2012年04期目次

  • Prokaryotic expression and purification of attachment protein fragment of human respiratory syncytial virus

    ZHU Chuan-feng,FU Sheng-fang,KOU Gui-ying,CHEN Han-quan,YU Li,ZHOU Xu(Lanzhou Institute of Biological Products Co.Ltd.,Lanzhou 730046,China)

    Objective To express the attachment protein(G) fragment of human respiratory syncytial virus(HRSV) in prokaryotic cells and purify the expressed product.Methods AA75-225 fragment of G gene was amplified from HRSV Lanzhou strain(subtype A) by PCR and cloned into prokaryotic expression vector pET-42b(+).The constructed recombinant plasmid pET-42b-G was transformed to E.coli BL21(DE3) and induced with IPTG.The expressed product was purified by nickel ion affinity chromatography,and identified by SDS-PAGE and Western blot.Results DNA fragment at a length of 453 bp was amplified by PCR.Restriction analysis and sequencing proved that recombinant plasmid pET-42b-G was constructed correctly.The expressed recombinant protein,with a relative molecular mass of 50 230,contained 25% of total somatic protein and existed in both inclusion body and soluble forms.The purified recombinant protein reached a purity of more than 95%,and showed specific binding to anti-RSV-G monoclonal antibody.Conclusion The G fragment of HRSV Lanzhou strain was successfully expressed in E.coli BL21(DE3),which provided a material for further preparation of RSV vaccine,the serological diagnosis of RSV infection and the development of diagnostic kit.

    2012 04 v.25 [Abstract][OnlineView][Download 187K]

  • Prokaryotic expression,purification and antigenic characters of heat shock protein ClpL of Streptococcus pneumoniae

    ZHONG Wen,ZHANG Qun,GONG Yi,ZHANG Yan-qing,CHEN Qian,CHEN Te,DONG Jie,WANG Hong,ZHANG Xue-mei(Key Laboratory of Medical Diagnostics of Ministry of Education,Department of Laboratory Medicine,Chongqing Medical University,Chongqing 400016,China)

    Objective To express heat shock protein ClpL of Streptococcus pneumoniae(S.pn) in prokaryotic cells,and evaluate the feasibility of expressed product as a candidate protein of S.pn vaccine.Methods Full-length ClpL gene of S.pn D39 was amplified by PCR and cloned into prokaryotic expression vector pET-28a(+).The constructed recombinant plasmid pET-28a(+)-ClpL was transformed to E.coli BL21(DE3) and induced with IPTG.The expressed product was purified by Ni-NTA affinity chromatography,with which BALB/c mice were immunized and determined for titer and subtype of induced polyclonal antibody against ClpL.The ClpL protein was analyzed for conservativeness in five common S.pn strains by Western blot,and for subcellular location in S.pn by flow cytometry and Western blot.Results Restriction analysis and sequencing proved that recombinant plasmid pET-28a(+)-ClpL was constructed correctly.ClpL protein was expressed in a soluble form in E.coli BL21(DE3),reached a purity of 90% and a concentration of 4.97 mg/ml respectively after purification,and induced highly specific IgG with high titer in mice,most of which were of subtypes IgG1 and IgG2b.ClpL was expressed in all the five common S.pn strains.However,ClpL was a secretory protein not expressed on surface of S.pn.Conclusion The ClpL protein of S.pn was expressed in prokaryotic cells and purified,which was conserved in S.pn and might be used as a satisfactory candidate protein of S.pn vaccine.

    2012 04 v.25 [Abstract][OnlineView][Download 270K]

  • A primary evaluation on effect of combined immunization with inactivated enterovirus 71 vaccine and inactivated hepatitis A virus vaccine

    YANG Ting,LI Hua,LONG Run-xiang,YANG Rong,DONG Cheng-hong,JIANG Rui-ju,CUI Ping-fang,BAI Hui-zhu,XIE Zhong-ping(Institute of Medical Biology,Chinese Academy of Medical Science & Peking Union Medical College,Yunnan Provincial Key Laboratory for Development of Vaccines against Major Infections Diseases,Kunming 650118,China)

    Objective To primarily evaluate the specific immune response level induced in mice by combined immunization with inactivated enterovirus 71(EV71) vaccine and inactivated hepatitis A virus(HAV) vaccine as well as the interaction of EV71 and HAV antigens.Methods Combined vaccine was prepared by formulation of inactivated HAV and inactivated EV71 vaccines at various dosage ratios,with which ICR mice were immunized and determined for serum anti-HAV titer by ELISA,for serum neutralizing antibody titer against EV71 by virus fixation-serum dilution method,for percentage of lymphocyte subsets CD4+/CD8+ in spleen by flow cytometry,and for IL-4 and IFNγ levels secreted by lymphocytes in spleen by ELISPOT.Results All the positive rates of neutralizing antibody against EV71 in sera of mice in various groups reached 100% 28 d after primary immunization and 7 d after booster immunization,while the neutralizing antibody titers were dose-dependent.Protective HAV antibody was induced in sera of mice 28 d after primary immunization,which increased after booster immunization.The antibody levels induced by combined vaccine showed no significant difference with those by the corresponding monovalent vaccine(P > 0.05),and no interaction between EV71 and HAV was found.The percentages of CD8+ cells in spleens of mice in various groups were significantly higher 28 d after primary immunization(P < 0.001),while was significantly lower 7 d after booster immunization,than those of CD4+ cells(P < 0.001).Neither whole HAV nor EV71 polypeptide stimulated the secretion of cytokines by splenocytes of mice in various groups 28 d after primary immunization,however,7 d after booster immunization,the IL-4 levels secreted by splenocytes of mice in various groups were significantly higher than those of IFNγ(P < 0.05).Conclusion Combined immunization with HAV and EV71 vaccines at certain dosages induced high humoral immune response and relatively low cellular immune response.However,no interference or synergism was observed between the two vaccine antigens.

    2012 04 v.25 [Abstract][OnlineView][Download 230K]

  • Effect of Dermatophagoides farinae extract at various dosages on CD4+CD25+ regulatory T cells of mice with asthma

    WU Xue-jun,HUANG Ying,HAN Jie,WANG Mo-kui,WANG Ying,WANG Li-jia(Children's Hospital of Chongqing Medical University,Key Laboratory of Developmental Disease in Childhood,Ministry of Education,Chongqing 400014,China)

    Objective To investigate the effect of Dermatophagoides farinae(Derf) extract at various dosages on CD4+CD25+ regulatory T cells(Tregs) of mice with asthma and optimize the maintenance dosage of the extract for immune therapy.Methods Mouse model of anaphylactic asthma was established by injecting i.p.C57BL/6 mice with the extract of Derf.Thirty-two model mice were divided into one control and three test groups.The mice in test groups 1,2 and 3 were injected i.p.with Der f at low(100 μg/mouse),moderate(1 mg/mouse) and high(2 mg/mouse) dosages respectively,while those in control groups with physiological saline.The contents of CD4+CD25+ T cells and Foxp3+CD4+CD25+ Tregs in splenocytes of mice were determined by flow cytometry,while the IL-10 and TGF-β1 levels in sera by ELISA.Results The percentages of CD4+CD25+T cells in CD4+T cells and the percentages of Foxp3+CD4+CD25+Tregs in CD4+CD25+ T cells as well as serum IL-10 and TGF-β1 levels were significantly lower in control group and test group 1 than in test groups 2 and 3(each P < 0.05),while showed no significant difference in test groups 2 and 3(each P > 0.05).Conclusion The optimal maintenance dosage of Der f extract for specific immune therapy of asthma in mice was 1 mg/mouse.

    2012 04 v.25 [Abstract][OnlineView][Download 228K]

  • Effect of NKR-P1+ cells on synergism of interleukin-12 and interleukin-18 in pathogenesis of experimental autoimmune neuritis

    LI Hu-lun,YAN Bin-bin,LI Guo-zhong,LIU Xi-jun,WANG Guang-you,XU Xin,SUN Bo,CAO Jing-yan,WANG Dan-dan,WANG Shu-chen,JIN Lian-hong(Department of Neurobiology,Harbin Medical University,Heilongjiang Provincial Key Laboratory of Neurobiology,Harbin 150081,China)

    Objective To investigate the role of synergism of interleukin(IL)-12 and IL-18 in pathogenesis of experimental autoimmune neuritis(EAN) as well as the effect of NKR-P1+ cells on the synergism.Methods Rat model of EAN was established by immunization of Lewis rats with 200 μl of immune emulsion containing 230 μg of specific peptide P2 53-78,2 mg of tuberculin,100 μl of PBS and 100 μl of incomplete Freund adjuvant.The lymph nodes of rats at peak incidence were isolated,from which monocyte suspension was prepared and,in presence or absence of NKR-P1 + cells,co-cultured with IL-12(20 ng/ml),IL-18(25 ng/ml) and IL-12(20 ng/ml) + IL-18(25 ng/ml) respectively.The monocyte suspension in each group was stimulated with 20 μg/ml P2 53-78 for 24 h,and transferred to normal Lewis rats at a dosage of 1 × 107 live monocytes per rat.The rats were observed for signs of disease daily and subjected to clinical score,of which the sciatic nerves were subjected to histopathological examination.The secretion levels of IFNγ,TNF-α and IL-4 in culture supernatant of monocytes were determined by ELISA,while the proliferative reaction of P2 53-78 specific lymphocytes by lymphocyte proliferation test.Results P2 53-78 specific lymphocytes stimulated with IL-12 or IL-18 alone caused moderate EAN,while those co-stimulated with IL-12 and IL-18 caused aggressive EAN.However,the lymphocytes with NKR-P1+ cells deleted caused severe EAN and,after co-culture with IL-12 + IL-18,induced low IFNγ level and inhibited Th1 differentiation,of which the auto-reactivity was inhibited.Conclusion The co-stimulation with IL-12 and IL-18 enhanced the anto-reactivity of P2 53-78 specific lymphocytes,induced high levels of IFNγ and promoted the differentiation of Th1 cells partially through NKR-P1 + cells.

    2012 04 v.25 [Abstract][OnlineView][Download 331K]

  • Effect of p115 gene silence on migration and invasion abilities of human gastric cancer BGC-823 cells and relevant mechanism

    YAN Tian-jing,YI Yong-fen,DENG Wei,WEN Xue,QU Yu-ling(Department of Pathology,Molecular Medicine and Tumor Research Center,Chongqing Medical University,Chongqing 400016,China)

    Objective To investigate the effect of Golgi-vesicular transport protein p115 gene silence on migration and invasion abilities of human gastric cancer BGC-823 cells as well as the relevant mechanism.Methods BGC-823 cells transfected with plasmid pGPU6/GFP/Neo/p115(p115 shRNA) and negative control plasmid(shNC) in mediation of liposome respectively,using those untransfected as blank control.Forty-eight hours after transfection,the expressions of p115,macrophage migration inhibitory factor(MIF),matrix metalloproteinase-2(MMP-2) and MMP-9 genes and proteins in various groups were determined by RT-PCR,Western blot and IFA,while the invasion ability of cells by wound healing test and Transwell test.Results Compared with those in shNC and blank control groups,the expression levels of p115,MIF,MMP-2 and MMP-9 gene and protein as well as the healing ability and the number of transmembrane cells in p115 shRNA group decreased significantly(each P < 0.01).Conclusion Inhibition of P115 expression in BGC-823 cells inhibited the invasion ability of tumor cells by down-regulating the expressions of MIF,MMP-2 and MMP-9 genes.

    2012 04 v.25 [Abstract][OnlineView][Download 442K]

  • Effect of COL1A1 gene interference on proliferation of human colon cancer HCT-8 cells

    LI Peng,MA Yan-jiao,LI Jian-hua,GONG Peng-tao,YANG Ju,LI He,OUYANG Hong-sheng,ZHANG Xi-chen(Institute of Animal Science and Technology,Heilongjiang Bayi Agriculture University,Daqing 163319,Heilongjiang Province,China)

    Objective To investigate the effect of COL1A1 gene interference on proliferation of human colon cancer HCT-8 cells.Methods HCT-8 cells were transiently transfected with COL1A1 RNA interfering plasmid pGPU6/GFP/Neo-COL1A1(RNAi COL1A1) and negative control plasmid pGPU6/GFP/Neo-shNC(PGNsN) respectively,using those untransfected as blank control.The transcription levels of COL1A1 mRNA in HCT-8 cells 48 ～ 72 h after transfection were determined by semi-quantitative RT-PCR,while the proliferation activity of HCT-8 cells by MTT method.The HCT-8 cells in various groups were inoculated into the epithelial tissue of BALB/c mice,and the tumor sizes were measured every 6 d.Results The transcription level of COL1A1 mRNA in HCT-8 cells of RNAi COL1A1 group was significantly lower than PGNsN and blank control groups(P < 0.01).The proliferation activity of cells in RNAi COL1A1 was lower than those in PGNsN and blank control group,which showed significant difference on days 4 ～ 7(P < 0.05 on days 4 ～ 7 and P < 0.01 on days 5 and 6).Compared with those in PGNsN and blank control groups,the tumor sizes of mice in RNAi COL1A1 group showed no significant difference within 18 d(P > 0.05),while were significantly small more than 24 d after inoculation(P < 0.05 on day 24 and P < 0.01 on day 30).Conclusion COL1A1 after interference inhibited the proliferation of HCT-8 cells,which provided an experimental basis for serving COL1A1 as a candidate gene for therapy of cancer.

    2012 04 v.25 [Abstract][OnlineView][Download 221K]

  • Increase of expression level of human proinsulin in Pichia pastoris by double promoter co-expression system

    LI Hong-liang,CHEN Yong,CHEN Hai-rong,HUANG Cheng,FENG Yi-jian,MEI Xiang,HE Yue,FAN Kai(School of Pharmacy and Bioengineering,Chongqing University of Technology,Chongqing 400054,China)

    Objective To increase the expression level of human proinsulin in Pichia pastoris by double promoter(AOX1 and GAP) co-expression system.Methods Plasmid pMD18-T-HuPIDesB30 was digested with EcoRⅠand NotⅠ,and the cDNA fragment of human mini-proinsulin des B30(HMPIDesB30) containing short C peptide AAK,in which the Thr(T) gene at site 30 of B chain was deleted,was recovered and inserted into vector pGAPZαA.The constructed recombinant plasmid pGAPZαA-HMPIDesB30 was transformed to proinsulin-secreting FJ-H1 strain integrated by another recombinant plasmid pPIC9K-HMPIDesB30,i.e.pPIC9K-HMPIDesB30/GS115,based on which the strain for high expression of proinsulin was screened with antibiotic and subjected to fermentation test.Results Restriction analysis and sequencing proved that recombinant plasmid pGAPZαA-HMPIDesB30 was constructed correctly.FJ-H3 strain for high expression of proinsulin was obtained by screening with Zeocin.The expression level of proinsulin in FJ-H3 strain fermented in 30 L fermenter reached 1.6 g/L,which was 1.6 times higher than that in FJ-H1 strain and 5.3 times higher than that in FJ-H2 strain(pGAPZαA-HMPIDesB30/GS115).Mass spectrometry showed that the relative molecular mass of target protein digested with trypsin was consistent with the theoretical value.Conclusion HMPIDesB30 was highly expressed in P.pastoris by using AOX1 and GAP double promoters.

    2012 04 v.25 [Abstract][OnlineView][Download 233K]

  • Prokaryotic expression of intracellular pathogen resistance 1 gene

    SHE Qian,XU Lei,HE Yong-lin,JIN Zhi-dong,ZHANG Dan,YANG Chun(Department of Pathogen Biology,Chongqing Medical University,Chongqing 400016,China)

    Objective To construct a prokaryotic expression for intracellular pathogen resistance 1(Ipr1) gene and express in E.coli.Methods Ipr1 gene was amplified by PCR using plasmid pMD19-T simple-Ipr1 as a template and cloned into vector pET-32a(+).The constructed recombinant plasmid pET-32a(+)-Ipr1 was transformed to E.coli BL21(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(+)-Ipr1 was constructed correctly.The relative molecular mass of expressed recombinant protein was about 70 000.The expression level reached the maximum after induction with 1.5 mmol/L IPTG for 4 h,which was about 16% of total somatic protein.Recombinant Ipr1 protein showed specific binding to monoclonal antibody with His tag.Conclusion The prokaryotic expression vector for Ipr1 gene was successfully constructed,which laid a foundation of further study on function of Ipr1 protein and construction of recombinant BCG with Ipr1.

    2012 04 v.25 [Abstract][OnlineView][Download 189K]

  • Effect of over-expression of BRIT1 gene on apoptosis of cervical cancer HeLa cells

    HU Ren-zhi,SONG Fang-zhou,YUAN Cheng-fu,GOU Xiao-yan,BU You-quan,YI Fa-ping,LIU Ge-li,JI Ying(Research Center of Molecular Medicine and Tumor,Chongqing Medical University,Chongqing 400016,China)

    Objective To investigate the effect of over-expression of BRIT1 gene on apoptosis of cervical cancer HeLa cells.Methods Eukaryotic expression plasmid pcDNA3.1(-)/BRIT1 was identified by restriction analysis and sequencing and transfected to HeLa cells,using the cells transfected with empty plasmid and those untransfected as control.Forty-eight hours after transfection,the cells were determined for transcription and expression of BRIT1 mRNA by RT-PCR and real-time PCR,for expression of BRIT1 protein by Western blot,and for apoptosis by flow cytometry.Results Restriction analysis and sequencing proved that recombinant plasmid pcDNA3.1(-)/BRIT1 was constructed correctly.Both the expressions of BRIT1 mRNA and protein were up-regulated effectively in HeLa cells 48 h after transfection with the constructed plasmid.However,the apoptosis rate of HeLa cells transfected with plasmid pcDNA3.1(-)/BRIT1[(12.37 ± 0.19)%] was significantly higher than those transfected with empty plasmid[(1.81 ± 0.22)%] and those untransfected[(2.06 ± 0.10)%](P < 0.05).Conclusion Over-expression of BRIT1 gene induced the apoptosis of HeLa cells in vitro,which laid a foundation of further study on the role of BRIT1 gene in apoptosis of cervical cancer cells and the relevant mechanism.

    2012 04 v.25 [Abstract][OnlineView][Download 244K]

  • Effect of islet neogenesis associated protein-pp on secretion level of insulin by human islet cells cultured in vitro

    REN Li-li,QI Hui,CHEN Li-juan,LI Fu-rong(Clinical Research Center of Shenzhen People's Hospital,Shenzhen 518020,Guangdong Province,China)

    Objective To investigate the effect of islet neogenesis associated protein-pp(INGAP-pp) on secretion level of insulin by human islet cells cultured in vitro.Methods The pancreas tissues of adult patients with carcinoma of head of pancreas were collected by surgery,based on which islets were isolated from the normal tissue at tail of pancreas,then purified,cultured in vitro and determined for biological activity.The secretion levels of insulin by islets added with and without INGAP-pp were compared.Results The isolated and purified islets showed biological activity.After culture in vitro for 21 d,the islets without addition of INGAP-pp lost its ability in secretion of insulin gradually,of which the morphology changed significantly,and the cell death appeared gradually after disaggregation of cell aggregates.However,the islets added with INGAP-pp were of normal morphology,by which the secretion levels of insulin were significantly higher than those by the islets without addition of INGAP-pp starting from day 6 after culture(P < 0.05 or P < 0.01).Conclusion INGAP-pp significantly promoted the secretion of insulin by human islet cells cultured in vitro.

    2012 04 v.25 [Abstract][OnlineView][Download 214K]

  • Screening of high effective siRNAs targeting high mobility group box 1 protein gene

    LI Xiao-feng,XIA Yun,SU Xiao-yan,WANG Hui-juan(The First Affiliated Hospital,Chongqing Medical University,Chongqing 400016,China)

    Objective To screen the high effective siRNAs targeting the gene encoding high mobility group box protein 1(HMGB1),a late mediator of inflammation,and determine its biological effect in vitro.Methods Three siRNA sequences specific to HMGB1 mRNA were selected and synthesized by T7 transcription system in vitro.RAW264.7 cells were divided into five groups.The cells in intervention groups 1,2 and 3 were transfected with synthesized HMGB1 siRNAs 1～3 respectively and,6 h later,stimulated with 500 ng/ml lipopolysaccharide(LPS) for 24 and 48 h,while those in LPS group were untransfected and only stimulated with LPS,and those in control group were only added with medium.The transcription levels of HMGB1 mRNAs in cells of various groups were determined by RT-PCR,while the HMGB1 contents in culture supernatants by ELISA.Results The RNA fragments each at a length of 21 bp were observed on agarose gel electrophoretic profile of synthesized HMGB1 siRNAs 1 ～ 3.After stimulation with LPS for 24 and 48 h,both the transcription levels of HMGB1 mRNAs and HMGB1 contents in culture supernatants of cells in three intervention groups were significantly higher than those in control group.All the three HMGB1 siRNAs inhibited the transcription of HMGB1 mRNA in cells and decreased the HMGB1 content in cell culture supernatant,of which siRNA1 showed satisfactory effect as compared with siRNA2 and siRNA3.Conclusion LPS stimulated RAW264.7 cells to release a large quantity of HMGB1 and increased the transcription level of mRNA in the cells.HMGB1 siRNA decreased the protein and mRNA levels of HMGB1,which might provide a new method for controlling the progress of late inflammation.

    2012 04 v.25 [Abstract][OnlineView][Download 197K]

  • Roles of interferon,interleukin-6 and interleukin-1β in enterovirus 71 infection

    FAN Sheng-tao,WANG Li-chun,ZHAO Heng,LIU Long-ding,LI Qi-han(Department of Basic Medicine,Kunming Medical College,Kunming 650031,China)

    Objective To investigate the roles of interferon(IFN),interleukin-6(IL-6) and interleukin-1β(IL-1β) in enterovirus(EV)71 infection.Methods Vero and KMB17 cells were infected with EV71-FY23 strain in presence of IFNα1b,IFNλ1 and IFNγ separately,and the protective effects of IFNs on cells were observed.Suckling mouse model of EV71 infection was established,based on which the antiviral effects of IFNα1b,IFNλ1,IFNγ,IL-6 and IL-1β were observed.Results Both IFNα1b and IFNλ1 showed significantly protective effects on cells infected with EV71,while IFNγ showed no significantly protective effect.IFNα1b showed good protective effect on suckling mice infected with EV71,with which the pre-treatment before EV71 infection showed more significant effect.IL-1β also showed a certain protective effect on suckling mice infected with EV71.However,the pre-treatment with IL-6 showed no protective effect while accelerated the death of suckling mice.Conclusion IFN and IL-1β showed protective effect against EV71 infection,while IL-6 showed no protective effect.

    2012 04 v.25 [Abstract][OnlineView][Download 328K]

  • Cloning and eukaryotic expression of human interleukin-29 gene

    CHEN Wei,YU Ming-lei,ZHENG Hai-jun,YU Xin,WU Min-chen,WU Jing(School of Medicine and Pharmaceutics,Jiangnan University,Wuxi 214122,Jiangsu Province,China)

    Objective To clone human interleukin-29(IL-29) gene and express in eukaryotic cells.Methods Total RNA was extracted from human peripheral blood mononuclear cells(PBMCs) and reversely transcribed into cDNA as a template with which IL-29 gene was amplified by PCR and inserted into eukaryotic expression vector pPIC9K.The constructed recombinant plasmid pPIC9K-29 was transformed to Pichia pastoris GS115 and induced with methanol,and the expressed product was identified by SDS-PAGE and Western blot.Results The sequence of amplified IL-29 gene was identical to that reported in GenBank(NM_172140).Restriction analysis and sequencing proved that recombinant plasmid pPIC9K-29 was constructed correctly.Specific protein bands with relative molecular masses of about 30 000 and about 27 000 respectively were observed on SDS-PAGE profile of expressed product,both of which showed specific reactions with goat anti-human IL-29 polyclonal antibody.Conclusion Human IL-29 gene was cloned and expressed in P.pastoris GS115,which laid a foundation of further study on biological activity and application of IL-29.

    2012 04 v.25 [Abstract][OnlineView][Download 154K]

  • Changes of free calcium ion concentration and mitochondrial membrane potential during apo-ptosis of human gallbladder carcinoma GBC-SD cells induced by tea polyphenols

    LIAN Chao-qun,ZHANG Jing,CHEN Zhen-dong,XIA Jun(Medical Inspection Department,Bengbu Medical College,Bengbu 233030,Anhui Province,China)

    Objective To investigate the changes of free calcium ion concentration(i) and mitochondrial membrane potential(ΔΨm) during apoptosis of human gallbladder carcinoma GBC-SD cells induced by tea polyphenols(TP).Methods The effect of TP on proliferation of GBC-SD cells was determined by MTT method,while that on apoptosis of GBC-SD cells by laser scanning cofocal microscopy(LSCM) after Annexin V-FITC/PI staining,and that oni and ΔΨm by LSCM after Fluo-3 AM and Rhodamine123 staining.Results TP inhibited the proliferation and induced the apoptosis of GBC-SD cells significantly,both in time-and dose-dependent patterns.Meanwhile,TP increased thei and decreased ΔΨm,both in time-and dose-dependent patterns.Conclusion TP inhibited the proliferation and induced the apoptosis of GBC-SD cells by a possible mechanism of up-regulating the intracellular i and decreasing the ΔΨm.

    2012 04 v.25 [Abstract][OnlineView][Download 456K]

  • Effect of wasabi extract on proliferation of colon carcinoma SW480 cells and transcription level of mRNA of tumor suppressor gene P16INK4a

    MAO Chun-mei,YANG Lan-lan,LIU Xian-jun(Molecular Medicine and Cancer Research Center,Chongqing Medical University,Chongqing 400016,China)

    Objective To investigate the effect of wasabi extract on proliferation of colon carcinoma SW480 cells as well as transcription level of mRNA of tumor suppressor gene P16INK4a.Methods SW480 cells were treated with wasabi extract at various concentrations for 48 h,then determined for proliferation level by MTT method,for apoptosis and distribution of cell cycle by flow cytometry,and for transcription level of P16INK4a mRNA by RT-PCR.Results Wasabi extract showed dose-dependent inhibitory effect on proliferation of SW480 cells,with an IC50 of about 130 μmol/L.The early and late apoptosis rates of SW480 cells showed a dose-dependent increase,of which the cell cycle was mainly arrested at G2 phase.However,the transcription level of P16INK4a mRNA showed a dose-dependent decrease.Conclusion Wasabi extract inhibited and proliferation and induced the apoptosis of SW480 cells,and arrested the cell cycle at G2 phase by inhibiting the transcription of P16INK4a mRNA.

    2012 04 v.25 [Abstract][OnlineView][Download 271K]

  • Influence of Bortezomib on apoptosis of acute myelogenous leukemia cells as well as on SALL4 gene and Wnt/β-catenin signaling pathway

    FU Lei-hua,WANG Lan,YU Yi-chuan,HU Cheng-lin,CHEN Lin(Department of Hematology,The Second Affiliated Hospital of Chongqing Medical University,Chongqing 400010,China)

    Objective To investigate the influence of Bortezomib(Bor) on proliferation and apoptosis of acute promyelocyte leukemia NB4 cell strain and acute erythroleukemia TF1 cell strain as well as on expressions of SALL4 gene and C-mye and CCND1 genes downstream in Wnt/β-catenin signaling pathway.Methods NB4 and TF1 cells were treated with 10,30 and 50 nmol/L Bor for 12,24 and 48 h separately,using those untreated as control,then determined for proliferation activity by MTT method,for apoptosis rate by flow cytometry,and for expressions of SALL4,C-mye and CCND1 genes by RT-PCR and fluorescent quantitative PCR.Results Bor showed time-and dose-dependent inhibitory effect on proliferation of NB4 and TF1 cells,with IC50 values of 23.97 and 25.36 nmol/L respectively 48 h after treatment.Bor induced the apoptosis of two kinds of cells in dose-dependent mode.The apoptosis rates of NB4 cells 24 after treatment with 30 and 50 nmol/L Bor and TF1 cells 24 h after treatment with 50 nmol/L Bor showed significant difference with those in control group(both P < 0.05).Bor inhibited the expressions of SALL4,C-mye and CCND1 genes in the two kinds of cells significantly.Twenty-four hours after treatment with 50 nmol/L Bor,the expression levels of the three genes in both NB4 and TF1 cells were significantly higher than those in control group(each P < 0.05).The expression of SALL4 gene was closed related to those of C-mye and CCND1 genes(rs = 0.857,0.929,both P < 0.01).Conclusion The inhibition of SALL4 gene as well as target genes C-mye and CCND1 downstream in Wnt/β-catenin signaling pathway may play an important role in induction of apoptosis of NB4 and TF1 cells.

    2012 04 v.25 [Abstract][OnlineView][Download 321K]

  • Preparation and application of monoclonal antibody against Japanese encephalitis virus SA14-14-2 and P3 strains

    ZHOU Zhi-jun,SHI Jin-rong,ZHU Hua-song,WANG Ping,YU Mo-song(Wuhan Institute of Biological Products Co.Ltd.,Wuhan 430060,China)

    Objective To prepare and preliminarily apply the monoclonal antibody(McAb) against Japanese encephalitis(JEV) SA14-14-2 and P3 strains.Methods BALB mice were immunized with live attenuated JEV vaccine prepared with SA14-14-2 strain and P3 antigen respectively,and boosted crossly.The splenocytes of immunized mice were fused with SP2/0 cells,and positive hybridoma cells were screened by indirect ELISA.The clones negative for human serum albumin(HSA) but positive for JEV antibody were selected and subcloned for 2 times,based on which ascites was prepared and purified,and McAb and hybridoma were identified.Capture ELISA method was developed with McAbs against SA14-14-2 strain and used for determination of JEV antibody,while competitive inhibition ELISA and double antibody sandwich ELISA methods for determination of JEV antigen content.Results After 3 times of fusion,four hybridoma cell strains secreting McAb against SA14-14-2 strain and three hybridoma cell strains secreting McAbs against P3 strain were obtained,but no hybridoma cell strains secreting McAbs with common antigenic determinants of SA and P3 strains were obtained.All the McAbs secreted by the seven hybridoma cell strains were IgG1 with high specificity but without neutralizing activity.In the order of relative affinity,the McAbs against SA14-14-2 strain were 3D1,5E3,6H3 and 4F12,all of which recognized the same SA14-14-2 antigenic epitopes;while the McAbs against P3 strain were 1C7,5H12 and 3C4,all of which recognized the same P3 antigenic epitopes.The titers of McAbs prepared with the seven hybridoma cell strains 6 months after storage in liquid nitrogen and with those after continuous culture for 3 months in vitro were stable.By using McAb 5E3 against SA14-14-2 strain,the capture ELISA method for determination of JEV antibody as well as competitive inhibition ELISA method and double antibody sandwich ELISA methods for JEV antigen were developed.Conclusion The McAbs against SA14-14-2 and P3 strains of JEV were prepared.The capture ELISA method developed with McAb against SA14-14-2 strain was more simple and sensitive than indirect ELISA for determination of JEV antibody.However,double antibody sandwich ELISA was superior to competitive inhibition ELISA in determination of samples with high antigen contents.

    2012 04 v.25 [Abstract][OnlineView][Download 294K]

  • Construction and expression of prokaryotic expression vector for culture filtrate protein 10-pentose-5-phosphate-3-epimerase 68 fusion gene of Mycobacterium tuberculosis

    DONG Zhi-ling,HE Yong-lin,XU Lei,WANG Jing-xian,ZHANG Zhuang-miao,YANG Jing,FENG Xin,YANG Chun(Department of Microbiology,Chongqing Medical University,Chongqing 400016,China)

    Objective To construct a prokaryotic expression vector for culture filtrate protein 10(CFP10)-pentose-5-phosphate-3-epimerase 68(PPE68) fusion gene of Mycobacterium tuberculosis and express in E.coli.Methods CFP10 and PPE68 genes were amplified by PCR respectively using the genomic DNA of M.tuberculosis H37Rv strain as template,based on which fusion gene CFP10-PPE68 was amplified by gene SOEing,and cloned into vector pET-32a(+).The constructed recombinant plasmid pET-32a(+)-CFP10-PPE68 was transformed to E.coli BL21(DE3) and induced with IPTG,and the expressed product was identified by SDS-PAGE and Western blot.Results Restriction analysis and sequencing proved that recombinant plasmid pET-32a(+)-CFP10-PPE68 was constructed correctly.The expressed fusion protein,with a relative molecular mass of 68 630,contained 41.8% of total somatic protein,mainly existed in a soluble form,and showed specific reaction with sera of rabbits immunized with PPE68 rBCG.Conclusion The prokaryotic expression vector for CFP10-PPE68 fusion gene of M.tuberculosis was constructed successfully,and fusion protein was expressed in E.coli BL21(DE3),which laid a foundation of application of the fusion protein to serological diagnosis of tuberculosis.

    2012 04 v.25 [Abstract][OnlineView][Download 187K]

  • Prokaryotic expression and purification of human cystatin C and preparation of its polyclonal antibody

    CHEN Te,HUANG Mei-rong,WANG Peng,LIU Yu-si,WANG Hong,ZHANG Xue-mei,XU Wen-chun(Key Laboratory Medical Diagnostics,Ministry of Education,Department of Laboratory Medicine,Chongqing Medical University,Chongqing 400016,China)

    Objective To construct the prokaryotic expression vector for human cystatin C(Cys C) gene,express and purify thioredoxin(Trx)-Cys C fusion protein,and prepare polyclonal antibody against human Trx-Cys C.Methods A DNA fragment encoding full-length Cys C was designed and synthesized based on optimization of synonymous codon bias of E.coli,without modification of amino acid sequence,and inserted into prokaryotic expression vector pET-32a(+).The constructed recombinant plasmid pET-32a(+)-Cys C was transformed to E.coli BL21(DE3) and induced with IPTG at low temperature.The expressed fusion protein was purified by Ni-Sepharose 6FF chromatography and immunized to New Zealand rabbits,and the prepared polyclonal antibody against human Cys C was identified by indirect ELISA and Western blot.Results Restriction analysis and sequencing proved that recombinant plasmid pET-32a(+)-Cys C was constructed correctly.The obtained gene sequence with optimized synonymous codon was completely consistent with that expected.Recombinant Cys C protein with a relative molecular mass of about 35 000 was expressed in soluble form,which contained about 50% of total somatic protein.The purified recombinant protein reached a purity of more than 90%,and was recognized by clinical detection kit for Cys C.The prepared polyclonal antibody reached a titer of more than 1 ∶(5.12 × 106),and recognized commercial Cys C protein specifically.Conclusion Soluble recombinant Cys C protein was successfully expressed in prokaryotic cells,and its polyclonal antibody with high titer was prepared,which laid a foundation of development of immunological method for detection of Cys C.

    2012 04 v.25 [Abstract][OnlineView][Download 216K]

  • Relationship between blood group antibody IgG titer of pregnant women and ABO hemolytic disease of newborn in ten provinces and cities in China

    ZHU Ye-hua,WU Wei-jian,MA Chun-hui,GUO Ru-hua,YU Jin-lin(Foshan Blood Center,Foshan 528000,Guangdong Province,China)

    Objective To analyze the relationship between blood group antibody IgG titer of pregnant women and ABO hemolytic disease of newborn(HDN).Methods Three hundreds of pregnant women in Foshan City as well as their husbands were subjected to ABO blood grouping,while the pregnant women were determined for anti-A and/or anti-B IgG titers in sera.From the newborns with clinical indications of HDN,the blood samples were collected for ABO blood grouping as well as Coomb test,free antibody test and antibody release test.The relationship between blood group antibody IgG titer of pregnant women and HDN in ten provinces and cities including Foshan City in China was analyzed.Results All the 300 pregnant women in Foshan City were of blood group O,while their the husbands were of blood groups A,B or AB.In all the ten provinces and cities,the incidence rates of HDN increased with the increasing blood antibody IgG titers of pregnant women.However,the incidence rate showed significant difference in the newborn delivered by pregnant women with the same IgG titers in various provinces and cities(each P < 0.05) and in the newborns delivered by pregnant women with various IgG titers(each P < 0.05).The blood group antibody IgG titers of pregnant women was positively related to the incidence rate of HDN(r = 0.866,P < 0.05).The increasing fold of incidence rate of HDN in newborns delivered by pregnant women with IgG titers of 32 ～ 64 was 440%,which was higher than those by pregnant women with other IgG titers.Conclusion The blood group IgG titer of pregnant woman might be used as one of the indexes,but not the only index,for evaluation of risk of HDN.The risk should be evaluated by the IgG titer combined with other test results and clinical status of pregnant women.

    2012 04 v.25 [Abstract][OnlineView][Download 139K]

  • Development of a method for large-scale purification of rotavirus

    ZHANG Biao,TONG Lin,YI Shan,ZHANG Guang-ming,XIE Tian-hong,LI Hong-jun,SUN Mao-sheng(Department of Histology and Embryology,Gaungdong Medical College,Zhanjiang 524023,Guangdong Province,China)

    Objective To develop a method for large-scale purification of rotavirus(RV).Methods The feasibility of centrifugation-microfiltration-ultrafiltration-chromatography(module method) for purification of RV was evaluated using cesium chloride density gradient centrifugation as control.RV was observed for morphology by transmission electron microscopy,then determined for infectious titer by microtitration method,and for protein contents before and after purification by Bradford method,based on which the recovery rate of protein was calculated.BALB/c mice were immunized with RV purified by the two methods,and determined for neutralizing antibody titer in sera by micro-neutralization assay.Results RV purified by module method showed intact structure,of which the infectious titer reached(7.15 ± 0.10) lgCCID50.More than 95% of protein was removed,and the recovery rate of protein was(2.58 ± 0.06)%.The purified RV induced neutralizing antibody titer in mice.However,the recovery rate of protein,infectious titer and the induced neutralizing antibody titer of the RV purified by module method were significantly higher than those by cesium chloride density gradient centrifugation(P < 0.05 or P < 0.01).Conclusion The developed module method may be used for effective purification of RV,which provides an experimental basis for development of procedure for large-scale purification of inactivated RV vaccine.

    2012 04 v.25 [Abstract][OnlineView][Download 184K]

  • Development of capillary isoelectric focusing electrophoresis method for analysis of isoelectric point of fusion protein FP3

    LI Xiang,GAO Xiang-dong,TIAN Hong,RAO Chun-ming(National Institutes for Food and Drug Control,Beijing 100050,China)

    Objective To develop a capillary isoelectric focusing electrophoresis(cIEF) method for analysis of isoelectric point of fusion protein FP3.Methods A cIEF method was developed by using neutral-coated capillary tube at an effective length of 20 cm,a total length of 30 cm and an inner diameter of 50 μm.The temperature for separation was 20℃;the wavelength for detection was 280 nm;the pressure and time for loading were 20 Psi and 99 s respectively;the voltage and time for separation focusing were 20 kV and 20 min,while those for chemical migration were 25 kV and 25 min,respectively.The effects of various ampholytes,kinds(PharmalyteTM 3-10 and AmpholineTM 3.5-10) and concentrations of solubilizers and voltages for focusing(15,20 and 30 kV) on separation of test samples were compared,based on which the suitability and stability of system as well as reproducibility of the method were verified.Results By the developed cIEF method,the charge-based isoforms of test samples were separated effectively,with a separation degree(USP) of 1.249 between the main and the secondary peaks.By using AmpholineTM 3.5-10,various charge-based heterogeneous components of test samples were separated effectively.The urea at a concentration of 6 mol/L provided a good environment of solubilization.The separation effect was satisfactory at a voltage for focusing of 20 kV.The relative standard derivations(RSDs) of migration times of main and secondary peaks of test samples in five consecutive loadings were 1.29% and 1.32% respectively.The isoelectric points of main bands of test samples ranged from 6.33 to 6.90,and the detection result was stable within 24 h.Conclusion The cIEF method for analysis of isoelectric point of fusion protein FP3 was developed,which showed high separation degree,reproducibility,stability and accuracy and provided a more effective tool for quality control of products of the same kind.

    2012 04 v.25 [Abstract][OnlineView][Download 208K]

  • Development of TaqMan probe-based fluorescent quantitative PCR method for porcine cir-covirus type 2

    CHEN Rong,WANG Yan,WEI Jian-zhong,LIU Jun-jun,SUN Pei,YIN Zong-jun,LI Yu(College of Animal Science and Technology,Anhui Agricultural University,Hefei 230036,China)

    Objective To develop a TaqMan probe-based fluorescent quantitative PCR method for porcine circovirus type 2(PCV2).Methods Two pairs of specific primers and TaqMan probe were designed according to the relatively conserved sequence of PCV2 ORF2 for preparation of standard for recombinant plasmid.The reaction system and condition were optimized,based on which the standard curve was plotted,and a TaqMan probe-based fluorescent quantitative PCR method was developed and verified for sensitivity,specificity and reproducibility.Fifty clinical samples were determined by the developed method,and the results were compared with those by routine PCR method.Results PCR and sequencing proved that the standard for recombinant plasmid was constructed correctly.The Ct value of plotted standard curve showed good linearity to the log of copy number of template,with a R2 value of 0.993 85.The sensitivity of developed method was 6 × 101 copies/μl,which was two orders of magnitude higher than that of routine PCR method.No amplification curves were obtained from porcine reproductive and respiratory syndrome virus(PRRSV),porcine pseudorabies virus or attenuated lapinized classical swine fever virus.The variation coefficients of determination results of three PCV2-positive templates by the developed method were 0.07% ～ 0.50%.The positive rate of clinical samples by the developed method(64%) was significantly higher than that by routine PCR method(44%)(P < 0.05).Conclusion The developed TaqMan probe-based fluorescent quantitative PCR method showed high sensitivity,specificity and reproducibility,which was suitable for rapid detection of trace PCV2 in clinical samples as well as study on tissue tropism and cell culture characteristics of PCV2.

    2012 04 v.25 [Abstract][OnlineView][Download 241K]

  • Development of one-step RT-PCR method for identification of wild virus strain and gene-deleted attenuated vaccine virus strain of porcine reproductive and respiratory syndrome virus

    MEI Lin,GAO Ying-jie,LIANG Zhi-xuan,ZHAO Jian-zeng [Sinovet(Beijing) Biotechnology Co.Ltd,Beijing 100085,China]

    Objective To develop a one-step RT-PCR method for identification of wild virus strain and gene-deleted attenuated vaccine virus strain TJM-F92 of porcine reproductive and respiratory syndrome virus(PRRSV).Methods A pair of primers were designed according to the nsp2 gene sequences of highly pathogenic PRRSV TJ strain and gene-deleted attenuated vaccine virus strain TJM-F92,based on which a one-step RT-PCR method for identification of various wild and gene-deleted attenuated strains of PRRSV was developed and verified for specificity,sensitiveness,sensitivity and reproducibility.Results The gene fragment at a length of 1 100 bp was amplified from TJ strain,while that at a length of 740 bp from TJM-F92 strain,indicating that the two strains were identified correctly.The developed one-step RT-PCR method showed high sensitiveness,by which PRRSV-TJ vaccine strain at 101 TCID50/ml was detected.Both the coincidence rates of determination results of known positive and negative serum samples of piglets were 100%,indicating high sensitivity of the method.The method also showed high reproducibility.Conclusion The developed one-step RT-PCR method might be used for clinical identification of wild PRRSV infection and PPRSV vaccination in pigs inoculated by vaccine prepared with TJM-F92 strain,which was helpful to evaluation of immune effect of vaccine and early diagnosis of wild PRRSV infection.

    2012 04 v.25 [Abstract][OnlineView][Download 155K]

  • Optimization of fermentation procedure for recombinant E.coli pUC118-ski/DH5α

    PENG Yan,LI Ping,LIU Ping,ZHOU Yuan-guo(Molecular Biology Center,State Key Laboratory of Trauma,Burns and Combined Injury,Research Institute of Surgery and Daping Hospital,Third Military Medical University of Chinese PLA,Chongqing 400042,China)

    Objective To optimize the fermentation procedure for recombinant E.coli pUC118-ski/DH5α.Methods Recombinant E.coli pUC118-ski/DH5α with high copy number of plasmid was screened,of which the fermentation parameters,including component of medium,temperature for fermentation,matrix component and mode for fed batch and time for culture,were optimized.Three consecutive batches of recombinant E.coli pUC118-ski/DH5α were fermented in 50 L fermenter under the optimized condition,based on which the reproducibility of the optimized procedure was verified.Results The fermentation parameters were optimized as follows: the recombinant E.coli pUC118-ski/DH5α was inoculated to the medium using glycerol as carbon source and fermented at 25℃,supplemented with 50% glycerol by gradient feeding at a constant rate and cultured for 12 h.The wet weights and plasmid contents of three batches of recombinant E.coli pUC118-ski/DH5α fermented under the optimized condition reached 52.7 ～ 60.3 g/L and 1.79 ～ 1.95 mg/g wet bacteria respectively,while the plasmid copy numbers were 1012 copies/μl,and the proportion of supercoiled plasmid were more than 90%.Conclusion The fermentation procedure for recombinant E.coli pUC118-ski/DH5α was optimized,which laid a foundation of large-scale production and downstream purification of recombinant plasmid.

    2012 04 v.25 [Abstract][OnlineView][Download 220K]

  • Optimization of condition for expression of long chain Arg3 human insulin-like growth factor-1 in Pichia pastoris

    ZHANG Wen-ming,HONG Jing,SUN Tian-wei,HUANG Guo-tuan,LIANG Jun,WANG Da-li,LIN Dian-hai(Zhejiang Biotechnology Engineering Co.,LTD,Anji 313300,Zhejiang Province,China)

    Objective To optimize the condition for expression of long chain Arg3 human insulin-like growth factor-1(LR3IGF-1) in Pichia pastoris.Methods The effects of methanol concentration(0.25%,0.50%,0.75%,1.00%,1.25% and 1.50%),temperature(25,28 and 30℃),pH value(4.75,5.00,5.25,5.50,5.75 and 6.00) and additives(0.1% Arg,Gly,His,Tween-20 and Tween-80) on expression of LR3IGF-1 in P.pastoris were investigated,based on which the condition for expression of LR3IGF-1 was optimized.The expression of LR3IGF-1 in fermenter was induced under the optimized condition,and the expression level was determined and compared with that under original condition.Results The expression levels of LR3IGF-1 after induction with 0.75% methanol and at 25℃ were 76.5 and 98 mg/L respectively.The expression level of LR3IGF-1 was high after induction at pH 5.25 for 24 h,with low degradation.After addition of Tween-20,the expression level increased to 271 mg/L.The expression level of LR3IGF-1 under optimized condition reached 497 mg/L,which increased by nearly 9 folds as compared with that before optimization of condition for expression,while the time for induction was shortened from 48 h to 24 h.Conclusion The condition for expression of LR3IGF-1 in P.pastoris was optimized,which laid a foundation of large-scale production of LR3IGF-1.

    2012 04 v.25 [Abstract][OnlineView][Download 164K]

  • Development of inactivation procedure for virus in chitosan by sodium hydroxide ethanol solution and γ irradiation

    LI Bo,ZHANG Jia-li,XIA Wen-shui(State Key laboratory of Food Science and Technology,School of Food Science and Technology,Jiangnan University,Wuxi 214122,Jiangsu Province,China)

    Objective To develop an inactivation procedure for virus in chitosan by sodium hydroxide ethanol solution and γ irradiation.Methods Bovine viral diarrhea virus(BVDV)and porcine parvovirus(PPV)as indicators in chitosan were inactivated by sodium hydroxide ethanol solution and γ irradiation separately,and the two methods were optimized.A two-step inactivation procedure was developed under optimized condition,by which the inactivated samples were determined for virus titer,relative molecular mass of chitosan,degree of deacetylation and ethanol content.Results After treatment with 10% ethanol solution containing 8 mol/L sodium hydroxide at 35℃ for 1 h followed by lyophilization and irradiation with γ-ray at a dosage of 5 kGy,both BVDV and PPV titers in chitosan decreased by more than 4 lgTCID50,while the relative molecular mass of chitosan decreased by only 8.4%,the degree of deacetylation showed no significant change,and no residual ethanol was detected.Conclusion The two-step inactivation procedure consisting of treatment with sodium hydroxide ethanol solution and γ irradiation was effective for inactivation of indicator viruses in chitosan,while showed little effect on chitosan,which ensured the safety of chitosan as a biomaterial and a medicinal subsidiary material.

    2012 04 v.25 [Abstract][OnlineView][Download 236K]

  • Isolation,culture and identification of neural tissue committed stem cells from bone marrow

    LI Lin,ZHANG Xing-xiu,WANG Jian,JIANG Rong,ZHENG Min(Department of Stem Cells and Tissue Engineering,Department of Histology and Embryology,Chongqing Medical University,Chongqing 400016,China)

    Objective To isolate,culture and identify neural tissue committed stem cells(NTCSCs) from bone marrow of SD rats.Methods NTCSCs were isolated and cultured from rat bone marrow by using serum-free DMEM/F12(1︰1) conditional medium,then identified for differentiation by immunohistochemical assay,and determined for transcriptions of Nestin,CXCR4,CD31,βⅢ-Tubulin and GFAP mRNAs by RT-PCR.Results Cell spheres in suspension were isolated and cultured,in which Nestin and CXCR4 as markers of neural stem cells were expressed.βⅢ-Tubulin as neuron marker and GFAP as gliacyte marker were expressed in the cells after differentiation.The transcriptions of Nestin,CXCR4,CD31,βⅢ-Tubulin and GFAP mRNAs were proved in the cells.Conclusion NTCSCs with neurobiological characters were successfully isolated from bone marrow and might be used as cell seeds,which provided a basis for therapy of diseases and repair of wound in central nervous system.

    2012 04 v.25 [Abstract][OnlineView][Download 256K]

  • Determination of formaldehyde content in influenza virus subunit vaccine by acetylacetone spectrophotometry

    TIAN Wen-li,YANG Jiang-shan,WAN Ya-fen,QI Ji,YANG Xu,LI Xiao-qiang[Tasly-Jenner Biotechnology(Tianjin) Co.,Ltd,Tianjin 300410,China]

    Objective To determine the formaldehyde content in influenza virus subunit vaccine by acetylacetone spectropho-tometry.Methods The formaldehyde content in influenza virus subunit vaccine was determined by acetylacetone spectrophotometry,based on which the detection wavelength,concentrations of acetylacetone,acetic acid and ammonium acetate as well as time for maintaining and lowering the temperature were optimized,and the method was verified.Results The optimal reaction system consisted of 500 μl of test sample and 4.5 ml of acetylacetone.The optimal concentrations of acetylacetone,acetic acid and ammonium acetate were 0.125%,1.5% and 25% respectively,while the optimal time for maintaining(at 40℃) and lowering the temperature were both 30 min,and the optimal detection wavelength was 414 nm.Under the optimized condition,the determination result was satisfactory.The standard curve showed good linearity within a concentration range of 1 ～ 50 μg/ml,while the recovery rate and minimum detection limit were 101.9% and 1 μg/ml respectively.Conclusion Acetylacetone spectrophotometry was simple,rapid,accurate and sensitive for determination of formaldehyde content in influenza virus subunit vaccine,which was suitable for the routine determination of formaldehyde content in laboratory.

    2012 04 v.25 [Abstract][OnlineView][Download 153K]

  • Removal of phenol from group A meningococcal polysaccharide by ultrafiltration

    HU Jing,XUE Hong-gang,GUO Rong,CHEN You-xi,QU Ming-xia,CHEN Hao-jun(Wuhan Institute of Biological Products Co.Ltd.,Wuhan 430060,China)

    Objective To remove phenol from group A meningococcal polysaccharide by ultrafiltration.Methods The materials of group A meningococcal polysaccharide were ultrafiltrated by using Omega OS010C10(type C screen,10 KD,filtration area: 0.1 m2) and Omega OS010T12(type T screen,10 KD,filtration area: 0.1 m2) membranes respectively,based on which the effects of various pressures on membrane filter velocity were compared,the recovery rates of membrane water purification filter velocities before and after ultrafiltration were determined,and the phosphorus,phenol and total solid contents in samples before and after ultrafiltration and dialysis were analyzed.Results As compared with those of OS101C10 membrane,the increased tangential velocity and pressure of OS010T12 membrane influenced the filtration rate significantly,and the filter velocity was high at low pressure difference and low transmembrane pressure.At steps of concentration and washing filter,the mean filter velocity of OS101T12 membrane increased by about 1.3 folds as compared with that of OS101C10 membrane.Conclusion The water purification filter velocity of OS101T12 membrane after washing recovered to the level before test.Both the phenol contents in ultrafiltrates by using the two membranes met the requirements in Chinese Pharmacopoeia(Volume Ⅲ,2010 edition),while the recovery rates of polysaccharide reached more than 90%.However,the time-consumed for ultrafiltration decreased by at least 46 h as compared with that for dialysis,while the water-consumed of the former was only 20% of that of the latter.

    2012 04 v.25 [Abstract][OnlineView][Download 139K]

  • Progress in research on combined vaccine home and abroad

    WANG Li-chan,HOU Qi-ming,ZHANG shu-min(National Institutes for Food and Drug Control,Beijing 100050,China)

    Along with the implementation of the expanded programme on immunization(EPI) as well as the development and application of novel vaccines,children need to be vaccinated with more and more doses,which not only increases the pain of children,but also brings many inconvenience to the parents and medical workers.It is necessary to develop and apply combined vaccine which prevents several diseases by a single vaccination.This article describes the latest progress in research on combined vaccine home and abroad.

    2012 04 v.25 [Abstract][OnlineView][Download 145K]

  • Progress in research on PEGylation of protein and polypeptide drugs

    HUI Xi-wu,CHEN Hong,HUANG Bing-ren(National Laboratory of Medical Molecular Biology,Institute of Basic Medical Sciences,Chinese Academy of Medical Sciences & Peking Union Medical College,Beijing 100005,China)

    Along with the development of genetic engineering technology,the clinical application of protein and polypeptide drugs are paid more and more attentions.However,the disadvantages such as immunogenicity,toxicity and short half-life of protein and polypeptide drugs limit their further application and development.Since polyethylene glycol(PEG) can give a number of relevant advantages to the conjugated protein,such as half-life prolongation in vivo,a reduction or an abolishment of immunogenicity,and a reduction of aggregation,the application of protein PEGylation technology is of a wide prospect in biotechnology and biomedicine.The PEGylation of protein and polypeptide drugs,the development of PEG modification technology,the main ways of PEG modification as well as the present situation and prospects of PEG-drug are briefly described in this review.

    2012 04 v.25 [Abstract][OnlineView][Download 218K]


@2012 Editorial Department of "Chinese Journal of Biologicals"