and reproducibility and might be used for quantitative determination of antigen in EV71 vaccine during production and in final product.

    2010 01 v.23 [Abstract][OnlineView][Download 328K]

  • Development of Nucleic Acid Probe Method for Determination of Staphylococcus aureus

    XUE Li-gang△,LIU Jin-hua,WANG Quan-kai,et al (△College of Animal Science and Technology,Jilin Agriculture University,Changchun 130118,China)

    Objective To develop a nucleic acid probe method for rapid determination of Staphylococcus aureus in foods.Methods Staphylococcus aureus was determined by actinide ester (AE)-labeled specific DNA probe,based on which the maximum and minimum concentrations of probe were defined,and the specificity and sensitivity of the developed method were verified and compared with those of traditional national standard method.Results The maximum and minimum concentrations of probe for the developed method were 2 and 0.25 pmol∕50 μl respectively.The developed method showed high specificity,and the minimum detection limit of bacterial colony in pure culture was 106 cfu∕ml.The determination result by the developed method was highly consistent with that by national standard method.Conclusion The developed nucleic acid probe method may be used for the rapid determination of Staphylococcus aureus in foods.

    2010 01 v.23 [Abstract][OnlineView][Download 162K]

  • Preparation of Heparolysate and Verification of Procedure for Inactivation /Removal of Virus

    GU Zheng-ping,WANG Bo-chu,KUANG Yu(Bioengineering College of Chongqing University,Chongqing 400044,China)

    Objective To prepare heparolysate and verify its virus inactivation /removal procedure.Methods The Newcastle disease virusn(NDV)and infectious bursal disease virus(IBDV)as indicators were inactivated by boiling at 100℃,pH 6.0 ～ 7.0 for 20 min,then removed by ultrafiltration using hollow fiber column with a cut-off relative molecular mass of 10 000 at feeding pressure of 0.15 MPa,retention pressure of 0.05 MPa,and at feeding pressure of 0.20 MPa,retention pressure of 0.10 MPa,3 cycles for each.The virus inactivation /removal efficacies were verified by using SPF chick embryo and chick fibroblast.Results No indicator viruses were detected after boiling.The decreases of titers of NDV and IBDV were not less than 7.4 logEID50 /0.2 ml and not less than 5.45 log CCID50 /0.1 ml respectively.No IBDV was detected after ultrafiltration,and its titer decreased by not less than 5.43 logCCID50 /0.1 ml.After blind passage and recovery to normal,no virus was detected in chick embryos.The polypeptide content in prepared heparolysate was more than 20 mg /ml.Conclusion Both the procedures for virus inactivation /removal during preparation of heparolysate were effective,and the quality of obtained heparolysate was stable.

    2010 01 v.23 [Abstract][OnlineView][Download 126K]

  • Safeties of PICKCa as An Adjuvant and Rabies Vaccine Using The Adjuvant

    LIN Hai-xiang△,YU Yong-xin(△Beijing Yishengxingye Science and Technology Co.Ltd,Beijing 102600,China)

    As a conjugate of poly I,poly C,kanamycin(K)and calcium chloride(Ca),PICKCa is a safe and effective interferon inducer.The safety of rabies vaccine prepared using PICKCa as an adjuvant met the requirements in Chinese Pharmacopoeia,and its toxicity as well as antigen and preservative contents were lower than those of the current rabies vaccine for human use.The safeties of PICKCa as an adjuvant and rabies vaccine using the said adjuvant are reviewed in this paper.

    2010 01 v.23 [Abstract][OnlineView][Download 104K]

  • Progress in Research on Reassortment of Influenza Virus

    SHEN Juan,CHEN Yuan-ding (Institute of Medical Biology,Chinese Academy of Medical Sciences and Peking Union Medical College,Kunming 650118,China)

    Influenza virus is an enveloped single negative stranded RNA virus with segmental genome.The RNA fragments of influenza virus are reassorted frequently during natural infection,resulting in rapid variation of virus antigenicity especially those of surface glycoprotein HA and NA,which makes the virus escape the immune monitoring,causes infection and pathogenesis and brings difficult to prevention of influenza by vaccination.Based on the character of natural reassortment (i.e.traditional reassortment)of virus gene fragments or reverse genetic technique,the influenza virus strains with reassortment of natural genes or test genes were screened to obtain attenuated strains with high titer,immunogenicity and immune protection,which are of important social and economic significances in controlling the outbreak and epidemic of influenza in humans or animals.The progresses in research on traditional and reverse genetic methods for reassortment of influenza virus as well as their applications are reviewed in this paper.

    2010 01 v.23 [Abstract][OnlineView][Download 132K]

  • Progress in Research on GnRH-Ⅰ Vaccine

    FANG Fu-gui,ZHANG Xiao-rong (College of Animal Science and Technology,Anhui Agricultural University,Hefei 230036,China)

    As an immunological preparation with function of regulating breeding,gonadotropin releasing hormone-Ⅰ(GnRH-Ⅰ) vaccine is widely used and has an unexampled superiority in castration and contraception in animals and immunotherapy in humans.GnRH-Ⅰ vaccine includes chemical synthetic peptide vaccine,gene engineering vaccine and DNA vaccine,of which DNA vaccine is the focus of current study.This paper reviews the progress in research on above-mentioned vaccines.

    2010 01 v.23 [Abstract][OnlineView][Download 139K]

  • Advance in Research on Production of Edible Transgenic Plant Vaccine by Using Plant Bioreactor

    QU Jing △,WANG Pi-wu,FAN Yu-guang (△College of Life Science,Jilin Agricultural University,Changchun 130118,China)

    Large-scale production of edible vaccine using transgenic plant as a bioreactor is a novel field of research on life science.Along with the development of biotechnique,plant bioreactor will become an effective route to vaccine production.This paper describes the advantage,immune mechanism and current state of edible transgenic plant vaccine as well as the problems in research.

    2010 01 v.23 [Abstract][OnlineView][Download 126K]


@2012 Editorial Department of "Chinese Journal of Biologicals"