2013年01期目次

  • Prokaryotic expression and immunogenicity of coxsackievirus group A type 16 VP1

    YANG Yong-juan,HAO Chun-sheng,LI Yi,SONG Dong-mei,LIU Yu,MA Shu-hua,TIAN Long,LI Xiu-ling National Vaccine & Serum Institute,Beijing 100024,China

    Objective To express coxsackievirus group A type 16(CA16) in prokaryotic cells and determine its immunogenicity.Methods VP1 gene was amplified from Qingdao strain of CA16 by RT-PCR and cloned into prokaryotic expression vector pET-43.1a(+).The constructed recombinant plasmid pET43.1a-VP1 was transformed to E.coli Rossatte(DE3) and induced with IPTG.The expressed recombinant VP1 protein was purified by nickel ion affinity chromatography.BALB / c mice were immunized with the purified VP1 protein at various dosages(5,10,20 and 40 μg) and determined for specific IgG,IgG1,IgG2a,IgG2b and IgG3 titers in sera by ELISA,and neutralizing antibody titer by micro-cytopathic effect inhibition assay.Results Both restriction analysis and sequencing proved that recombinant plasmid pET43.1a-VP1 was constructed correctly.The expressed CA16 VP1 protein,with a relative molecular mass of about 34 000,mainly existed in a form of inclusion body and contained 15% of total somatic protein.The purified VP1 protein reached a purity of more than 95% and showed specific reaction with monkey antiserum against CA16.The recombinant VP1 at various dosages induced specific antibody against CA16 in BALB / c mice.The titers of total IgG,IgG1,IgG2a,IgG2b and IgG3 titers in sera of immunized mice were significantly higher than those in control group,which were dose-dependent at a certain degree.However,all the neutralizing antibody titers in sera of mice immunized with VP1 at various dosages were less than 1 ∶ 8.Conclusion The CA16 VP1 protein was successfully expressed in E.coli,and induced specific humoral immune response in mice after purification,which laid a foundation of further study on structure and function of CA16 and development of the relevant vaccines.

    2013 01 v.26 [Abstract][OnlineView][Download 284K]

  • Development of a novel procedure for purification of acellular pertussis vaccine

    WU Teng-jie,ZHANG Bin,ZHANG Qing,ZHENG Li-hua,QU Ai-dong Shanghai Institute of Biological Products Co.,Ltd.,Shanghai 200052,China

    Objective To develop a novel procedure suitable for large-scale production of acellular pertussis vaccine(APV).Methods Bordetella pertussis strain was resuscitated and subcultured.The pH value of fermentation liquid of B.pertussis was adjusted to weak acidity before harvest.Supernatant was collected after centrifugation,concentrated by ultrafiltration and subjected to desalting,from which the target components were purified by cation exchange chromatography.The purified protein was identified by SDS-PAGE and Western blot,based on which the condition for purification was optimized.Three successive batches of APV were purified by the developed procedure and tested for various quality indexes to verify the reproducibility of the procedure.Results Adjusting the pH value of fermentation liquid to 5.8 ～ 6.0 before harvest increased the content of major protective antigen in supernatant significantly.By strong cation exchange chromatography,the capture of target protein and purification of various components were completed in a single step.All the purities of various target protein components were more than 85%,while the recovery rates were more than more than 90%.The developed procedure showed high reproducibility.Conclusion A novel purification procedure which might be scaled up linearly and were suitable for large scale production of APV was developed.It laid a foundation of improvement of quality standard of vaccine.

    2013 01 v.26 [Abstract][OnlineView][Download 201K]

  • Determination of osmolality of live attenuated measles,mumps and rubella combined vaccine

    DING Rui,JI Hong,HU Qin,CHEN Si,Liu Yi-ming,ZHOU Chang-ming Beijing Institute for Drug Control,Beijing 100035,China

    Objective To determine the osmolality of live attenuated measles,mumps and rubella combined vaccine,and provide a reference for improve the quality standard of the vaccine.Methods The osmolality of 31 batches of live attenuated measles,mumps and rubella combined vaccine were determined by freezing point osmometer according to requirements in Chinese Pharmacopoeia(Volume III,2010 edition).Results The osmolality of 31 batches of vaccine ranged from 620 to 774 mOsmol / kg,which showed a certain difference.Conclusion It is necessary to include the determination of osmolality into the quality control of live attenuated measles,mumps and rubella combined vaccine.

    2013 01 v.26 [Abstract][OnlineView][Download 119K]

  • Effect of angiotensin-(1-7) on signal transduction of angiotensinⅡ in rat cardiac fibroblasts and relevant mechanism

    KAO Guo-ying,FAN Jin-qi,ZHANG Xiao-ge,CUI Kun,TAN Li,SU Li,YIN Yue-hui Department of Cardiovascular Medicine,The Second Affiliated Hospital of Chongqing Medical University, Chongqing 400010,China

    Objective To investigate the effect of angiotensin(Ang)-(1-7) on signal transduction of AngⅡin rat cardiac fibroblasts(CFs) as well as the relevant mechanism.Methods Primary CFs of neonatal SD rats were isolated,cultured and identified,then divided into blank control,AngⅡ,Ang-(1-7),AngⅡ+ Ang-(1-7),AngⅡ+ Ang-(1-7) + A-779,AngⅡ+ Ang-(1-7) + PAO,Ang-(1-7) + PAO and AngⅡ + PAO groups.The expressions of extracellular signal regulated kinase 1 / 2(ERK1 / 2) and phosphorylated ERK1 / 2(p-ERK1 / 2) were determined by Western blot,while the activity of Src-homology domain 2 containing protein tyrosine phosphatase-1(SHP-1) by immunoprecipitation capture assay,and the transcription levels of transforming growth factor-β1(TGF-β1),collagenⅠ(ColⅠ) and ColⅢ mRNAs by real-time fluorescent quantitative PCR.Results AngⅡincreased the expression level of p-ERK1 / 2 and the pERK / ERK ratio,while,while Ang-(1-7) antagonized the effects.Ang-(1-7) activated the SHP-1 activity by binding to Mas receptor,and antagonized AngⅡ-induced decrease of SHP-1 activity.After inhibition of SHP-1 activity,the antagonistic effect of Ang-(1-7) on AngⅡ-induced increases of p-ERK1 / 2 expression level and p-ERK / ERK ratio were inhibited.Ang-(1-7) showed no significant effect on transcription levels of TGF-β1,ColⅠ and ColⅢ mRNAs,while inhibited AngⅡ-induced increases of above-mentioned transcription levels.Conclusion Ang-(1-7) activated SHP-1 by binding to Mas receptor,which was significantly associated with the antagonism of AngⅡ-induced increases of p-ERK1 / 2 expression level and p-ERK / ERK ratio.Ang-(1-7) also inhibited the up-regulation of expressions of TGF-β1,ColⅠand ColⅢinduced by AngⅡ.These phenomena may represent a protective mechanismin cardiac fibroblasts whereby potentially deleterious effects of AngⅡ are antagonized by Ang-(1-7).

    2013 01 v.26 [Abstract][OnlineView][Download 280K]

  • Construction and identification of RNAi lentiviral vector for human breast cancer anti-estrogen resistance 1

    HUANG Wei,FAN Xiao-qing,WANG Ru-wen,DENG Bo,JIANG Yao-guang,ZHAO Yun-ping,GUO Wei Thoracic Surgery Department,Institute of Surgery Research,Daping Hospital,Third Military Medical University,Chongqing 400042,China

    Objective To construct and identify the RNAi lentiviral vector for human breast cancer anti-estrogen resistance 1(BCAR1) gene.Methods Four siRNA sequences,i.e.PLVT350,PLVT351,PLVT352 and PLVT353,and a negative control sequence PLVT4 were designed and synthesized according to the target sequence of human BCAR1 gene and,after annealing,inserted respectively into lentiviral vector pMagic 4.1 digested with Age Ⅰ and EcoR Ⅰ.The constructed recombinant plasmids were transformed to competent E.coli DH5α,and positive clones were screened by PCR and DNA sequencing,from which recombinant RNAi plasmids were extracted and co-infected to 293T cells with helper plasmid for packaging.The obtained lentivirus was concentrated and determined for titer by gradient dilution.A549 cells were infected with the recombinant lentivirus particles and observed for infection efficacy by fluorescent microscopy.The recombinant lentivirus particles with satisfactory interfering effect were screened and infected to A549 cells in which the expression level of BCAR1 was determined by Western blot.Results Four RNAi lentiviral vectors were successfully constructed.The titers of PLVT350,PVLT351,PLVT352 and PLVT353 were 3.45 × 108,2.13 ×108,3.01 × 108 and 2.47 × 108 TU / ml,respectively.The infection efficiency of A549 cells was more than 90% 3 d after infection with recombinant lentivirus.Except PLVT350,all the other three siRNA sequences were effective targets.The silencing rates of PLVT351,PLVT352 and PLTV353 were 82%,73% and 82% respectively.The expression levels of BCAR1 gene in A549 cells transfected with recombinant RNAi plasmids containing the three sequences were significantly lower than those untransfected and those transfected with recombinant plasmid containing PLVT4(P < 0.001).However,the expression level of BCAR1 protein in A549 cells infected with recombinant lentivirus containing PLVT351 sequence was significantly lower than those uninfected and those infected with lentivirus containing PLVT4 sequence(P < 0.01).Conclusion RNAi lentiviral vector for human BCAR1 gene as well as A549 cell strain for stable expression were successfully constructed,which laid a foundation of further study on role of BCAR1 in lung cancer as well as the relevant mechanism.

    2013 01 v.26 [Abstract][OnlineView][Download 336K]

  • Isolation and biological characteristics of CD34+ cell population from leukemia K562 cell strain

    LIU Jun,ZHANG Xian-ping,CAI Shi-zhong,XU Chun-yan,WANG Ya-ping,LIN Xue-mei Department of Histology and Embryology,Laboratory of Stem Cell and Tissue Engineering,Chongqing Medical University,Chongqing 400016,China

    Objective To isolate CD34+ cell population from leukemia K562 cell line,analyze its biological characteris-tics and provide an experimental basis for stem cells-based treatment of leukemia.Methods CD34+ cell population was isolated from K562 cells by magnetic cell sorting(MACS),and determined for activity by trypan blue dye exclusion method,for percentage and cell cycle of CD34+ cells by flow cytometry,for self-renewal ability by single cell clone culture,and for transcription levels of erythropoietin(EPO) and granulocyte macrophage colony stimulating factor(GM-CSF) mRNAs by RT-PCR.Meanwhile,the expression of P-glycoprotein(P-gp) in CD34+ cell population was determined.Results CD34+ cell population was effectively isolated by MACS,of which the activity was 99% ～ 100%.The isolated CD34+ cells accounted for 78.5% ～ 85.3% of total cells,of which the percentage at phases G0 / G1 was about 80%,significantly higher than that of K562 cells before MACS.The CD34+ cells showed ability in formation of colonies,in which the transcription levels of EPO and GM-CSF mRNAs decreased significantly as compared with those in K562 cells(P < 0.01),while P-gp was expressed.Conclusion CD34+ cell population was successfully isolated from K562 cell strain,which showed abilities in self-renewal and multi-directional differentiation.

    2013 01 v.26 [Abstract][OnlineView][Download 305K]

  • Construction of eukaryotic expression vector for human hepatocyte growth factor and its expression in human bone marrow mesenchymal stem cells

    CUI Jie,JIANG Ming-de,MEI Zhe-chuan,CHEN Li,ZENG Wei-zheng,ZHENG Shu-mei,WANG Zhao Department of Gastroenterology,General Hospital,Chengdu Military Command,Chengdu 610083,Sichuan Province,China

    Objective To construct a eukaryotic expression vector for human hepatocyte growth factor(hHGF),and to determine its expression in human bone marrow mesenchymal stem cells(BMSCs) as well as its effect on cell growth.Methods BMSCs were isolated from human bone marrow by density gradient centrifugation and determined for phenotype by flow cytometry,of which the adipogenic and osteogenic differentiations were induced.The hHGF gene was amplified by PCR and cloned into vector pEGFP-N1.The constructed recombinant plasmid pEGFP-N1 / hHGF was transfected to BMSCs by electroporation.The expression of enhanced green fluorescent protein(EGFP) was observed by fluorescent microscopy,while the transcription of hHGF mRNA by RT-PCR,the expression of hHGF by Western blot,and the effect of hHGF on proliferative activity of BMSCs by MTT method.Results CD29 and CD44 were highly expressed in BMSCs,while CD34 and CD45 were unexpressed.Adipogenic and osteogenic differentiations were induced in vitro.Restriction analysis and sequencing proved that recombinant plasmid pEGFP-N1-hHGF was constructed correctly.The expression of EGFP was observed in BMSCs 48 h after transfection.RT-PCR and Western blot showed expression of hHGF in BMSCs.The proliferative activity of BMSCs transfected with hHGF was significantly higher than those untransfected and those transfected with empty vector(P < 0.05).Conclusion The eukaryotic expression vector for hHGF was successfully constructed and expressed in BMSCs,and the expressed hHGF promoted the proliferation of BMSCs.

    2013 01 v.26 [Abstract][OnlineView][Download 372K]

  • Co-expression of bone morphogenetic protein 9-2 induces differentiation of multipotential stem cells C3H10 into osteoblasts

    CHEN Cong,LUO Qing,TIAN Wen,DIE Xiao-hong,KANG Quan Ministry of Education Key Laboratory of Child Development and Disorders,Key Laboratory of Pediatrics in Chongqing,Chongqing International Science and Technology Cooperation Center for Child Development and Disorders,Children's Hospital of Chongqing Medical University,Chongqing 40014,China

    Objective To investigate the capacity of co-expression of bone morphogenetic protein 9-2(BMP9-2) in inducing the differentiation of multipotential stem cells C3H10 into osteoblasts.Methods C3H10 cells were divided into four groups,and infected with recombinant adenoviruses with BMP9-2,BMP2,BMP9 and GFP respectively.The induction of differentiation of C3H10 cells to osteogenesis by co-expression of BMP9-2 was observed by alkaline phosphatase(ALP) staining,alkaline phosphatase quantitation and calcium-alizarin red staining.Results The co-expression of BMP9-2 induced the expression of ALP in C3H10 cells.The positive rate of ALP in BMP9-2 group was 1.6 times higher than that in BMP2 group,2.5 times higher than that in BMP9 group,and 4 times higher than that in GFP group.The activities(A520) of ALP in BMP9-2 group 5,7 and 9 d after infection were higher than those in BMP2,BMP9 and GFP groups.The co-expression of BMP9-2 induced the sedimentation of calcium salt in C3H10 cells.Calcified tuberculum was observed in C3H10 cells 14 d after infection with BMP9-2 by calcium-alizarin red staining.The sedimentation level in BMP9-2 group was 1.4 times higher than that in BMP2 group,1.3 times higher than that in BMP2 group,and 5.2 times higher than that in GFP group.Conclusion The co-expression of BMP9-2 may induce the differentiation of C3H10 cells into osteoblasts,of which the ability in inducing osteogenesis is higher than those of BMP2 and BMP9.

    2013 01 v.26 [Abstract][OnlineView][Download 311K]

  • Screening of virulence-associated genes of Edwardsiella tarda based on disturbance of transcription machinery

    WANG Xiao-bo,YE Jiang,SONG Shan-shan,WANG Ke-ping,ZHANG Hui-zhan State Key Lab of Bio-reactor and Engineering,East China University of Science and Technology, Shanghai 200237,China

    Objective The whole transcription process was disturbed by over-expression of σE factor of transcription machinery(RNA polymerase) so as to screen the virulence-associated genes of Edwardsiella tarda.Methods The rpoE gene encoding the σE factor of RNA polymerase was amplified from E.tarda EIB202 by PCR and inserted into vector pACYC184,based on which a recombinant E.coli strain for over-expression of rpoE gene was established.Mutation was introduced during cloning of rpoE gene to inactivate the gene expression,based on which a recombinant E.coli strain with shift mutation was established.The LD50 of various strains were determined by infection of zebra fish model,while the transcription levels of rpoE,degP,fkpA,plsB,lpxD and yfiO mRNAs by RT-PCR.Results Restriction analysis and sequencing proved that recombinant plasmid pACYC184-rpoE was constructed correctly.The virulences of recombinant E.coli strain for over-expression of rpoE gene,at concentrations of 102,103,104 and 105 CFU / g,showed no significant difference with those of E.coli transfected with empty vector(P > 0.05).The transcription level of rpoE mRNA in the recombinant E.coli strain for over-expression was 9.5 times higher than that in the E.coli strain transfected with empty vector.However,as compared with those in recombinant E.coli strain with shift mutation,the transcription levels of degP,fkpA,plsB,lpxD and yfiO mRNAs increased by 6.9,2.64,2.85,2.38 and 1.34 folds respectively.Conclusion Recombinant E.coli strain for over-expression of rpoE gene of E.tarda as well as that with shift mutation were successfully established,and degP,fkpA,plsB,lpxD and yfiO was preliminarily identified as virulence-associated genes of E.tarda.

    2013 01 v.26 [Abstract][OnlineView][Download 223K]

  • Effect of Apg-2 gene silence on proliferations of BaF3-MIGR1 and BaF3-P210 cells

    CHEN Xi,ZHONG Liang,XIAO Qing,LI Chun-li,LI Ya-juan,WANG Hai-xia,FENG Wen-li Department of Clinical Hematology,Key Laboratory of Laboratory Medical Diagnostics of Education Ministry, Chongqing Medical University,Chongqing 400016,China

    Objective To investigate the effect of Apg-2 gene silence on proliferations of BaF3-MIGR1 cells transfected with empty vector pMIGR1 and BaF3-P210 cells for stable expression of Bcr-Abl fusion gene,and lay a foundation of further study on role of Apg-2 in Bcr-Abl positive CML cells.Methods Three shRNA sequences Hspa41,Hspa42 and Hspa43 targeting to nucleotides 609 ～ 629,845 ～ 865 and 2 110 ～ 2 130 of Apg-2 gene respectively,as well as negative control sequence HspaHK,were synthesized and transfected to BaF3-MIGR1 and BaF3-P210 cells by electroporation.The cell strains stably inhibiting Apg-2 gene were screened with G418,and determined for expressions of Apg-2 at mRNA and protein levels by RT-PCR and Western blot respectively,for proliferation by Am-blue method,for clone formation ability by methyl cellulose assay,and for cell cycle by flow cytometry.Results Of the three shRNA plasmids,shRNA-Hspa42 shwoed a satisfactory interfering effect.After Apg-2 expression was inhibited,both the proliferation and clone forming ability of BaF3-P210 cells decreased significantly(P < 0.05),while the percentage of cells at G1 phase increased signifi-cantly(P < 0.05) and those at S phase decreased significantly(P < 0.05).However,both the proliferation and clone for-mation ability of BaF3-MIGR1 cells increased significantly(P < 0.05),while the percentage at S phase increased signifi-cantly(P < 0.05).Conclusion The interference of Apg-2 gene expression inhibited the proliferation of Bcr-Abl positive BaF3-p210 cells while promoted the proliferation of Bcr-Abl negative BaF3-MIGR1 cells.

    2013 01 v.26 [Abstract][OnlineView][Download 214K]

  • Eukaryotic expression of domain deletion mutants of UHRF2

    BAI Lu,WANG Xiao-hui,HU Bin,ZHOU Dan-lin,DUAN Chang-zhu Department of Cell Biology and Genetics,Molecular Medicine & Cancer Research Center, Chongqing Medical University,Chongqing 400016,China

    Objective To construct domain deletion mutants of UHRF2 and express in HEK293 cells.Methods Five domain deletion mutants of UHRF2 were designed according to the location of domains.The genes encoding △UBL,△RING and △YDG + △RING were amplified directly by PCR using recombinant plasmid pCMV-3xFlag-UHRF2 as a template,while those encoding △PHD and △YDG by overlapping PCR.The amplified genes were cloned into eukaryotic expression vector pCMV-3xFlag.The constructed recombinant plasmids were transfected to HEK293 cells in which the expression of recombinant protein was identified by Western blot.Results The length of PCR product of △UBL was 2 018 bp,while those of upstream,downstream and upstream + downstream △PHD were 987,1 152 and 2 272 bp,those of upstream,downstream and upstream + downstream △YDG were 1 232,692 and 1 827 bp,and those of △RING and △YDG + △RING 2 163 and 1 287 bp,respectively.Restriction analysis and sequencing proved that all the expression vectors for domain deletion mutants were constructed correctly.All the relative molecular masses of expressed proteins in HEK293 cells transfected with the recombinant plasmids were consistent with those in theory.Conclusion Domain deletion mutants of UHRF2 were successfully expressed in HEK293 cells,which laid a foundation of further study on functions of various domains of UHRF2 as well as their interaction sites with other proteins.

    2013 01 v.26 [Abstract][OnlineView][Download 291K]

  • Prokaryotic expression of F41 pilus protein of enterotoxigenic Escherichia coli from bovine origin and preparation of its polyclonal antibody

    NI Hong-bo,LANG Xiu-yan,SHAO Guang-xi,ZHOU Yu-long,YAN Qiu-long,LIU Yin-bing, GONG Da-qing,WANG Chun-ren College of Animal Science Veterinary Medicine,Heilongjiang Bayi Agricultural University,Daqing 163319, Heilongjiang Province,China

    Objective To express F41 pilus protein of enterotoxigenic Escherichia coli(ETEC) from bovine origin in prokaryotic cells and prepare its polyclonal antibody.Methods F41 pilus gene was amplified by PCR using the genome of ETEC as a template,and inserted into prokaryotic expression vector pQE30.The constructed recombinant plasmid was transformed to E.coli XL1-Blue for expression under induction of IPTG.The expressed protein was identified by SDS-PAGE,purified by cutting the gel and inoculated to New Zealand rabbits.The prepared polyclonal antibody was determined for titer by indirect ELISA and for reactogenicity by Western blot.Results Restriction analysis and sequencing proved that recombinant plasmid pQE-30-F41 was constructed correctly.The expressed F41 pilus protein existed in a form of inclusion body,with a relative molecular mass of about 32 000,which contained about 35% of total somatic protein and reached a purity of 92% after purification.The prepared antiserum reached a titer of more than 1 ∶ 2.56 × 106,and showed good reactogenicity as proved by Western blot.Conclusion Recombinant F41 pilus protein was successfully expressed in prokaryotic cells and purified,and high titer polyclonal antibody was prepared,which laid a foundation of preparation of monoclonal antibody and development of immunological method for determination of E.coli F41 pilus protein.

    2013 01 v.26 [Abstract][OnlineView][Download 238K]

  • Construction and identification of recombinant adenovirus vector for expression of shRNA targeting phospholipase C epsilon gene

    ZHANG Yan-yi,OU Li-ping,LIU Qi,TAO Jia,WU Xiao-hou,LUO Chun-li College of Laboratory Medicine,Key Laboratory of Laboratory Medical Diagnostics of Education Ministry,Chongqing Medical University,Chongqing 400016,China

    Objective To construct the recombinant adenovirus vector for expression of shRNA targeting phospholipase C epsilon(PLCε) gene,with a green fluorescent protein(GFP) tag,and lay a foundation of gene therapy of bladder cancer by gene silencing technique.Methods The U6 expression promoter and shRNA sequences of interfering plasmid pGenesil-PLCε and negative control plasmid pGenesil-NP were cloned to shuttle plasmid pAdTrack with enhanced GFP(EGFP) report gene.The constructed recombinant shuttle plasmid pAdTrack-U6-PLCε was identified by restriction analysis and sequencing,then linearized with PmeⅠand transformed to competent AdEasier.The constructed recombinant plasmid pAdEasy-U6-PLCε was linearized with PacⅠ and transfected to HEK-293 cells for packaging.The obtained recombinant adenovirus was propagated in a large quantity and determined for titer.The expressions of PLCε in T24 cells were determined by RT-PCR and Western blot.Results Restriction analysis and sequencing proved that recombinant plasmids pAdTrack-U6-PLCε and pAdEasy-U6-PLCε were constructed correctly and transfected to HEK-293 cells successfully.The titer of recombinant adenovirus was 1.5×1012.Both the expression levels of PLCε mRNA and protein in T24 cells infected with the recombinant adenovirus decreased significantly(P < 0.05).Conclusion The recombin antadenovirus vector for expression of shRNA targeting PLCε gene was successfully constructed,which laid a foundation of study on role of PLCε in ongoing of bladder cancer by gene silencing technique.

    2013 01 v.26 [Abstract][OnlineView][Download 281K]

  • Effect of Ezrin gene knockdown and over-expression on invasion of glioma U87 cells

    LIU Nai-jie,QIN Zhi-gang,SUN Li-bo,JIN Xing-yi,YE Bao-guo,ZHANG Jin-nan,ZHU Qing-san China-Japan Union Hospital,Jilin University,Changchun 130031,China

    Objective To study the relationship between Ezrin gene and infiltrative growth of glioma through Ezrin gene knockdown and over-expression.Methods According to Ezrin gene sequence in GenBank,primers were designed using Prime Primer 5.0 software,with which the gene fragment encoding CDS region of Ezrin gene was amplified from U87 cells and cloned into expression vector pEGFP-1.U87 cells were transfected with the constructed recombinant plasmid pEGFP-C1/Ezrin as well as plasmids shRNA-EZrin-2 and pEGFP-C1 in mediation of LipofectimineTM 2000 respectively,then determined for expression of Ezrin protein by Western blot,and for migration by scarification test.Results The homologies of mRNAs of cloned Ezrin gene were 99% to those of homo sapiens ezrin(EZR) and transcript variant 1 reported in GenBank.The homologies of amino acids encoding by the cloned gene was 99% to that of ezrin [Homo sapiens](Sequence ID: ref-NP_003370.2-,Length:586),with variations of S66P,K258R,P265L and K577R,while the opening read frame was correct.The relative expression level(1.17) of Ezrin protein in U87 cells transfected with recombinant plasmid pEGFP-C1 / Ezrin was higher those transfected with shRNA-Ezrin-2(0.47) and pEGFP-C1(0.82).Scarification test showed migration of a small quantity of U87 cells transfected with shRNA-Ezrin-2 to the wound.However,in pEGFP-C1 / Ezrin transfection group,the wound was almost filled with cells.Conclusion Ezrin gene knockdown blocked,while the over-expression promoted the migration of U87 cells,indicating that Ezrin gene involved in the invasive growth of U87 cells.

    2013 01 v.26 [Abstract][OnlineView][Download 1020K]

  • Effect of adiponectin on expression and function of ATP binding cassette transporter A1 in macrophages

    AI Qing,MA Kang-hua College of Basic Medicine,Chongqing Medical University,Chongqing 400016,China

    Objective To investigate the effect of adiponectin on expressions of ATP binding cassette transporter A1(ABCA1) and its upstream regulatory factor liver X receptor α(LXRα) in macrophages,as well as the possible action mechanisms of ABCA1 and LXRα in reverse cholesterol transport(RCT) and anti-atherosclerosis(AS).Methods RAW264.7 cells were cultured with adiponection at various concentrations(0,1,5 and 10 μg / ml) in vitro for 24 h respectively,then determined for transcription levels of ABCA1 and LXRα mRNAs by RT-PCR,for expression levels of ABCA1 and LXRα proteins by Western blot,and for cholesterol efflux by scintillation counting method.Results Adiponectin showed significantly dose-dependent up-regulating effect on ABCA1 and LXRα mRNA and protein levels in RAW264.7 cells(P < 0.05),while increased the cholesterol efflux in a dose-dependent pattern(P < 0.05).Conclusion Adiponectin up-regulated the transcription and translation levels of ABCA1 gene by LXRα route,promoted reverse cholesterol transport,and delayed the occurrence and development of atherosclerosis.

    2013 01 v.26 [Abstract][OnlineView][Download 230K]

  • Preparation of human enterovirus 71 immunoglobulin (pH 4) for intravenous injection

    LIU Lan-jun,YANG Chun,WU Zhi-qiang,QIN Ting-ting,LI Xiao-jiao,LIU Rui-xi, LIU Bo,ZHANG Xue-mei,YANG Hui-chuan Chengdu Ronsen Pharmaceuticals Co.Ltd.,Chengdu 610061,China

    Objective To prepare human enterovirus 71 immunoglobulin(pH 4) for intravenous injection(EV71-IVIG) used for treatment of severe hand,foot and mouth disease(HFMD).Methods The EV71 neutralizing antibody titer(EV71-NT) in raw plasma was determined by cytopathic effect(CPE) inhibition assay,based on which the standard for screening of plasma containing EV71 antibodies was established.The plasma containing high neutralizing antibody titer against EV71 was screened from healthy human plasma,with which three consecutive batches of EV71-IVIG were prepared according to the requirements in Chinese Pharmacopoeia(Volume Ⅲ,2010 edition),then evaluated for therapeutic effect in suckling mouse model,and determined for neutralizing activity to clinical isolates of EV71 and CA16 virus.Results About 3.8 tons of plasma containing EV71 antibody was screened from 90 batches of raw plasma,with which the three batches of prepared EV71-IVIG met the requirements for intravenous human immunoglobulin(pH 4) in Chinese Pharmacopoeia(Volume Ⅲ,2010 edition).The EV71-NT in prepared EV71-IVIG was 4 ～ 5 times higher than that in domestic common IVIG.The protection rate of EV71-IVIG at a dosage of 0.5 mg to the suckling mice challenged with 10 LD50 EV71 was 100%.EV71-IVIG showed high neutralizing activity against recent clinical EV71 isolates in China mainland.Conclusion EV71-IVIG was successfully prepared,which provided a promising drug option for the therapy of patients with severe hand,foot and mouth disease(HFMD).

    2013 01 v.26 [Abstract][OnlineView][Download 167K]

  • Influence of FTY720 on differentiation of osteoclast precursor cells RAW264.7 induced by polyethylene particles

    ZHOU Yuan-dong,WANG Xin,LI Ping,WAN Rui,CHENG Ting-mei,ZHANG Jian Department of Orthopaedics,The First Affiliated Hospital of Chongqing Medical University,Chongqing 400016,China

    Objective To observe the influence of FTY720 on differentiation of osteoclast precursor cells(RAW264.7) induced by polyethylene particles,and investigate the possibility of prevention and treatment of aseptic loosening of artificial joint with FTY720.Methods Transwell Millipore co-culture model systems of osteoclasts(RAW264.7)-osteoblasts(MC3T3) and osteoclasts-bone piece were established,then FTY720 was used to intervene the differentiation of RAW264.7 cells irritated by polyethylene wearing particles,and the morphology of differentiated cells was observed under invert microscope.The osteoclasts were counted by tartrate-resistant acid phosphtas(TRAP) staining,and observed for general morphology and bone adsorption effect by scanning electron microscopy(SEM).The secretion levels of tumor necrosis factor-α(TNF-α) and interleukin-6(IL-6) in co-culture system were determined by ELISA.The transcription levels of receptor activator of NFκB(RANK) and TRAP mRNAs were determined by RT-PCR.Results The RAW264.7 cells treated with polyethylene particles were large in size and rich in cytoplasm and nuclei,of which the border was irregular and in cloudy shape.The count of TRAP positive osteoclasts treated with FTY720 was significantly smaller than that with polyethylene particles(P < 0.01).The osteoclasts were round,which were connected each other by fibroid foot processes.SEM showed attachments of osteoclasts on bone pieces.As compared with those in polyethylene particle group,both the number of absorption trapped concave and area of bone absorption in FTY720 group were significantly small(P < 0.01).The secretions of TNF-α and IL-6 increased significantly in polyethylene particle group,while were inhibited in FTY720 group.Both the transcription levels of TRAP and RANK mRNAs on surface of osteoclasts in FTY720 group were significantly lower than those in polyethylene particle group(P < 0.01).Conclusion FTY720 effectively inhibited the differentiation of osteoclast precursor cells(RAW264.7),decreased the formation of osteoclasts as well as bone osteolysis absorption and the secretion of inflammatory factors such as TNF-α and IL-6,and down-regulated the transcriptions of RANK and TRAP mRNAs,thus might be used as a drug for prevention and treatment of aseptic loosening of artificial joint.

    2013 01 v.26 [Abstract][OnlineView][Download 416K]

  • Development of a method for quality control of humanized monoclonal antibody against epithelial growth factor receptor

    TAO Lei,RAO Chun-ming,WANG Lan,HAN Chun-mei,LI Xiang,GAO Kai,WANG Jun-zhi Key Laboratory of Ministry of Health for Research on Quality and Standardization of Biotech Products,National Institute for Food and Drug Control,Beijing 100050,China

    Objective To develop a method for quality control of humanized monoclonal antibody(McAb) against epithelial growth factor receptor(EGFR).Methods Humanized McAb against EGFR was determined for biological activity by in vitro cell growth inhibition test,and for relative molecular mass by mass spectrometry and reduced SDS-PAGE.After deblocking by Edman degradation,the amino acids at N-terminus of the McAb were sequenced,while the peptide map was analyzed by RP-HPLC,and the residual exogenous DNA was determined by quantitative PCR and slot hybridization.Other tests were carried out according to requirements in Chinese Pharmacopoeia(Volume Ⅲ,2010 edition).Results The relative biological activities of bulk and final product of McAb against EGFR were(100 ± 20) % and(94 ± 14) % respectively.The relative molecular masses of light and heavy chains of the McAb reference measured by ESI-MS were 23 370.75 and 50 341.00,with relative errors of 0.005% and 0.006%,while those measured by reduced SDS-PAGE were 27 000 and 53 400,respectively.All the amino acid sequences at N-terminuses of light and heavy chains were consistent with those in theory.The purity of bulk of the McAb were 98.5% by reduced SDS-PAGE and(98.39 ± 0.05)% by SEC-HPLC.However,the purity of final product of the McAb determined by SEC-HPLC was(98.17 ± 0.04)%.The peptide map was consistent with that of reference.The residual exogenous DNA content determined by quantitative PCR was less than 100 pg / dose.Other quality indexes met the requirements in Chinese Pharmacopoeia(Volume Ⅲ,2010 edition) and other relevant requirements.Conclusion A method for quality control of McAb against EGFR was successfully developed,which might be used for the routine quality control of the product.

    2013 01 v.26 [Abstract][OnlineView][Download 286K]

  • Effect of liposome nanoparticles of hydrazinocurcumin on biological behavior of mouse breast cancer 4T1 cells

    TIAN Wen-xia,ZHANG Xi-wen,DANG Wei-qi,TANG Hao,WANG Lin,CAO Hong,CHEN Ting-mei Key Laboratory of Laboratory Diagnostic Medicine Designated by Ministry of Education,College of Laboratory Medicine,Chongqing Medical University,Chongqing 400016,China

    Objective To investigate the effect of liposome nanoparticles of hydrazinocurcumin(HC-NPs) on proliferation,apoptosis,invasion and migration of mouse breast cancer 4T1 cells.Methods Liposome HC-NPs were prepared by film-sonication method,and determined for size by transmission electron microscopy.4T1 cells were divided into three groups and treated with 10%(v / v) PBS,10%(v / v) NP and 10%(v / v) HC-NPs for 24 h respectively,then determined for proliferation activity by MTT method,for cell cycle and apoptosis rate by flow cytometry,for morphological change by Liu staining,for invasion and migration abilities by Transwell test,and for expressions of STAT3 and proteins related to cell proliferation,apoptosis,invasion and migration by Western blot.Results Liposome HC-NPs each at a diameter of about 150 nm were observed under electron microscope.The inhibitory rate of HC-NPs to proliferation of 4T1 cells was significantly higher than that of NPs(P < 0.01).Compared with those in PBS and NPs groups,the morphology of cells in HC-NPs was irregular,while the cell cycle was arrested in G2 / M phase,the apoptosis rate increased significantly(P < 0.01),and the invasion and migration of cells as well as activation of STAT3 were inhibited significantly(P < 0.01).The expressions of CyclinD1,c-Myc,Bcl-2,Survivin,MMP-9 in cells treated with NC-NPs were down-regulated,while that of Bax was up-regulated.Conclusion HC-NPs may promote the apoptosis of 4T1 cells by inhibiting the activation of STAT3 as well as proliferation,invasion and migration of cells.

    2013 01 v.26 [Abstract][OnlineView][Download 350K]

  • Analysis of quality of cryoprecipitate-reduced plasma

    MA Li,SUN Pan,LIN Fang-zhao,DIAO Ge,LI Jian-ping,ZHANG Hong-yi,LIU Jian-qiang, ZHANG Xin-sheng,BAI Ze-rong,ZHOU Jing-yu,LI Mei-jun,LI Chang-qing Institute of Blood Transfusion,Chinese Academy of Medical Sciences,Chengdu 610052,Sichuan Province,China

    Objective To compare the levels of coagulation factors,fibrinogen(Fib) and Von Willebrand factor cleaving protease,a disintegrin-like and metalloprotease with thrombospondin-1 repeats 13(ADAMTS13),in cryoprecipitate-reduced plasma(CRP).Methods CRP was prepared with the fresh frozen plasma(FFP) from 140 donors.The coagulation-promoting activities of FⅡ,FⅣ,FⅦ,FⅧ,FⅨ,FⅩ,FⅪ and FⅫ in FFP and CRP were determined by one-stage Biggers method,while the activities of Fib and Von Willebrand factor antigen(vWF ∶ Ag) by immune turbidimetry.The activity and antigen contents of ADAMTS13 in FFP and CRP were determined by fluorescence resonance energy transfer(FRET) assay.Results As compared with those in FFP,the levels of FⅧ ∶ C,FⅤ ∶ C,FⅦ ∶ C,FⅨ ∶ C,FⅩ ∶ C,FⅪ ∶ C,Fib,vWF ∶ Ag decreased significantly(P < 0.05 or P < 0.001),while the antigen content and activity of FⅡ ∶ C,FⅫ ∶ C and ADAMTS13 showed no significant difference(P > 0.05).Conclusion CRP may be used for treatment of thrombotic thrombocytopenic purpura(TTP) instead of CRP,while is unsuitable for complementary treatment of FⅧ,Fib and vWF deficiency.

    2013 01 v.26 [Abstract][OnlineView][Download 127K]

  • Effect of tamoxifen on proliferation of human endometfial carcinoma RL-95-2 cells and expression of let-7g

    LIU Zu-cui,WEI Sha-li,CHEN Guo-qing,CHEN Yu,LIU Ge-li Laboratory of Stem Cell and Tissue Engineering,Chongqing Medical University,Chongqing 400016,China

    Objective To investigate the effect of tamoxifen(TAM) on proliferation of human endometfial carcinoma RL-95-2 cells and expression of let-7g.Methods The expression of estrogen receptor(ER) in RL-95-2 cells was determined by immunofluorescence staining.RL-95-2 cells were respectively treated with TAM at various concentrations(1 × 10-11,1 × 10-9,1 × 10-7,1 × 10-5 and 1 × 10-4 mol / L for 48 and 72 h,and determined for proliferation activity by CCK-8 method.RL-95-2 cells were treated with 1 × 10-7 mol / L TAM for 72 h and determined for cell cycle by flow cytometry.RL-95-2 cells were treated with TAM at various concentrations for 72 h and determined for expression of let-7g by real-time quantitative PCR(RT-qPCR).The target gene of let-7g was predicted with the help of bioinformatics tools and analyzed for biological function.Results RL-95-2 cells were positive for ER.The treatment with TAM at a concentration of 1 × 10-7 mol / L for 48 h or with those at 1 × 10-9 ～ 1 × 10-5 mol / L for 72 h promoted the cell proliferation and increased the expression of let-7g,of which the effect of TAM at a concentration of 1 × 10-7 mol / L was the most satisfactory.TAM promoted the change of cell cycle of RL-95-2 cells from G0 / G1 phase to S phase.The possible target genes of let-7g included UHRF2,RB1,GAS7,PLAGL2,NAB1 and ZNF282.Conclusion The TAM at an appropriate concentration promoted the proliferation and the change of cell cycle from G0 / G1 phase to S phase of RL-95-2 cells by a possible mechanism partially associated with the up-regulation of 1et-7g expression.

    2013 01 v.26 [Abstract][OnlineView][Download 267K]

  • Expression and antiserum preparation of structural protein of Chinese sacbrood virus

    SHEN Ke-fei,ZHANG Yi-fan,CAO Lan,WANG Rui-sheng,YANG Jin-long,YANG Liu,DAI Rong-guo Chongqing Academy of Animal Sciences,Chongqing 402460,China

    Objective To express the structural protein of Chinese sacbrood virus(CSBV) in prokaryotic cells and prepare its antiserum.Methods The gene fragment encoding structural protein of CSBV was amplified by RT-PCR and cloned into vector pGEX-4T-1.The constructed recombinant plasmid pGEX-CSBV was transformed to E.coli BL21(DE3) for expression under induction of IPTG.The expressed recombinant protein was purified by Gluthathione-Sepharose 4B affinity chromatography and used for immunization of mice.The prepared antiserum as identified by Western blot.Results Colony PCR proved that recombinant plasmid pGEX-CSBV was constructed correctly.The expressed recombinant protein,with a relative molecular mass of about 52 000,existed in a soluble form and reached a purity of more than 95% after purification.The prepared antiserum recognized the structural protein of natural CSBV.Conclusion The structural protein of CSBV was successfully expressed in E.coli,which laid a foundation of further study on interaction between sacbrood virus and its host as well as development of serological diagnostic method.

    2013 01 v.26 [Abstract][OnlineView][Download 226K]

  • Pneumonia vaccination coverage and influencing factors among part of community elderly in Chaoyang District,Beijing City,China

    ZHANG Guo-hui,ZHENG Dong-yi,SHI Nian-min,AI Xing,BAI Yun-hua Center for Disease Prevention and Control of Chaoyang District,Beijing City,Beijing 100021,China

    Objective To analyze the pneumonia vaccination coverage as well as the influencing factors among part of community elderly in Chaoyang District,Beijing City,China,so as to improve the pneumonia vaccination and reduce the harm of pneumonia infection in the population.Methods A questionnaire survey was performed on partial community elderly at ages of not less than 60 years in Chaoyang District by cluster sampling method.The data on basic information,physical status,medical behavior and vaccination of the subjects were collected,based on which the influencing factors of pneumonia vaccination of community elderly were analyzed.Results The result showed the pneumonia vaccination rate of the community elderly was 2.1%.Logistic regression analysis showed that the main influencing factors of pneumonia vaccination rates were age,education background and physical status(P < 0.05).Conclusion Effective measures should be taken to increase the pneumonia vaccination coverage in target population including elderly.Intervention should be focused on the elderly relatively young and those with better physical status and at relatively low education level.

    2013 01 v.26 [Abstract][OnlineView][Download 120K]

  • Screening of mutation site of LEPRE1 gene in patients with osteogenesis imperfecta

    XU Chao,REN Xiu-zhi,WANG Yan-zhou,HAN Jin-xiang,WANG Zi-qiang,LU Yan-qin Shandong Medicinal Biotechnology Centre,Shandong Academy of Medical Sciences,Jinan 250062,China

    Objective To screen autosomal-recessive pathogenic gene LEPRE1 in patients with osteogenesis imperfecta(OI) who were negative in COL1A1 or COL1A2 gene mutant.Methods Peripheral blood samples were collected from patients with OI,from which genomic DNA was extracted for amplification of LEPRE1 gene by PCR.The PCR product was tested for mutation by direct sequencing.The effects of mutation on function of protein were analyzed by PolyPhen,Align GVGD and SIFT functional prediction software.Results A heterozygous mutation(c.1045G>A,p.Gly349Arg) in exon 5 of LEPRE1 gene were found in one patient as well as her mother and maternal grandfather,while were unfound in other healthy family members and 200 control samples from healthy persons.All the PolyPhen,Align GVGD and SIFT predictions supported that the mutation of p.Gly349Arg might affect the normal function of protein.Conclusion Substitution of G with A in site 1045 of LEPRE1 gene might be a potential pathogenic mutation in the reported patient with OI.

    2013 01 v.26 [Abstract][OnlineView][Download 297K]

  • Optimization of fermentation condition of recombinant E.coli for expression of glutamate decarboxylase

    XUE Zheng-lian,ZHANG Ke Institute of Biologic & Chemical Engineering of Anhui Polytechnic University,Wuhu 241000,Anhui Province,China

    Objective To optimize the fermentation condition of recombinant E.coli for expression of glutamate decarboxylase(GAD).Methods The time point,IPTG concentration,time duration and temperature for induction of recombinant E.coli B3C1 for expression of GAD were optimized by single factor test,while the fermentation condition,including the contents of sodium chloride,peptone,yeast powder,shaking speed of incubator shaker,initial pH value,culture time,MOI and liquid volume,by response surface assay.Results The optimal time point,concentration,time duration and temperature for induction with IPTG were 4 h after inoculation,0.8 mmol / L,4 h and 35℃ respectively.The optimal contents of yeast powder,sodium chloride,peptone,culture time,shaking speed of incubator shaker,initial pH value,MOI and liquid volume for fermentation of recombinant E.coli B3C1 were 1.01%,0.6%,1.5%,11.25 h,250 r / min,6.5,3.0% and 11.70 ml respectively.The activity of GAD expressed in E.coli under the fermentation condition reached 1 227.33 U,which increased by 12.29% as compared with that before optimization.Conclusion The fermentation condition of recombinant E.coli for expression of GAD was successfully optimized,which laid a foundation of industrial production of GAD.

    2013 01 v.26 [Abstract][OnlineView][Download 310K]

  • Optimization of culture condition of enterovirus 71 in Vero cells

    CHEN Li,JIANG Yuan-xiang,XIA De-zhen,YANG Zhi-jian,ZHANG Gao-xia Vaccine Research and Development Center,Shanghai Zerun Biotechnology Co.Ltd.,Shanghai 201203,China

    Objective To optimize the culture condition of enterovirus 71(EV71) in Vero cells.Methods EV71 was inoculated to Vero cells by adsorption method and direct inoculation method respectively for propagation and determination of titer.The virus was inoculated by the optimal method,based on which the effects of serum concentration,harvest method,MOI and harvest time on virus titer were evaluated.Results After EV71 was inoculated directly to serum-free medium at a MOI of 0.02 ～ 0.2 and cultured for 60 h,the virus harvested in supernatant reached a high titer.Conclusion The culture condition of EV71 in Vero cells was optimized,which laid a foundation of large-scale culture of EV71 and development of EV71 vaccine.

    2013 01 v.26 [Abstract][OnlineView][Download 272K]

  • Graph pattern of Hansenula polymorpha-derived hepatitis B surface antigen purified by hydrophobic interaction chromatography

    XU Ning,ZHANG De-you,MA Rui,FNEG Xiao-lei,ZHANG Xu,YANG Xu-qin,LI Cai-mei,LIU Qiang, SHEN Yong-cai,LI Jin Beijing Tiantan Biological Products Co.,Ltd,Beijing 100024,China

    Objective To investigate the relationship of graph pattern of Hansenula polymorpha(HP)-derived hepatitis B surface antigen(HBsAg) purified by hydrophobic interaction chromatography(HIC) to HBsAg and protein contents.Methods Eight batches of the HP-HBsAg samples for small-scale preparation,five batches of samples for small-scale verification test and five batches of Saccharomyces cerevisae(SC)-derived HBsAg as control were purified by hydropobic chromatography,and determined for protein and HBsAg contents by Lowry method and electrochemiluminescent immunoassay(ECLIA),based on which the recovery rate of HBsAg was calculated.The UV-monitored graph was geometrically divided into several sections,of which the area was integrated by digital planimeter.The main and side peaks of one batch of samples for small-scale verification test were collected for electron microscopy.Results The mean recovery rate of eight batches of HP-HBsAg samples for small-scale preparation was 70%,while that of five batches for small-scale verification test was 54%.The HIC pattern consisted of breakthrough peak and target(HBsAg) peak,while the target peak appeared as a main peak and a side peak.The pattern of control samples appeared as breakthrough peak and target peak.Further quantitative analysis of the target peak showed that the area ratios of main,side and target peaks were related to those of collected liquid,protein and HBsAg contents.Electron microscopy showed that the sizes of HBsAg particles were even in the collected main peak,while were uneven in the side peak.Conclusion HIC was effective for purification of HP-derived HBsAg.By comparison of peak area graphed by UV-monitor,the contents of collected liquid,protein and HBsAg were calculated.It provided a reference for further purification of HBsAg.

    2013 01 v.26 [Abstract][OnlineView][Download 331K]

  • Trichloroacetic acid precipitation method for determination of 125I-rhIL-12 content in vivo of cynomolgus monkeys

    LI Shu-sui,LI Ruo-bing,LI Ru-bing,LI Jian,CHEN Xin,SUN Zhi-ping,ZHANG Yi-jun,FU Yong-hang Department of Pharmacy,Maternal and Child Health Hospital in Tianhe District,Guangzhou City,Guangzhou 510630,Guangdong Province,China

    Objective To preliminarily apply and verify trichloroacetic acid(TCA) precipitation method for determination of 125I-rhIL-12 content in vivo of cynomolgus monkeys.Methods The 125I-rhIL-12 contents in vivo of cynomolgus monkeys were by Na 125I and TCA precipitation.The developed method was verified for linearity,precision and recovery rate.Cynomolgus monkeys were injected s.c.with a single dose of 2 μg / kg 125I-rhIL-12,of which urine and stools were collected 4,8,24,32,48,72,96,120,144 and 168 h after injection,and determined for radioactivity by the developed method to calculate the percentage of output radioactivity in input radioactivity.Results The titer of 125I-rhIL-12 showed no significant difference with that unlabeled.The 125I-rhIL-12 contents in various tissues / body liquids,at a range of 0.02 ～ 200 ng-equivalent / g,showed good linear relationship to total radioactivity and TCA precipitation radioactivity,with most of CV values of less than 20%.The 125I-rhIL-12 mainly existed in TCA precipitate.The radioactivity of 125I-rhIL-12 was mainly excreted by urine,a small part of which by stool,and was completely excreted 168 h after subcutaneous injection.Conclusion 125I was combined stably with rhIL-12 in vivo,and TCA precipitation method was suitable for determination of 125I-rhIL-12 content in vivo of cynomolgus monkeys.

    2013 01 v.26 [Abstract][OnlineView][Download 346K]

  • Development of light induced chemiluminescent immunoassay for prostate specific antigen

    LIU Zhi-yong,REN Jie,LI Chan,LI Xiao-lei,CHEN Xin-ying,LI Hui-qiang Department of Immunology,Tianjin Medical University,Tianjin 300070,China

    Objective To develop a light induced chemiluminescent immunoassay(LICA) for quantitative determination of prostate specific antigen(PSA).Methods A LICA kit was developed,which consisted of the receptor particles coated with polyclonal antibody against PSA,the monoclonal antibody against PSA coated with biotin,and the donor particles coated with streptavidin.The condition for determination was optimized,and the developed method was verified.A total of 82 clinical samples were determined by the developed kit and electrochemiluminescent(ECL) immunoassay kit(Roche),and the results were compared.Results The analytical sensitivity of the developed method was 0.31 ng / ml.The intra-and inter-coefficients of variation of determination results were 4.66% ～ 6.75% and 5.68% ～ 8.42% respectively,while the recovery rate was 95.1% ～ 105.2%.The determination result by LICA showed high correlation with that by ECL immunoassay,of which the R2 value was 0.981 5.Conclusion The developed homogeneous chemiluminescent immunoassay method may be used for the quantitative detection of serum PSA,of which the performance meets the clinical requirements.

    2013 01 v.26 [Abstract][OnlineView][Download 179K]

  • Determination of content of nonoxynol-9 as a cleavage reagent in influenza virus subunit vaccine by two methods

    TIAN Wen-li,YANG Jiang-shan,WAN Ya-fen,QI Ji,YANG Xu Tasly Jenner Bio-tech(Tianjin) Co,.Ltd.,Tianjin 300410,China

    Objective To compare the determination results of content of nonoxynol-9 as a cleavage reagent in influenza virus subunit vaccine by HPLC and UV spectrophotometry.Methods The nonoxynol-9 contents in intermediate and final product of influenza virus subunit vaccine were determined by HPLC and UV spectrophotometry separately,and the intermediate was analyzed by SDS-PAGE.Results The nonoxynol-9 contents in intermediate and final product,especially in intermediate,of influenza virus subunit vaccine determined by UV spectrophotometry increased by more than 10% as compared with those by HPLC.SDS-PAGE showed the nucleoprotein(NP) contents were significantly different in intermediates 1,2 and 3,which was the highest in intermediate 3.Conclusion HPLC may be used for determination of nonoxynol-9 content in influenza virus subunit vaccine instead of UV spectrophotometry.

    2013 01 v.26 [Abstract][OnlineView][Download 104K]

  • Optimization of procedure for spray drying of Bacillus subtilis from swine origin

    LIU Yu,LI Dan,WANG Yan,WU Bo,WANG Shuang,ZHU Dan-dan,SHI Tong-rui,ZHU Zhan-bo Veterinary Research Institute in Heilongjiang Province,Qiqihar 161006,Heilongjiang Province,China

    Objective To optimize the procedure for spray drying of Bacillus subtilis from swine origin and increase the survival rate of bacteria after spray drying.Methods The test was designed by using the Quadratic Orthogonal Rotation Combination Design in SAS V8.0 software,and the effect of various factors on response value was analyzed.Three repeat spray drying tests were performed by the optimized procedure to verify the accuracy of predicted result.The effect of germ formation rate on survival rate of bacteria after spray drying was evaluated.Results The optimal outlet air temperature,inlet air temperature,stabilizer concentration and liquid feed rate were 90 ℃,150 ℃,20% and 1 200 ml / h respectively.The mean survival rate of bacteria in three repeat spray drying tests was 75.7%,which was basically consistent with the predicted value(77%).The germ formation rate showed significant effect on the survival rate of bacteria after spray drying.Conclusion The procedure for spray drying of Bacillus subtilis from swine origin was successfully optimized,which laid a foundation of industrial production of B.subtilis spray.

    2013 01 v.26 [Abstract][OnlineView][Download 332K]

  • Development of reverse HPLC for monosialotetrahexosylganglioside sodium salt content in injection

    HE Dan,YANG Lin,ZHANG Jing-qing Medicine Engineering Research Center,Chongqing Key Laboratory of Biochemical & Molecular Pharmacology, Chongqing Medical University,Chongqing 400016,China

    Objective To develop a reversed phase high performance liquid chromatography(RP-HPLC) method for determination of monosialotetrahexosylganglioside sodium salt(GM1) content in injection and provide a reference for quality control of GM1 injection.Methods The chromatographic condition was as follows: Hypersil ODS C18 column(4.6 mm × 150 mm,5 μm) was selected,and acetonitrile-0.03% triethylamine solution(77 ∶ 23,the pH value was adjusted to 7.5 with phosphoric acid) was served as mobile phase;the detection wavelength,flow rate and volume of samples loaded were 205 nm,1.0 ml / min and 10 μl respectively;the number of theoretical plate calculated according to GM1 peak was not less than 2 000.The developed method was determined for linear range by linear regression of peak area against concentration,and verified for precision,stability,reproducibility and recovery rate.Three batches of GM1 injection were determined by the developed method.Results The developed method showed good linearity within a concentration range of GM1 of 50 ～ 800 μg / ml(r = 0.999 2).The method showed high reproducibility as well as high precisions within one day and on various days.The control solutions were basically stable within 8 h,and the mean recovery rate of samples was 99.74%.The GM1 contents in three batches of samples determined by the method were 96.80%,98.06% and 97.32% respectively.Conclusion A RP-HPLC method for determination of GM1 injection was developed,which was accurate,simple,rapid,reliable and suitable for quality control of GM1 injection.

    2013 01 v.26 [Abstract][OnlineView][Download 147K]

  • Progress in research on high expression of recombinant antibody(Ⅰ)

    HU Di-chao,ZHANG Ai-hua,YANG Xiao-ming Wuhan Institute of Biological Products Co.Ltd.,Wuhan 430060,China

    The high expression of recombinant antibody in mammalian cells is dependent on the construction of high expression vector,optimization of antibody gene sequence,screening of stable antibody-expressing cell strains,modification of host cells as well as optimization of cell culture process.The progress in research on above-mentioned problems is reviewed in this paper.

    2013 01 v.26 [Abstract][OnlineView][Download 214K]

  • Application of thimerosal as a preservative to vaccines for human use

    HE Peng,LIANG Zheng-lun National Institutes for Food and Drug Control,Beijing 100050,China

    Thimerosal is a preservative widely used in biological products such as vaccines.Safety issues of vaccines containing thimerosal has been argued since the end of last century.Safety related researches,position of authorized agencies,thimerosal-removing actions and problems remained to be solved on the usage of thiomersal in vaccines for human use is discussed in this review.

    2013 01 v.26 [Abstract][OnlineView][Download 182K]

  • Progress in research on New Delhi metallo-lactamase-1

    XIA Li-liang,SUN Yang,FENG Shu-zhang College of Animal Science and Veterinary Medicine,Jilin University,Changchun 130062,China

    A new metallo-lactamase,New Delhi metallo-lactamase-1(NDM-1),has been discovered,which can hydrolyze carbapenems.With a high hydrolytic activity,NDM-1 is resistant to almost all the antibiotics,which has been a threat to the health of humans.This paper reviews the prevalence of bacteria carrying NDM-1 as well as the gene arrangement,protein structure and enzyme activity site of NDM-1.

    2013 01 v.26 [Abstract][OnlineView][Download 400K]
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