2012年07期目次

  • Expression of glycoprotein of rabies virus in silkworm chrysalis by using Bac-to-Bac baculovirus expression system

    ZHAO Li-li*,SUN Wei-yang,FENG Hao,HU Gui-qiu,LI Ling,Li Lu,GUO He,ZHAO Ping-sen,WANG Hua-lei,YANG Song-tao,XIA Xian-zhu(*College of Animal Science and Veterinary,Jilin University,Changchun 130062,China)

    Objective To highly express the glycoprotein(G)of rabies virus(RABV)in silkworm chrysalis by using Bac-toBac baculovirus expression system.Methods The gene was amplified from total RNA of rabies virus CVS strain and inserted into plasmid pFastBacⅠvector.The constructed recombinant plasmid pFastBacⅠ-G was transformed to E.coli DH10Bac,and the resulted recombinant bacmid shuttle plasmid Bacmid-G was transfected to BmN cells.Silkworm chrysalis was infected with the obtained recombinant baculovirus,of which the hemolymph was collected and analyzed by Western blot.Results PCR and proved that recombinant baculovirus shuttle plasmid Bacmid-G was constructed correctly.The G protein of rabies virus was expressed correctly in BmN cells infected with recombinant baculovirus,which showed specific binding to the monoclonal antibody with His-tag and rabies virus-positive sera.Conclusion Recombinant baculovirus for expression of G protein of rabies virus was constructed successfully and expressed in silkworm chrysalis,and the expressed product showed excellent antigenicity.

    2012 07 v.25 [Abstract][OnlineView][Download 257K]

  • Stability of immune complex as enhanced hepatitis B vaccine

    ZHANG Yun,HE Ya-li,SUN Jin-wen,CHEN Guo-ming,ZHANG De-you,ZHOU Gen-xi(Beijing Tiantan Biological Products Corporation Limited,Beijing 100024,China)

    Objective To study the stability of immune complex as enhanced hepatitis B(HB)vaccine.Methods Immune complex as enhanced HB vaccine was stored at 37℃ for 6 weeks(accelerated stability test)and at 2～8℃ for 30 months(long term stability test) respectively,and subjected to overall control tests,of which the potency(ED50)in mice was compared with that of routine recombinant HB(yeast)vaccine.Results All the quality indexes of immune complex as enhanced HB vaccine after storage at 37℃ for 6 weeks and at 2 ～ 8℃ for 30 months met the relevant temporary requirements.Compared with those of routine HB vaccine,the ED50 values of the immune complex in mice at various time points were low,of which the increasing rate was slow.The DE50 values of immune complex,except those after storage at 37℃ for 2 and 4 weeks,showed significant difference from those of routine HB vaccine(P < 0.05).Conclusion Immune complex as enhanced HB(HB)vaccine showed high stability as compared with routine HB vaccine,of which the potency in mice was superior to that of routine HB vaccine.It provided an experimental basis for determination of validity period of immune complex as enhanced HB vaccine.

    2012 07 v.25 [Abstract][OnlineView][Download 169K]

  • Construction and identification of recombinant human adenovirus type 5 with E2 glycoprotein gene of classical swine fever virus

    YANG Yang*,GAO Bo,GONG Ting,LI Ye-wei,SUN Cheng-long,ZHANG Shou-feng,LIU Ye,HU Rong-liang(*College of Animal Science and Veterinary Medicine,Jilin University,Changchun 130062,China)

    Objective To construct and identify recombinant human adenovirus type 5 with E2 glycoprotein gene of classical swine fever virus(CSFV).Methods The full-length E2 gene sequence was amplified from live CSFV vaccine by nested PCR and cloned to the polyclonal site of shuttle plasmid pacAd5 CMVK-NpA.The constructed recombinant shuttle plasmid pacAd5 CMVKNpA-E2 was linearilized and co-transfected to 293AD cells with backbone plasmid pacAd59.2-100 for pacakging.After appearance of CPE,the transcription of E2 mRNA was determined by RT-PCR.The recombinant adenovirus was determined for titer by CPE method,and for stability after subculture.The expression of E2 protein was determined by Western blot.Kunming mice were immunized i.p.with the recombinant adenovirus at a dosage of 105.82 TCID50,and determined for antibody titer against CSFV in sera by indirect ELISA.Results Restriction analysis and sequencing proved that recombinant shuttle plasmid pacAd5 CMVK-NpA-E2 was constructed correctly.Both transcription of E2 gene and expression of E2 protein were proved in 293AD cells transfected with recombinant adenovirus rAd5v-MxE2.The titer of rAd5v-MxE2 was 105.82 TCID50 / ml.Target gene fragments were amplified from rAd5v-MxE2 of passages 5 and 30,while the virus titer showed no significant change.The recombinant adenovirus induced antibody against CSFV in mice.Conclusion Recombinant human adenovirus type 5 with E2 gene of CSFV was successfully constrcuted and showed high immunogenicity,which laid a foundation of further preparation of recombinant CSFV vaccine.

    2012 07 v.25 [Abstract][OnlineView][Download 223K]

  • Construction and expression of recombinant eukaryotic expression vector for peroxiredoxin gene of Toxoplasma gondii

    HAO Hai-xia*,WANG Hai-long,YIN Li-tian,MENG Xiao-li,SHEN Jin-yan,YIN Guo-rong(*Department of Parasitology,Shanxi Medical University,Taiyuan 030001,China)

    Objective To construct the recombinant eukaryotic expression vector for peroxiredoxin(Prx)gene of Toxoplasma gondii(Tg)and express in HEK293T cells.Methods TgPrx gene was amplified by PCR using recombinant plasmid pET-30a(+)/ TgPrx as a template,and inserted into eukaryotic expression vector p3 × Flag-CMW-14.HEK293T cells were transfected with the constructed recombinant plasmid p3 × Flag-CMW-14 / TgPrx,and determined for transcription of TgPrx gene and expression of TgPrx protein by RT-PCR and Western blot respectively.Results TgPrx gene fragment at a length of 591 bp was amplified by PCR.PCR,restriction analysis and sequencing proved that recombinant plasmid p3 × Flag-CMW-14 / TgPrx was constructed correctly.Both the transcription of TgPrx gene and expression of TgPrx protein were observed in HEK293T cells transfected with the constructed recombinant plasmid.Conclusion Recombinant plasmid p3 × Flag-CMW-14 / TgPrx was successfully constructed and expressed in HEK293T cells,which laid a foundation of preparation of DNA vaccine.

    2012 07 v.25 [Abstract][OnlineView][Download 163K]

  • Specific humoral immune response induced in mice by eukaryotic expression plasmid for Neisseria gonorrhoeae surface protein A

    LI Guo-cai,JIANG Gui-hua,JIAO Hong-mei,CHEN Hong-ju,PAN Xing-yuan,YAN Hua,JI Ming-chun(Department of Pathogen Biology and Immunology,College of Medicine,Yangzhou University,Yangzhou 225001,Jiangsu Province,China)

    Objective To observe the immune response induced in mice by eukaryotic expression plasmid for Neisseria gonorrhoeae surface protein A(NspA)by various routes.Methods BALB / c mice were immunized with eukaryotic expression plasmid pcNspA carrying NspA gene by intramuscular,intranasal and intravaginal routes respectively at weeks 0,2,3 and 4.Sera as well as bronchial alveolar lavage fluids and vaginal lotions of mice in various groups were collected 2 weeks after the last immunization,and determined for antibody titer by indirect ELISA,based on which the antibody-mediated complement bacteriolytic activity was determined.Results Plasmid pcNspA by intranasal and intramuscular routes induced high specific IgG and IgA levels in mice.Specific sIgAs were detected in both bronchial alveolar lavage fluids and vaginal lotions of mice immunized by intranasal route.However,only low sIgA level was detected in vaginal lotion of mice immunized by intravaginal route.The sera of mice immunized by intranasal route showed bacteriolytic activity.Conclusion The eukaryotic expression plasmid for NspA induced specific antibody with antibacterial activity in mice,which might be more effective by intranasal route than by intramuscular and intravaginal routes.

    2012 07 v.25 [Abstract][OnlineView][Download 205K]

  • Cloning,expression and bioinformatics of NDM-1 gene

    XIA Li-liang*,SUN Yang,ZHANG Rui,CHEN Shuo,QIU Shao-fu,WANG Zhong-qiang,LIU Nan,LIU Jun,ZHU Ling-wei,JI Xue,SONG Hong-bin,FENG Shu-zhang(*College of Animal Science and Veterinary Medicine,Jilin University,Changchun 130118,China)

    Objective To clone the blaNDM-1 gene of Acinetobacter baumannii,analyze the bioinformatics of NDM-1 gene and protein,then express and purify recombinant NDM-1 protein.Methods The blaNDM-1 gene of A.baumannii was amplified by PCR and cloned into plasmid pMAL-p2X to construct recombinant plasmid pMAL-p2X ∶ ∶ NDM-1,based on which the bioinformatics of NDM-1 gene and protein were analyzed.The constructed recombinant plasmid was transformed to E.coli Tb1 and induced with IPTG,and the expressed recombinant protein MBP-NDM-1 was purified by affinity chromatography.Results Restriction analysis and sequencing proved that recombinant plasmid pMAL-p2X ∶ ∶ NDM-1 was constructed correctly.Bioinformatic analysis showed that the relative molecular mass and isoelectric point of NDM-1 were 28 487.86 and 5.89 respectively,while the signal peptide sequence was located in amino acids 1 ～ 28,and NDM-1 was low homologous to the metal β lactamase of other subtypes.The expressed recombinant protein,with a relative molecular mass of about 73 000,mainly existed in a soluble form,and reached a protein concentration of 1.72 mg / ml after purification.Conclusion Soluble recombinant protein MBP-NDM-1 was expressed and purified,which laid a foundation of further study on activity,function and inhibitor of NDM-1.

    2012 07 v.25 [Abstract][OnlineView][Download 445K]

  • Characteristics of gene encoding VP1 region of enterovirus 71 causing epidemic of hand, foot and mouth disease in Jilin Province,China in 2011

    FAN Ming*,ZHOU Jian-hui,ZHANG Yan,Wei Lei-lei,WANG Shuang,ZHANG Yun-xian,GOU Wei-min,MA Xue-jun,QI Zhong,SUN Yu,ZHAO Hai-xia,Sun Wei,CHEN Chuang(*Jilin Provincial Center for Disease Control and Prevention,Key Virus Laboratory of Health Bureau of Jilin Province,Changchun 130062,China)

    Objective To analyze characteristics of gene encoding VP1 region enterovirus 71(EV71)causing the epidemic of hand,foot and mouth disease(HFMD)in Jilin Province,China in 2011.Methods A total of 35 EV71 strains were isolated from the patients with HFMD in 8 cities of Jilin Province in 2011,of which the genes in VP1 regions were amplified and sequenced,and analyzed for homology and genetic relationship by Bioedit and MEGA4.0 softwares.Results All the 35 EV71 strains were of subgenotype C4a.The homology of nucleotide sequences in VP1 regions of the 35 strains was 95.9% ～ 100.0%,which belonged to 3 clades and formed 3 transmission chains.The genetic relationship of the 35 strains were close to those isolated in Fuyang,Anhui in 2008,with a homology of nucleotide of 97.6% ～ 99.3%,while was far to those isolated from Beijing in 2007 and from Zhejiang in 2003.Each transmission chain was epidemic in 5 cities.However,3,2 and 1 transmission chains were observed in 1,4 and 3 cities respectively.Conclusion The co-cycling of three transmission chains of EV71 of subgenotype C4a caused the HFMD epidemic in Jilin Province in 2011.However,no dominant transmission chain was found.

    2012 07 v.25 [Abstract][OnlineView][Download 230K]

  • Construction of suppression subtractive library of high-producing mutagenic strain of Mortierella isabellina

    WANG Dan-feng,YU Chang-qing(College of Food Science,Heilongjiang Bayi Agricultural University,Daqing 163319,Heilongjiang Province,China)

    Objective To construct the suppression subtractive library of high-producing mutagenic strain of Mortierella isabellina.Methods Subtractive cDNA library was constructed by suppression subtractive hybridization(SSH)technique using highproducing mutagenic strain and original strain of M.isabellina as materials.Two times of subtractive hybridization and two times of nested PCR were performed serving the cDNA of high-producing mutagenic strain as tester and that of original strain as driver,and the last PCR product was inserted into vector pMD18-T.The constructed recombinant plasmid was transformed to E.coli TG1,and the positive clones were screened by blue-white selection and identified by PCR.Results Most of the lengths of inserted fragments in library were 250 ～ 750 bp,and 91% of the formed clones were white.A total of 96 clones were identified by PCR,from which 78 positive ones were obtained.About 82% of the 78 clones showed a single band.Conclusion The suppression subtractive library of high-producing mutagenic strain of M.isabellina was successfully constructed by SSH technique,which laid a foundation of further screening and identification of differentially expressed genes in the mutagenic and original M.isabellina strains.

    2012 07 v.25 [Abstract][OnlineView][Download 196K]

  • Effect of over-expression of Golgi α-mannosidase Ⅱ on apoptosis of human gastric cancer BGC823 cells

    LI Fan,YANG Ya-ying,YI Yong-fen(Department of Pathology,Molecular Medicine and Cancer Research Center,College of Basic Medical,Chongqing Medical University,Chongqing 400016,China)

    Objective To investigate the effect of over-expression of Golgi α-mannosidase Ⅱ(GMⅡ)on the apoptosis of human gastric cancer BGC-823 cells.Methods Eukaryotic over-expression vector EX-E2372-M03 for GMⅡ gene was constructed and transfected to BGC-823 cells in mediation of Lipofectamine 2000.The stably transfected BGC-823 cells were screened with 400 mg / L G418,and determined for transcription level of GM Ⅱ mRNA by RT-PCR,for expression level of GM Ⅱ protein by Western blot,and for apoptosis after over-expression of GMⅡ by Hoechst 33258 fluorescent staining and flow cytometry(Annexin V / PI staining).Results Both the transcription level of GMⅡ mRNA and expression level of GMⅡ protein increased significantly in BGC-823 cells after transfection with EX-E2372-M03(P < 0.05),while the apoptosis rate of cells decreased significantly(P < 0.05).Conclusion The overexpression of GMⅡ gene might promoted the onset and progress of gastric cancer by inhibiting the apoptosis of gastric cancer cells.

    2012 07 v.25 [Abstract][OnlineView][Download 337K]

  • Cloning and prokaryotic expression of TvRab11C gene of Trichomonas vaginalis

    LIU Chang,DING He,GONG Peng-tao,LI Jian-hua,LI Shu-hong,LI He,ZHANG Guo-cai,ZHANG Xi-chen(College of Animal Science and Veterinary Medicine,Jilin University,Changchun 130062,China)

    Objective To clone the TvRab11C gene of Trichomonas vaginalis and express in prokaryotic cells.Methods The TvRab11C gene was amplified by PCR from T.vaginalis and inserted into prokaryotic expression vector pET-28a.The constructed recombinant plasmid pET-28a-TvRab11C was transformed to E.coli BL21(DE3) and induced by IPTG.The expressed product was analyzed for solubility by SDS-PAGE and for reactogenicity by Western blot.Results Both restriction analysis and sequencing proved that recombinant plasmid pET-28a-TvRab11C was constructed correctly.The expressed recombinant protein,with a relative molecular mass of about 30 000,mainly existed in a form of inclusion body,and was recognized by polyclonal antibody against T.vaginalis.Conclusion The TvRas gene of T.vaginalis was successfully cloned and expressed in E.coli BL21(DE3),which laid a foundation of further study on relationship of TvRas gene and protein to the parasitic ability and pathogenicity of T.vaginalis.

    2012 07 v.25 [Abstract][OnlineView][Download 154K]

  • Effect of hypoxia on epithelial-mesenchymal transition of human differentiated thyroid carcinoma KAT and WRO cells

    JIA Chao-li*,LIU Zhi-min,ZENG De-kang,YANG Jun-yan,LONG Fang-yi,LIU Xiao-hua(*Department of Biochemistry and Molecular Biology,Institute of Molecular Medicine and Cancer,College of Basic Medical Sciences,Chongqing Medical University,Chongqing 400016,China)

    Objective To investigate the effect of hypoxia on epithelial-mesenchymal transition(EMT)of human differentiated thyroid carcinoma KAT and WRO cells.Methods Hypoxia microenvironment was mimicked by using cobalt chloride(CoCl2),in which the KAT and WRO cells were determined for proliferative activities by MTT method,and the working concentration of CoCl2 was optimized.The morphologies of KAT and WRO cells in hypoxia microenvironment were observed by invert microscopy.The expression of hypoxia-inducible factor-1a(HIF-1a)in KAT and WRO cells 0,3,6 and 12 h,as well as those of E-cadherin and Vimentin in the cells 6 h after culture in hypoxia microenvironment were determined by Western blot.Results The optimal working concentration of CoCl2 was 150 μmol / L.The KAT and WRO cells showed the morphological characteristics of mesenchymal cells gradully in hypoxia microenvironment.The expression levels of HIF-1a in KAT and WRO cells 6 h after culture in hypoxia microenvironment reached the maximums,which showed significant difference with those before culture(P < 0.01).The expression of E-cadherin in KAT cells cultured in hypoxia microenvironment was significantly lower than that in normal microenvironment(P < 0.01),while the expression level of Vimentin in the former showed no significant difference with that in the latter(P > 0.05).However,the expression level of E-cadherin was significantly lower,while that of Vimentin significantly higher in the WRO cells cultured in hypoxia microenvironment than in normal microenvironment(both P < 0.01).Conclusion Hypoxia promoted the EMT of human differentiated thyroid carcinoma KAT and WRO cells through the down-regulation of E-cadherin and the up-regulation of Vimentin.

    2012 07 v.25 [Abstract][OnlineView][Download 207K]

  • Cloning and prokaryotic expression of virB8 gene of Brucella abortus

    ZHANG Rui*,WANG Xiu-ran,Xia Li-liang,LI Xiao-yan,WANG Xing-long,WANG Jing-long,RAN Hui-ling,QIAN Jing(*College of Animal Science and Veterinary Medicine,Jilin University,Changchun 130062,China)

    Objective To clone the virB8 gene of Brucella abortus and express in E.coli.Methods The virB8 gene was amplified by PCR from genomic DNA of B.abortus S19 and inserted into pEASY-E1 vector.The constructed recombinant plasmid pEASY-virB8 was transformed to E.coli BL21(DE3)for expression under induction of IPTG.The expressed product was purified by HisTrapTM FF chromatography and identified by SDS-PAGE and Western blot.Resultus The length of amplified virB8 gene was 720 bp.Restriction analysis and sequencing proved that recombinant plasmid pEASY-virB8 was constructed correctly.The expressed recombinant protein,with a relative molecular mass of about 30 000,contained 17.3% of total somatic protein and reached a purity of 85% after purification.At a protein concentration of 1.52 mg / ml determined by Lowry method,the expressed product showed specific binding to mouse monoclonal antibody against His.Conclusion The virB8 gene of B.abortus was successfully cloned and expressed in E.coli,which provided an effective candidate antigen for development of novel vaccines.

    2012 07 v.25 [Abstract][OnlineView][Download 197K]

  • Cloning and prokaryotic expression of trehalose synthase gene

    SHANGH Hong-li,GU Ying,FU Li,ZHANG Ting(College of Food Science and Engingeering,Liaoning Medical University,Jinzhou 121001,Liaoning Province,China)

    Objective To clone trehalose synthase(Tres)gene and express in E.coli.Methods Tres gene was amplified by PCR using the genomic DNA of Thermus thermophilus SH-110 as a template,and cloned into prokaryotic expression vector pET30a(+).The constructed recombinant plasmid pET-30a(+)-Tres was transformed to E.coli BL21(DE3) and induced with IPTG and lactose respectively.The expressed product was identified by SDS-PAGE,Western blot and thin layer chromatography(TLC).Results Both restriction analysis and sequencing proved that recombinant plasmid pET-30a(+)-Tres was constructed correctly.Both IPTG and lactose induced high expression of recombinant protein.The activity of crude Tres,with a relative molecular mass of about 110 000,was 10 times of that of wild Thermus thermophilus SH-110.The recombinant protein showed high activity of Tres as proved by TLC analysis of hydrolysate of maltose.Conclusion Recombinant Tres was successfully expressed in E.coli,which laid a foundation of large-scale production of trehalose.

    2012 07 v.25 [Abstract][OnlineView][Download 189K]

  • Effect of TPX2 gene silence on proliferation of human lung adenoma A549 cells

    TANG Xi,ZHANG Hui,LIU Ying(Department of Pathology,Research Centre of Molecular Medicine and Cancer,Chongqing Medical University,Chongqing 400016,China)

    Objective To investigate the effect of TPX2 gene silence on proliferation of human lung adenoma A549 cells.Methods Three shRNA plasmids for TPX2 gene were constructed and transfected to A549 cells.The transcription level of TPX2 mRNA was determined by RT-PCR.The cells transfected with the plasmid showing satisfactory interfering effect were screened,and determined for the transcription levels of mRNAs of proliferation-related genes CDK4 and CyclinD1 by RT-PCR,and for expression level of TPX2 protein by Western blot.The growth of cells was observed by microscopy,while the proliferation activity was determined by MTT method.Results TPX2 shRNA-3 plasmid showed a satisfactory interfering effect.After TPX2 gene was silenced effectively,both the transcription of TPX2 mRNA and expression of TPX2 protein in A549 cells were inhibited significantly(P < 0.01),with inhibitory rates of 73.8% and 82.8% as compared with those in control group respectively.Both the transcription levels CDK4 and CyclingD1 mRNAs decreased significantly(P < 0.01 or P < 0.05),with inhibitory rates of 76.7% and 67.3% as compared with those in control group respectively.The growth and proliferation of A549 cells were inhibited significantly,of which the inhibitory rate 72 h after transfection was 61.55%(P < 0.05).Conclusion The shRNA plasmid for TPX2 gene was successfully constructed,which inhibited the expression of TPX2 at both mRNA and protein levels in A549 cells,afterwards the growth and proliferation of A549 cells were inhibited.

    2012 07 v.25 [Abstract][OnlineView][Download 217K]

  • Inhibitory effect of p16 gene on osteosarcoma U-2OS cells

    XIA Yi-fan*,YU Xian,FENG Li,YUAN Bin(*Department of Orthopaedics,The Affiliated Hospital,North Sichuan Medical College,Nanchong 637007,Sichuan Province,China)

    Objective To investigate the inhibitory effect of p16 gene on osteosarcoma U-2OS cells.Methods U-2OS cells were transfected with recombinant plasmids pcDNA3-HA-p16 and pcDNA3-HA respectively,using those untransfected as negative control,and determined for proliferative activity by CCK-8 method,for cell cycle by PI staining,and for apoptosis by DNA ladder and PI staining.Results The p16 gene inhibited the proliferation of U-2OS cells significantly(P < 0.05),which was dose-dependent within a certain range.The p16 gene increased the percentage of U-2OS cells at G1 phase(P < 0.05)while decreased that at S phase significantly(P < 0.05).Meanwhile,p16 gene increased the apoptosis rate of U-2OS cells(P < 0.05).Typical DNA ladder was observed on agarose gel electrophoretic profile of DNA of U-2OS cells.Conclusion The p16 gene significantly inhibited the proliferation of U-2OS cells,arrested the cell cycle at G1 phase and promoted the cell apoptosis.

    2012 07 v.25 [Abstract][OnlineView][Download 158K]

  • Distribution of drug-resistance and mechanism of fluoroqninolones-resistance of Shigella isolates in Dali Region,Yunnan Province,China

    WANG Guo-fu,XUE Shi-peng,WU Li-xian(Department of Medical Microbiology of Dali College,Dali 671000,Yunnan Province,China)

    Objective To investigate the distribution of drug-resistance and mechanism of fluoroqninolones-resistance of Shigella isolates in Dali Region,Yunnan Province,China.Methods Fecal specimens were collected from various hospitals in Dali Region in 2010 ～ 2011,from which Shigella strains were isolated,cultured and identified for serotype.The resistances of Shigella isolates to 12 routine antibacterial drugs were analyzed by K-B agar diffusion test.The minimal inhibitory concentrations(MICs) of ceflriaxome and nalidixic acid to drug-resistant Shigella strains were determined by E-test.The gene mutations of drug-resistant strains were analyzed by PCR and DNA sequencing.The relationship between resistance to fluoroqninolones and R plasmid mediation was evaluated by R plasmid conjugation test.Results A total of 68 Shigella strains were isolated,including 41 Shigella flexner strains,25 Shigella sonnei strains and 2 Shigella boydii strains.The resistance rate of Shigella to nalidixic acid was the highest,followed by those to sulfalene and nalidixic.The Shigella isolates were relatively sensitive to ceftriaxone and cefotaxime,while showed a certain resistance to fluoroquinolones.The MICs of ciprofloxacin to Shigella strains ranged from 0.008 to 32 μg / m1.However,all the MICs of nalidixic acid to Shigella strains were more than 1 μg / m1,of which those to 4 strains more than 256 μg / m1.Substitutions of Ser83 → Leu in gyrA gene and Ser80 → Ile in parC gene were observed in 6,while those of Asp87 → Asn in gyrA gene in 2,Asp87 → Gly of gyrA gene in 2,and Gln91 → His of parC gene in 3 fluoroquinolones-resistant strains.No evidence suggested that R plasmid mediated the resistance to fluoroquinolones.Conclusion Ceftriaxone and cefotaxime were the first choice for treatment of bacterial dysentery.The fluoroquinolone-resistance of Shigella was closely associated with the mutations of gyrA and parC genes,while was unassociated with R plasmid mediation.

    2012 07 v.25 [Abstract][OnlineView][Download 195K]

  • Effect of miR-450a on development at early stage of mouse embryo cultured in vitro

    YANG Dan-dan,LIU Chen,JIANG Feng-bing,LI Bao-lin,BAI Hui-li,SHI Qiong(Key Laboratory for Clinical Laboratory Diagnostics,of Education Ministry,College of Laboratory Medicine,Chongqing Medical University,Chongqing 400016,China)

    Objective To investigate the effect of miR-450a on development at early stage of mouse embryo cultured in vitro.Methods The mouse embryos at stages of 2 ～ 8 cells were cultured in M16 media containing 0.01,0.1 and 1 ng / ml miR-450a respectively,using those in miR-450a-free medium as control.The development of mouse embryo was observed under inverted microscope,and the development rates of embryos at stages of 2,4 and 8 cells were calculated.Results The miR-450a inhibited the normal division of embryonated egg cells significantly and caused the abnormal development of mouse embryos.The development rate of embryos decreased with the increasing concentration of miR-450a(P < 0.05).Partial embryos died or degenerated during culture in media containing high miR-450a concentrations.Conclusion The miR-450a showed inhibitory effect on development at early stage of mouse embryo cultured in vitro,which provided a novel route for study on abnormal development of embryo.

    2012 07 v.25 [Abstract][OnlineView][Download 149K]

  • Effect of various types of newborn bovine sera on culture in vitro of BHK-21 cells

    SUN Zhao-jin*,CHEN Bing-qi,WANG Ling,YE He-jia,TONG Ju-yun,LUO Kai-jian(*Guangzhou South China Biological Medicine Co.Ltd,Guangzhou 511300,China)

    Objective To compare the effect of various types of newborn bovine sera on culture in vitro of BHK-21 cells.Methods BHK-21 cells were cultured with six kinds of newborn bovine sera,manufactured by various manufacturers or of different batches manufactured by the same manufacturer,respectively,and observed for morphology,increased fold in quantity,and survival time after subculture.Results The cells cultured with serum No.6 grew well,of which the increased fold in quantity was high and survival time was long as compared with those cultured with other newborn bovine sera.The culture efficacy of cells with serum No.3 was only second to that with serum No.6.However,no monolayer was formed in the cells on day 4 after culture with sera No.2,4 and 5,and little growth was observed in the cells cultured with serum No.1.Conclusion Significant differences were observed in culture efficacies of BHK-21 cells with newborn bovine sera manufactured by various manufacturers or of different batches manufactured by the same manufacturer.

    2012 07 v.25 [Abstract][OnlineView][Download 173K]

  • Specificity of ERC (N5) polypeptide in recognizing integrin αVβ3 and its effect on proliferation

    and apoptosis of HepG2 cells LIU Hai-tao,SUN Dong-dong,YANG Li-jun,ZHANG Dong,YANG Tao(Department of Biochemistry and Molecular Biology,Key Laboratory of Cellular Physiology,Ministry of Education,Shanxi Medical University,Taiyuan 030001,China)

    Objective To analyze the binding capacity of ERC(N5)polypeptide containing RGD sequence to integrin αVβ3 and investigate the effect of ERC(N5) on proliferation and apoptosis of hepatocellular carcinoma HepG2 cells.Methods The integrin αVβ3 content on surface of HepG2 cells as well as the competitive binding of ERC(N5)and FITC-αVβ3 antibody LM609 to HepG2 cells by flow cytometry.HepG2 cells were treated with ERC(N5),then determined for proliferative activity by CCK-8 method,observed for morphological change by optical microscopy,and analyzed for apoptosis rate by flow cytometry.Results The αVβ3 content on surface of HepG2 cells was 47%.The ERC(N5)at concentrations of 8,16,32 and 64 μmol / L showed dosedependent inhibitory effect on the binding of LM609 to HepG2 cells and the proliferative activity of the cells(P < 0.05).Compared with those in control group,the cells treated with 16 μmol / L ERC(N5)showed obvious morphological change,of which the apoptosis rate increased significantly.Conclusion ERC(N5)showed specific binding to integrin αVβ3,and inhibited the proliferation and induced the apoptosis of HepG2 cells by inhibiting the function of αVβ3.

    2012 07 v.25 [Abstract][OnlineView][Download 284K]

  • Screening of neutralizing antibody titers against enterovirus 71 in human intravenous immunoglobulin(pH 4)

    WANG Min-li*,WANG Wei,SUN Si-cai,DING Yong,Hou Ji-feng(*National Institutes for Food and Drug Control,Beijing 100050,China)

    Objective To screen the neutralizing antibody titers against enterovirus 71(EV71) in commercial human intravenous immunoglobulin(IVIG)(pH 4) and experimental intravenous EV71 immunoglobulin,and provide a basis for further passive immunotherapy of hand-foot-mouth disease(HFMD).Methods The neutralizing antibody titers against EV71 in domestic IVIG(pH 4) and in experimental intravenous EV71 immunoglobulin prepared with screened raw plasma were determined by microcytopathic effect(micro-CPE)method with two virus strains in two laboratories.Results The neutralizing antibody titers of 55 batches of domestic IVIG manufactured by 17 manufacturers determined with strain-01 in laboratory A were 104 ～ 256,of which 23.64%(13 / 55)were not less than 239,12.73%(7 / 55)were not less than 256,while the GMTs of neutralizing antibodies of 3 batches of experimental intravenous EV71 immunoglobulin were 1 203.However,the GMTs of neutralizing antibody against EV71 in 3 batches of experimental intravenous EV71 immunoglobulin(1 402) determined with strain-01 in NIFDC laboratory were significantly higher than those of 12 batches of IVIG manufactured by 8 manufacturers(P < 0.05).The GMTs of neutralizing antibody against EV71 in 16 batches of IVIG manufactured by 12 manufacturers determined with strain-02 were 332,which showed no significant difference with those determined with strain-01(P > 0.05).Conclusion All the neutralizing antibody against EV71 in domestic IVIG manufactured by various manufacturers were positive,of which the titers ranged from 104 to 630.However,the neutralizing antibody titers against EV71 in intravenous EV71 immunoglobulin prepared with the screened plasma were significantly higher than those in IVIG.

    2012 07 v.25 [Abstract][OnlineView][Download 164K]

  • Preparation and targeting ability in vitro of DR5-targeted lipid microbubble loaded with docetaxel

    YANG Jian*,KANG Juan,ZENG Yan,WU Xiao-ling,MEI Zhe-chuan,WANG Zhi-gang(*Departement of Gastroenterology,The Second Affiliated Hospital of Chongqing Medical University,Chongqing 400010,China)

    Objective To prepare the docetaxel-loaded lipid microbubbles(DLLM) coupled with DR5mAb against liver cancer,and determine its targeting ability in vitro.Methods DLLM were prepared by mechanical oscillation,based on which DR5targeted docetaxel-loaded lipid microbubbles(DR5-DLLM)were prepared by attaching biotinylated DR5mAb to the surface of DLLM via avidin-biotin interaction,and determined for size,concentration,Zeta potential,entrapment efficiency,drug-load,stability and targeting ability in vitro.Results The mean size,Zeta potential,mean concentration,entrapment efficiency and mean drug-load of DR5-DLLM were 1 232 nm,-9.86 mV,3.1 × 109 microbubbles / ml,73.5% and 25.3%,respectively.No obvious change was observed in appearance,entrapment efficiency or drug-load of DR5-DLLM before and after sterilization with 60Co and after storage at 4 and-20℃ for 14 d.DR5-DLLM were tightly conjugated with HepG2 cells,while DLLM showed no specific conjugation with the cells.Conclusion DR5-DLLM was successfully prepared,which were conjugated with HepG2 cells tightly,and might be used as a novel and highly effective liver cancer-targeted drug carrier for ultrasonund molecular imaging.

    2012 07 v.25 [Abstract][OnlineView][Download 206K]

  • Prokaryotic expression and activity of humanized single-chain variable fragment against chronic myelogenous leukemia cells

    ZHU Xiao-ying,WANG Dong,LI Shen-feng,ZHANG Li-jun,ZHONG Liang,FENG Wen-li(De partment of Clinical Hematology,Key Laboratory of Laboratory Medical Diagnostics,Ministry of Education,Chongqing Medical University,Chongqing 400016,China)

    Objective To express humanized single-chain variable fragmen(t hscFv)against chronic myeloid leukemia(CML) cells in prokaryotic cells and determine its antigen-binding activity.Methods The hscFv gene was amplified from recombinant plasmid pUC57-hscFv and inserted into prokaryotic expression vector pET-32a(+)with a 6 × His tag.The constructed recombinant plasmid pET-32a-hscFv was transformed to E.coli BL21(DE3)for expression under induction of IPTG.The expressed fusion protein was purified by Ni2+-NTA chromatography,then analyzed for purity by SDS-PAGE,for reactogenicity by Western blot,and for antigenbinding activity by indirect IFA.Results PCR,restriction analysis and sequencing proved that recombinant plasmid pET-32a-hscFv was constructed correctly.The expressed recombinant fusion protein,with a relative molecular mass of about 46 000,contained about 37% of total somatic protein and mainly existed in a soluble form,which reached a purity of 94% after purification and showed specific binding to mouse monoclonal antibody against 6 × His tag and the antigen on surface of K562 cells.Conclusion The hscFv against CML cells was successfully expressed in prokaryotic cells and purified,which laid a foundation of further application of hscFv for clinical molecular diagnosis and target biotherapy of CML.

    2012 07 v.25 [Abstract][OnlineView][Download 197K]

  • Development of dry chemistry method for alanine aminotransferase

    TIAN Yi,CHEN Han-yan,WANG Man(Shanghai Kehua Bio-Engineering Co.Ltd.,Shanghai 200233,China)

    Objective To develop a dry chemistry method for alanine aminotransferase(ALT)based on pyruvate oxidase method and prepare the test strip.Methods The test strip consisted of a sample spreading layer,a substrate layer and a color development layer,which was coated with reaction agent for determination of ALT activity by reflectance photometer.The prepared test strip was verified.Results The intra-CV of the test strip for low(48.7 U / L)and high(127 U / L)level quality controls were 7.4% and 4.9% respectively.The linear range of the test strip was 10 ～ 1 000 U / L.No significant difference was observed between the determination results of heparinized blood and plasma by the test strip(P > 0.05).The determination results of ALT were uninfluenced when the bilirubin content in sample was not more than 257 μmol / L,the ascorbic acid content was not more than 50 mg / L,and the pyruvate content was not more than 0.5 mmol / L.The developed dry chemistry method showed good relationship to the method recommended by International Federation of Clinical Chemistry(IFCC)(r = 0.988 9).The reference range of 95% confidence interval of the test strip was 0 ～ 40 U / L.Conclusion The developed dry chemistry method was rapid,accurate and simple,which was suitable for immediate determination of ALT.

    2012 07 v.25 [Abstract][OnlineView][Download 163K]

  • Expressions of hypoxia-inducible factor-1α,Twist and E-cadherin in human breast cancer tissue and their significances

    LONG Fang-yi*,YIN Guo-bing,LIU Zhi-min,WU Ting-ting,JIA Chao-li,YANG Jun-yan,LIU Xiao-hua(*Department of Biochemistry and Molecular Biology,College of Basic Medicine,Chongqing Medical University,Chongqing 400016,China)

    Objective To investigate the expressions of hypoxia-inducible factor-1α(HIF-1α),Twist(a zinc finger transcription inhibitor)and E-cadherin in human breast cancer tissue and their significance.Methods The expressions of HIF-1α,Twist and E-cadherin in breast cancer tissue specimens from 69 patients receiving no radiotherapy,chemotherapy or hormone therapy before surgery were determined by immunohistochemical assay with SP staining,between which the relationship was analyzed.Results The positive rates of HIF-1α,Twist and E-cadherin in the breast cancer tissue specimens were 71.01%(49 / 69),57.97%(40 / 69)and 50.72%(35 / 69)respectively,which were related to the TNM stage and axillary lymph node metastasis(P < 0.05 or P < 0.01),but unrelated to the age of patient as well as size and pathological type of tumor(P > 0.05).The expression of HIF-1α was positively related to that of Twist(r = 0.286,P < 0.05).However,the expression of Twist was negatively related to that of E-cadherin(r =-0.371,P < 0.01).Conclusion The expressions of HIF-1α,Twist and E-cadherin might be used as markers for monitoring the progress of breast cancer.

    2012 07 v.25 [Abstract][OnlineView][Download 206K]

  • Expression and distribution of prothymosin-alpha in human ovarian adenocarcinoma and its relationship to grading of tumor

    ZHANG Zhuo-mei*,HE Fei(*Department of Obstetrics and Gynecology,Chinese General Hospital of Armed Police,Beijing 100039,China)

    Objective To determine the expression and distribution of prothymosin-alpha(PTMA) in human ovarian adenocarcinoma and analyze its relationship to grading of tumor.Methods A total of 172 ovarian adnocarcinoma and 8 normal adjacent tissue specimens were obtained by tissue microarray slide and subjected to pathological grading and histological typing.The expression level of PTMA in ovarian adnocarcinomas and normal adjacent tissues were determined by Western blot,while the distribution of PTMA in ovarian adnocarinomas tissue by immunohistochemical staining.Results Of the 172 ovarian adnocarinoma tissue specimens,22,67 and 83 were of grades Ⅰ,Ⅱ and Ⅲ respectively.All the specimens were judged as papillary adenocarcinoma,except 4 as endometrial adenocarcinoma of grade Ⅱ.The expression level of PTMA was significantly lower in normal adjacent tissues than in ovarian adenocarcinoma tissue(P < 0.01).Of the 8 normal adjacent tissue specimens,5 were positive for nucleus staining,most of which appeared in ovarian epithelial cells,while the other 3 were negative.Of the 172 ovarian adenocarinoma tissue specimens,164(95.3%)were positive for PTMA expressions,including 44 with the expressions in nuclei,40 in cytoplasm,and 80 in both nuclei and cytoplasm.The distribution of PTMA was significantly related to the grade of tumors(P < 0.01).Mixed distributions were observed in most of ovarian adnocarcinoma tissues specimens of grade Ⅲ.The expression level of PTMA in ovarian adnocarcinoma of gradeⅠwas significantly higher than those of gradesⅡand Ⅲ(P < 0.01).Conclusion Increased PTMA expression was found in human ovarian adenocarcinoma compared with that in normal adjacent ovarian tissue.However,the distributions of PTMA expression were different in tumors of various grades.It laid a foundation of further study on clinical significance of abnormal expression of PTMA in ovarian adenocarcinoma tissue.

    2012 07 v.25 [Abstract][OnlineView][Download 268K]

  • Expressions of ATP binding cassette transporter A1 and Heme oxygenase-1 in human glioblastoma and their significances in clinic

    WANG Chen,YU Tian-ping,TENG Zhi-peng,LIU Bin,LI Yu(Department of Pathology,Chongqing Medical University,Chongqing 40016,China)

    Objective To investigate the expression of ATP-binding cassette transporter 1(ABCA1)and Heme oxygenase-1(HO-1) in human glioblastoma as well as their significances in clinic.Methods The expressions of ABCA1 and HO-1 in 44 giloblastoma tissue specimens and 14 normal adjacent tissue specimens were determined by immunohistochemical assay,based on which the relationship of ABCA1 and HO-1 expressions to clinical pathological characters of patients were analyzed.Results The expression rates of ABCA1 and HO-1 in glioblastoma tissue specimens were 77.2% and 75% respectively,which were significantly higher than those in normal adjacent tissue(both 7.1%)(each P < 0.001).Positive correlation was observed between the expressions of ABCA1 and HO-1(rS = 0.313,P < 0.05).However,the ABCA1 and HO-1 expressions showed no relationship to the sex,age,resected range in surgery,tumor size,recurrence,as well as postoperation radiotherapy and chemotherapy of patients.Conclusion High expressions of ABCA1 and HO-1 may play important roles in the tumorigenesis and progress of glioblastoma.

    2012 07 v.25 [Abstract][OnlineView][Download 201K]

  • Investigation on test method for abnormal toxicity of live attenuated varicella vaccine in guinea pigs

    CHEN Zhe-wen,JIN Yu-lan,MA Xiang-hu,ZHANG Jian-guang,ZHU Yu-mei,WANG Liang(Shanghai Institute of Biological Products Co.Ltd.,Shanghai 200052,China)

    Objective To investigate the test method for abnormal toxicity of live attenuated varicella vaccine in guinea pigs.Methods Forty-one batches of live attenuated varicella vaccine manufactured by Shanghai Institute of Biological Products Co.Ltd(SIBP)were subjected to abnormal toxicity test in guinea pigs according to the requirements in Chinese Pharmacopoeia(Volume Ⅲ,2010 edition).Abnormal toxicity tests were also performed on the vaccine manufactured by SIBP in various laboratories,the vaccines produced by four other manufacturers,various batches of vaccine manufactured by SIBP,and the vaccine manufactured by SIBP after injection into guinea pigs with various bodyweights.Each guinea pig was weighed before injection,of which the bodyweight should be 250 ～ 350 g.Each guinea pig was injected i.p.with 0.5 ml of test sample,and observed for 7 d.Results All the guinea pigs survived without abnormal reactions during the whole observation period,of which the bodyweights increased at the end of observation period.The bodyweight increases of guinea pigs injected with the same batch of vaccine and tested in the same laboratory showed difference.The bodyweight increases of guinea pigs tested in various laboratories showed difference,and weighing every day showed influence on the bodyweight increase.The bodyweights of most of guinea pigs decreased slightly on days 1 and 2 after injection,then increased gradually.Conclusion The live attenuated varicella vaccine manufactured by SIBP showed high safety.The guinea pigs above the clean level are recommended to be used for abnormal toxicity test.Meanwhile,the sex of guinea pig and observation period before injection shall be prescribed,and positive and negative controls shall be set up,so as to evaluate the safety of biologics more rationally.

    2012 07 v.25 [Abstract][OnlineView][Download 158K]

  • Screening and optimization of condition for fermentation of phospholipase A1-producing bacterial strains

    ZHAO Meng-meng,XUE Zheng-lian,SU Yan-nan,ZHAO Shi-guang,CHEN Tao(Institute of Biologic & Chemical Engineering of Anhui Polytechnic University,Microorganism Fermentation Engineering and Technology Research Center of Anhui Province,Wuhu 241000,Anhui Province,China)

    Objective To screen phospholipase A1(PLA1,EC3.1.1.32)-producing bacterial strains and optimize the condition for their fermentation.Methods PLA1-producing bacterial strains were screened from oil-rich soil.The strains with high PLA1 activity were subjected to molecular biological assay,of which the condition for fermentation was optimized.Results Of the five strains screened,Dx208 showed the highest PLA1 activity which reached 4.52 U / ml in shake flask.Dx208 strain was preliminarily identified as Streptomyces griseus based on its morphological characters and 16S rDNA sequence.The optimal initial pH value,temperature for culture,time for fermentation and shaking speed were 7.0,30℃,5 d and 200 r / min respectively.Under the optimal condition,the PLA1 activity in fermentation liquid was 7.15 U / ml,which was 1.58 times of that before optimization.Conclusion A highly PLA1-producing bacterial strain was screened,of which the condition for fermentation was optimized.It laid a foundation of production and application of PLA1.

    2012 07 v.25 [Abstract][OnlineView][Download 254K]

  • Establishment of neonatal mouse model of non-lethal Streptococcus pneumonia infection

    YANG Ting*,LUO Zheng-xiu,FU Zhou,LIU Chang-lin,WANG Li-jia,LUO Jian,LIU En-mei(*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,Department of Respiration,Children's Hospital,Chongqing Medical University,Chongqing 400014,China)

    Objective To investigate the method for establishment of neonatal BALB / c mouse model of non-lethal Streptococcus pneumonia infection.Methods SPF neonatal BALB / c mice were randomly divided into two test and two control groups,20 for each.The mice in test group 1 were infected once with 10 μl of S.pneumonia by intranasal drip on day 7 after birth at dosages of 107,108,109,and 1010 CFU respectively,while those in control group 1 with 10 μl PBS.However,the mice in test group 2 were infected twice with 10 μl of S.pneumonia by intranasal drip on days 6 and 7 after birth at dosages of 107,108,109,and 1010 CFU respectively,while those in control group 2 with 10 μl PBS.Ten mice in each group were killed 24 h after the last infection,of which the lung tissues were homogenized for bacterial culture and identification,and observed for pathological change by HE staining.The other 10 mice in each group were observed for change of bodyweights within 5 d after infection and survival.Results The mice infected twice with 109 CFU S.pneumonia were quiet,of which the skins were pale and the increases of bodyweights decreased.Pathological examination showed inflammatory change in lung,thickening of alveolar septum and a certain inflammatory cell infiltration around blood vessels and bronchus,most of which were neutrophils.Conclusion Infection with 109 CFU S.pneumonia on days 6 and 7 after birth might be an effective method for establishment of neonatal BALB / c mouse model of nonlethal S.pneumonia infection

    2012 07 v.25 [Abstract][OnlineView][Download 208K]

  • Dynamic monitoring of virulence stability of challenge strain of B. pertussis stored in liquid nitrogen

    WANG Li-chan*,WEI Chen,ZHANG Lu-min,XU Ying-hua,LUO Peng,ZHANG Shu-min,HOU Qi-ming(*Department of Serum,National Institutes of Food and Drug Control,Key Laboratory of the Ministry of Health for Research on Quality and Standardization of Biotech Products,Beijing 100050,China)

    Objective To dynamically monitor the stability of virulence of challenge strain,stored in liquid nitrogen,for determination of potency of pertussis vaccine.Methods After B. pertussis seeds was resuscitated and subcultured for 3 passages,the bacterial lawns were collected,diluted by turbidimetry,distributed and stored in liquid nitrogen.The virulence of the B. pertussis strain was monitored dynamically every year from 2008 ～ 2010 by modified challenge test in mouse brain,of which the stability was evaluated.Results No significant change was observed in the virulence of challenge strain of B. pertussis after storage in liquid nitrogen,idnicating high stability.Conclusion The virulence of challenge strain of B. pertussis was stable after storage in liquid nitrogen,indicating that liquid nitrogen was suitable for storage of challenge strain of B.pertussis for potency test of pertusssis vaccine.

    2012 07 v.25 [Abstract][OnlineView][Download 108K]

  • Isolation and identification of rabbit rib perichondrium-derived mesenchymal stem cells

    LI Min,LIU Jun,JIANG Rong,WANG Wei-Wei(Department of Histology and Embryology,Laboratory of Stem Cells and Tissue Engineering,Chongqing Medical University,Chongqing 400016,China)

    Objective To isolate and identify mesenchymal stem cells(MSCs)from rabbit-rib perichondrium.Methods Rib perichondrium cells were isolated from New Zealand white rabbits aged 3 and 4 weeks by two-step digestion method consisting of enzyme digestion and mechanical digestion,cultured in vitro and observed for growth and morphology,and identified for phenotype as well as and potential in differentiation into adipocyte and osteoblast.Results The primary cells isolated from rib perichondrium were round or polygon and assemble as conglobation.The cells were subcultured for 15 passages and grew well,and those from passage 2 were in spindle-shape and whirlpool arrangement,in which CD29 and CD90 were expressed while CD34 and collogen type Ⅱ were unexpressed.The cells from passage 3 were differentiated into osteoblast or adipose cells by induction.Conclusion Stem cells were isolated from rabbit rib perichondrium,of which the characters were similar to those of MSCs.

    2012 07 v.25 [Abstract][OnlineView][Download 240K]

  • Purification of HBsAg expressed in Hansenula polymorpha by hydrophobic interaction chromatography with WorkBeads series and C4 media

    LI Cai-mei*,ZHANG De-you,MA Rui,XU Ning,YANG Xu-qin,SHEN Yong-cai,WANG Xi,LIU Ying-wei,CHEN Xing,LI Jin(*National Vaccine & Serum Institute,Beijing 100024,China)

    Objective To compare the purification efficacy of HBsAg expressed in Hansenula polymorpha by hydrophobic interaction chromatography using columns 3146VL,3147L,3148H and 3149VH with various butyl densities in WorkBeads 40 / 10000 Bus low series of Bio-Work and CIM Monoliths C4 of BIA.Methods HBsAg was dissolved with various loading buffers,then loaded onto the column pre-filled with WorkBeads type 4 and C4 butyl chromatographic column,and eluated with various elution buffers.The flowthrough and elution peaks were collected and determined for HBsAg content,based on which the flow-through and elution rates were calculated,and the optimal loading and elution buffers were screened.Results The optimal column for purification of HBsAg was 3147L,while the optimal loading buffer was 20 mmol / L PB containing 4% ammonium sulfate.Neither 20 mmol / L PB nor 20 mmol / L PB containing 30% isopropanol was the ideal elution buffer.The optimal loading buffer on CIM Monoliths C4 was 50 mmol / L MOPS containing 3% ammonium sulfate and 20 mmol / L PB containing 4% ammonium sulfate,while the optimal elution buffer was 50 mmol / L MOPS containing 0.1% Triton X-100.Conclusion It is feasible to introduce WorkBeads series which may be used repeatedly and CIM Monoliths C4 which is resistant to high flow rate into the purification of HBsAg expressed in Hansenula polymorpha.

    2012 07 v.25 [Abstract][OnlineView][Download 241K]

  • Characteristics of biodistribution of routine gene transfer vectors

    MIAO Yu-fa,LI Bo(National Institutes for Food and Drug Control,Beijing 100176,China)

    Gene transfer vectors are divided into viral and non-viral vectors,of which the biodistribution is of important reference significance in searching for minicity target organ and evaluating the risk of germ-line transmission.This paper reviews the characteristics of biodistribution of various gene transfer vectors and provides a reference for selection and minicity evaluation of gene transfer vectors.

    2012 07 v.25 [Abstract][OnlineView][Download 195K]

  • Progress in research on biological characteristics of exosomes and therir roles in tumor immunotherapy

    Feng Ye-tong,Liu Peng-fei,Dong chao,ZHOU Yu-lai(Bioengineering Experimental Center,College of Pharma cology,Jilin University,Changchun 130021,China)

    Exosomes are membranous vesicles secreted by living cells into the extracellular environment,at diameters of 50 ～ 100 nm.There are lots of proteins and lipids on the surface of exosomes,which are closely related to their structure and function.Exosomes could be released by many kinds of cells.Exosomes have various important functions,especially in tumor immunotherapy.Exosomes from DCs and tumor cells showed good curative effect on tumors in recent studies,which have become a hotspot in the research of oncology.This review summarizes the biological origin,biological characteristics and physiological functions of exosomes as well as their action mechanism in diagnosis and immunotherapy of tumors.

    2012 07 v.25 [Abstract][OnlineView][Download 188K]

  • Application of metabolomics in study on hepatocellular carcinoma

    XIAO Zhong-hua,LI Jing(Chongqing Three Gorges Medical College,Chongqing 404120,China)

    Metabolomics is a systemic biology for study on organic metabolism status under interaction of the genome,proteome and survival environment of organisms.The development of hepatocellular carcinoma will inevitably bring about the change of metabolism.Metabolomics may reveal differential metabolites and their relationship panoramically and provide an idea for the clinical diagnosis and treatment of hepatocellular carcinoma.The application of metabolomics in study on hepatocellular carcinoma in recent year is reviewed in this paper.

    2012 07 v.25 [Abstract][OnlineView][Download 124K]


@2012 Editorial Department of "Chinese Journal of Biologicals"