2011年05期目次

  • Prokaryotic Expression and Purification of Structural and Nonstructural Proteins of Human Bocavirus and Preparation of Polyclonal Antibodies

    WANG Ya-ying,GUO Li,WU Chao,WANG Jian-wei,HONG Tao(State Key Laboratory of Molecular Virology and Genetic Engineering,Institute of Pathogen Biology,Peking Union Medical College & Chinese Academy of Medical Sciences,Beijing 100730,China)

    Objective To express the structural protein VP2 as well as nonstructural proteins NS1 and NP1 of human bocavirus(HBoV) in prokaryotic cells,purify the expressed product and prepare their polyclonal antibodies.Methods The VP2,NS1 and NP1 genes of HBoV were amplify by PCR and cloned into prokaryotic expression vector pET-30a(+).The constructed recombinant plasmid pET-30a-VP2,pET-30a-NS1 and pET-30a-NP1 were transformed into E.coli BL21(DE3) respectively for expression under induction of IPTG.The expressed proteins were purified by ion exchange chromatography and Ni2+-NTA affinity chromatography and identified by SDS-PAGE and Western blot.Polyclonal antibodies were prepared by immunizing BALB / c mice with the purified proteins and determined for titer by ELISA.Results Both restriction analysis and sequencing proved that recombinant plasmids pET-30a-VP2,pET-30a-NS1 and pET-30a-NP1 were constructed correctly.VP2 was experssed mainly in a form of inclusion body,while NP1 mainly in soluble form,and NS1 only in a form of inclusion body,which contained 20%,40% and 30% of total somatic protein,and reached purities of 85%,more than 95% and 80% after purification,respectively.The titers of polyclonal antibodies prepared by immunizing the mice with VP2 and NP1 were more than 1 ∶ 16 000,while that with VP1 was 1 ∶ 8 000.Conclusion The VP2,NS1 and NP1 of HBoV were successfully expressed,and high titer polyclonal antibodies were prepared,which provided materials for further study on pathogenic mechanism and epidemiologic investigation of HBoV.

    2011 05 v.24 [Abstract][OnlineView][Download 437K]

  • Construction and Preliminary Validation of a Phage Displayed Human Single-chain Antibody Library Derived from Patients with Psoriasis

    ZHANG Yin-chuan△,ZHANG Ai-hua,PAN Yong-bing,YU Ling,JIANG Gong-ping,FANG Zhi-zheng(△Wuhan Institute of Biological Products,Wuhan 430060,China)

    Objective To construct a phage displayed human single-chain antibody library derived from patients with psoriasis and screen the specific single-chain antibody against human tumor necrosis factor α(hTNFα).Methods The serum samples from 33 patients with psoriasis were analyzed for IgG against hTNFα by indirect ELISA.The pComb3X_scFv library was constructed by phage displayed technique using the separated peripheral blood lymphocytes(PBLs),and identified for capacity,accuracy and diversity.Microtiter plate was coated with hTNFα,based on which the solid phase biopanning of specific hTNFα antibody was performed by 4 cycles of"adsorption-elution-amplification".Results The anti-hTNFα IgG level in sera of patients with psoriasis was significantly higher than that in healthy individuals(P < 0.01).A phage displayed human single-chain antibody library with a capacity of about 4 × 106,a recombination rate of more than 90% and good diversity was successfully constructed using the gene materials of the specific antibody.Specific phage displayed antibody was enriched by biopanning of hTNFα.Conclusion Specific single-chain antibody against hTNFα was successfully screened by using the constructed phage displayed human single-chain antibody library derived from patients with psoriasis.

    2011 05 v.24 [Abstract][OnlineView][Download 474K]

  • Construction and Identification of Recombinant Adenovirus Ad5-hSH2-EGFP and Its Mutant

    SHI Jing,PENG Zhi,LUO Hong-wei,CAO Wei-xi,TAO Kun,LI Ya-juan,WANG Hai-xia,FENG Wen-li(Department of Clinical Hematology,Key Laboratory of Laboratory Medical of Education Ministry,Chongqing Medical University,Chongqing 400016,China)

    Objective To construct recombinant adenovirus Ad5-hSH2-EGFP for SH2 gene,containing enhance green fluorescent protein(EGFP) gene and HA tag,as well as its mutant Ad5-hSH2mt,and observe its effect on proliferation of K562 cells.Methods Primers were designed and synthesized chemically,with which hSH2 gene was amplified by RT-PCR,while hSH2mt gene by overlapping PCR,and cloned into vector pAdTrack-CMV separately.The constructed shuttle plasmids were digested with PmeⅠ and transformed to competent pAdEasy-BJ5183,based on which replication-defective recombinant adenovirus plasmids pAd5-hSH2-EGFP and pAd5-hSH2mt-EGFP were obtained by homologous recombination in bacteria,digested with PacⅠ,and transfected to AD293 cells for packaging.The obtained recombinant adenoviruses Ad5-hSH2-EGFP and Ad5-hSH2mt-EGFP were amplified and determined for titers,then identified by PCR and Western blot and infected to K562 cells.The proliferative activity of K562 cells was determined by MTT method.Results Restriction analysis proved that both recombinant adenovirus plasmids pAd5-hSH2-EGFP and pAd5-hSH2mt-EGFP were constructed correctly.Both the titers of recombinant adenoviruses Ad5-hSH2-EGFP and Ad5-hSH2mt-EGFP reached 1.6 × 1012.PCR proved that the both the recombinant adenoviruses were successfully packaged and amplified.Western blot showed that the target genes in recombinant adenoviruses were expressed in AD293 cells.MTT method proved significantly inhibitory effect of Ad5-hSH2-EGFP on proliferation of K562 cells.Conclusion Recombinant adenoviruses Ad5-hSH2-EGFP and Ad5-hSH2mt-EGFP were successfully constructed,which laid a foundation of further study on curative effect of SH2 gene on chronic myeloid leukemia.

    2011 05 v.24 [Abstract][OnlineView][Download 469K]

  • Expression of Virus-like Particles of Enterovirus 71 in Insect Sf9 Cells

    XU Jing-wen,LI Hong-jun,XIE Tian-hong,ZHANG Guang-ming,YIN Na,SUN Mao-sheng(Institute of Medical Biology,Academy of Medical Sciences and Peking Union Medical College,Kunming 650118,China)

    Objective To construct a chimeric baculovirus plasmid carrying P1 and 3CD genes of enterovirus 71(EV71) and investigate the cleavage of P1 protein with 3CD protease as well as the possibility of formation of virus-like particles(VLPs).Methods The P1 and 3CD genes of EV71 were cloned into plasmid pFAStBac Dual,and the constructed shuttle plasmid pFastBac Dual-P1-3CD was transformed to E.coli DH10.The obtained recombinant baculovirus plasmid Bac-P1-3CD was transfected to Sf9 cells to prepare recombinant baculovirus,and the expressed products of target genes were identified by SDS-PAGE,Western blot and electron microscopy.Results PCR proved that recombinant baculovirus plasmid Bac-P1-3CD was constructed correctly.Specific protein bands with relative molecular masses of 39 000,32 000 and 26 000 were observed on SDS-PAGE profile of Sf9 cells infected with recombinant baculovirus,which were consistent with those of VP1,VP0 and VP3 of EV71.Western blot showed a specific protein band with similar relative molecular mass to that of VP1 of EV71.The VLPs of EV71 were observed by electron microscopy.Conclusion The P1 and 3CD genes of EV71 may be expressed in insect cells using recombinant baculovirus.3CD protease may digest P1 into VP1 antigen and form VLPs.

    2011 05 v.24 [Abstract][OnlineView][Download 514K]

  • Construction of Eukaryotic Expression Vector for Human P115 Gene and Its Effect on Proliferation of Hepatocarcinoma Cells

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

    Objective To construct a eukaryotic expression vector for human P115 gene and investigate the effect of the expression on proliferative activity of human hepatocarcinoma HepG2 cells.Methods Human P115 gene was amplified by RT-PCR from HepG2 cells and inserted into plasmid pEGFP-N1 Vector(+).The constructed recombinant plasmid pEGFP-N1 Vector(+)-USO1 was transfected to HepG2 cells.The transfection efficacy was determined by IFA and flow cytometry.The transfected cells were determined for expressions of P115 mRNA and protein by RT-PCR and Western blot,for proliferative activity by MTT method,and for cell cycle by flow cytometry.Results Restriction analysis and sequencing proved that recombinant plasmid pEGFP-N1 Vector(+)-USO1 was constructed correctly.The transfection efficacy of HepG2 cells with the recombinant plasmid was 84.83%.The expression levels of P115 gene and protein as well as proliferative activity of HepG2 cells transfected with the recombinant plasmid were significantly higher than those with empty vector and untransfeted.The percentage of transfected cells at G1 / G2 phases decreased significantly,while that at S phase increased significantly.Conclusion A recombinant eukaryotic expression vector for human P115 gene was successfully constructed and over-expressed in hepatocarcinoma cells,and the expressed P115 promoted the proliferation of hepatocarcinoma cells.

    2011 05 v.24 [Abstract][OnlineView][Download 408K]

  • Effect of Water Extracts from Dendrobium huoshanense on Proliferation and Apoptosis of Human Laryngeal Carcinoma Hep-2 Cells and Relevant Mechanism

    ZHANG Jing△,LIAN Chao-qun,WU Shou-wei,HU Ming-jie,CHEN Chuan-hao(△Bioscience Department,Bengbu Medical College,Bengbu 233030,Anhui Province,China)

    Objective To investigate the effect of water extracts from Dendrobium huoshanense(WEDH) on proliferation and apoptosis of human laryngeal carcinoma Hep-2 cells and the relevant mechanism.Methods Hep-2 cells were treated with 3.75,7.50,15.00,30.00 and 60.00 mg / ml WEDH respectively and observed for proliferation level by MTT method,for morphology by invert microscopy and for apoptosis by laser scanning confocal microscopy after stained with Annexin V-FITC / PI,then determined for free calcium ion(i) concentration by laser scanning confocal microscopy after stained with Fluo-3 AM and for caspase-3 activity by spectrophotometry.Results WEDH showed significantly time-and dose-dependent inhibitory effect on proliferation of Hep-2 cells.The IC50 of WEDH to Hep-2 cells 24,48 and 72 h after treatment were 48.54,26.67 and 14.93 mg / ml respectively.The morphology of Hep-2 cells 48 h after treatment with WEDH changed significantly,in a dose-dependent mode,and showed typical characteristics of apoptosis.Bothi concentration and caspase-3 activity of Hep-2 cells were significantly higher than those in negative control group(P < 0.05).Conclusion WEDH inhibited the proliferation and induced the apoptosis of Hep-2 cells significantly by a potential mechanism which might be associated with increasing the i concentration and activating caspase-3.

    2011 05 v.24 [Abstract][OnlineView][Download 259K]

  • Expression,Purification and Activity of Pituitary Adenylate Cyclase-activting Polypeptide 38 and Its Derivative

    ZHANG Hong-dan,XIAO Lei,ZHAO Li-fen,WANG Xiao-xi,HUANG Jing,WU Zi-rong(School of Life Science,East China Normal University,Shanghai 200062,China)

    Objective To improve the in vivo stability and prolong the half-life of bioactivity of pituitary adenylate cyclase-activting polypeptide 38(PACAP38).Methods The amino acid sequence of PACAP38 was modified to obtain a derivative named cPACAP38.The gene fragments encoding PACAP38 and cPACAP38 were designed and synthesized according to the codon bias of E.coil and cloned into vector pET-32a(+).The constructed recombinant plasmids were transformed to E.coli BL21(DE3) for expression under induction of IPTG.The expressed fusion proteins were purified by nickel ion affinity chromatography and digested with enterokinase,by which two target polypeptides were obtained and tested for anorexigenic activities in mice.Mice were treated with the polypeptides for 10 d,then determined for intraperitoneal glucose-resistance as well as serum triglyceride,HDL,LDL and cholesterol contents.Results Fusion proteins Trx-PACAP38 and Trx-cPACAP38 were expressed in soluble forms,which contained 22% and 25% of total somatic proteins and reached purities of 90% and 98% after purification respectively.Both PACAP38 and cPACAP38 showed anorexigenic activities in mice.However,the anorexigenic activity of cPACAP38 was higher than that of PACAP38.No significant changes were observed in intraperitoneal glucose-resistance as well as serum triglyceride,HDL,LDL and cholesterol contents of mice 10 d after treatment with the fusion proteins.Conclusion The derivative of PACAP38 with higher in vivo activity was successfully expressed,which laid a foundation of basic study and application of PACAP38.

    2011 05 v.24 [Abstract][OnlineView][Download 304K]

  • Mechanism of Ginsenoside Rh2 in Enhancing Sensitivity of Human Liver Cancer HepG2 Cells to Betulinic Acid

    LI Qing△,WANG Xiao-yu,LI Yang,JIN Ying-hua(△Key Laboratory for Molecular Enzymology and Engineering of the Ministry of Education,Jilin University,Changchun 130012,China)

    Objective To investigate the mechanism of ginsenoside Rh2(G-Rh2) in enhancing the sensitivity of human liver cancer HepG2 cells to betulinic acid(Bet A).Methods The HepG2 cells in logarithmic growth phase were divided into four groups.The cells in G-Rh2,Bet A and G-Rh2 + Bet A groups were treated with 7.5 μg / ml G-Rh2,10 μg / ml Bet A and 7.5 μg / ml G-Rh2 + 10 μg / ml Bet A respectively,while those in control group were untreated.The cells were observed for morphological change by fluorescent microscopy,for apoptosis by flow cytometry,determined for caspase-3 activity,then analyzed for breakage of poly(ADP-ribose) polymerase(PARP) and activation of caspase-9 by Western blot.Results Compared with those in G-Rh2 and Bet A groups,more than 75% of cells in G-Rh2 + Bet A group showed the characteristics of apoptosis,in which the percentage of cells at subG1 stage and caspase-3 activity increased,while obvious breakages of PARP and caspase-9 were observed.Conclusion G-Rh2 enhanced the sensitivity of HepG2 cells to Bet A through mitochondrion-associated cell apoptosis.

    2011 05 v.24 [Abstract][OnlineView][Download 325K]

  • Expressions and Significances of Golgi α-Mannosidase Ⅱ and E-Cadherin in Normal Liver Tissue and Liver Cancer Tissues

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

    Objective To investigate the expressions and significances of Golgi α-mannosidase Ⅱ(GMⅡ) and E-cadherin in normal liver tissue and liver cancer tissues.Methods The expressions of GMⅡ and E-cadherin mRNAs in normal liver tissue and in highly and lowly differentiated liver cancer tissues were determined by RT-PCR,while those of proteins by SP immunohistochemical assay and Western blot,based on which the differential expressions of GMⅡ and E-cadherin were analyzed for correlation.Results RT-PCR and Western blot showed that the expression levels of GMⅡ mRNA and protein were low in normal liver tissue,high in moderately and lowly differentiated liver cancer tissues,and moderate in highly differentiated liver cancer tissues.However,the expressions of E-cadherin was contrary to that of GMⅡ.SP immunohistochemical assay showed that GMⅡ was mainly expressed in the cytoplasma of normal liver tissue and liver cancer tissue,of which the positive rates were 40% in normal liver tissue,64% in highly differentiated liver cancer tissue,and 86% in moderately and lowly differentiated liver cancer tissue.However,E-cadherin was expressed in both cell membrane and cytoplasma of normal liver tissue and in cytoplasma of liver cancer tissue,of which the positive rates were 70% in normal liver tissue,33% in highly differentiated liver cancer tissue,and 9% in moderately and lowly differentiated liver cancer tissues.The expression levels of GMⅡ and E-cadherin mRNAs and proteins in various groups showed significantly difference(P < 0.05).The Pearson correlation coefficients(r) of GMⅡ and E-cadherin expressions was-0.415,while the P value was 0.01.Conclusion The expression of GMⅡ in liver cancer tissue was negatively related to that of E-cadherin,and GMⅡ might play a role in the onset and progress of liver cancer.

    2011 05 v.24 [Abstract][OnlineView][Download 345K]

  • Prokaryotic Expression and Purification of Rat 1,25(OH)2D3-Membrane Associated Rapid Response Steroid Binding Protein

    XU Jing,ZHANG Zhen-shu,WANG Can,LIU Fu-qiang,BAI Lan(Department of Digestive Disease,Nanfang Hospital,Southern Medical University,Guangzhou 510515,China)

    Objective To express rat 1,25(OH)2D3-membrane associated rapid response steroid binding(1,25D3-MARRS)protein in prokaryotic cells and purify the expressed product.Methods 1,25D3-MARRS gene was amplified by RT-PCR and cloned into prokaryotic expression vector pET-32a(+).The constructed recombinant plasmid pET-32a-1,25D3-MARRS was transformed to E.coli BL21(DE3) for expression under induction of IPTG.The expressed product was purified by Ni-NTA affinity chromatography and identified by Western blot.Results Both restriction analysis and sequencing proved that recombinant plasmid pET-32a-1,25D3-MARRS was constructed correctly.The expressed recombinant protein,with a relative molecular mass of about 77 000,mainly existed in a form of inclusion body,contained about 20% of total somatic protein,reached a purity of more than 90% and showed good reactogenicity.Conclusion Recombinant 1,25D3-MARRS protein was successfully expressed,which laid a foundation of further study on its property,function and characteristics of distribution.

    2011 05 v.24 [Abstract][OnlineView][Download 308K]

  • Prokaryotic Expression and Purification of Vibrio vulnificus Hemoolysin Protein

    ZHANG Zai-wen△,GUO Jian-wei,WEI Jie,MA Cong,QIAN Yang-hui,ZHNAG Yun(△Department of Clinical Laboratory,Navy General Hospital,Beijing 100048,China)

    Objective To construct a prokaryotic expression vector for Vibrio vulnificus hemoolysin(VVC) gene,and express and purify recombinant protein.Methods VVC gene was amplified from genomic DNA of V.vulnificus and inserted into prokaryotic expression vector pET-30a(+).The constructed recombinant plasmid pET-32a(+)-VVC was transformed to E.coli BL21(DE3) for expression under induction of IPTG.The expressed recombinant protein was purified by nickel ion metal chelating column chromatography and identified by SDS-PAGE and Western blot.Results Restriction analysis and DNA sequencing proved that the VC gene inserted into recombinant plasmid pET-32a(+)-VVC was at a length of 1 371 bp,of which the homology was 99% to that reported in GenBank.The expressed fusion protein,with a relative molecular mass of about 71 000,mainly existed in a form of inclusion body and contained 50% of total somatic protein.The purified recombinant protein reached a purity of more than 90% and showed specific binding to sera of mice immunized with V.vulnificus.Conclusion VVC protein was successfully expressed in E.coli and purified,which provided a material for preparation of diagnostic kit of V.vulnificus and laid a foundation of further study on structural domain and biological function of VVC as well as pathogenic mechanism of V.vulnificus.

    2011 05 v.24 [Abstract][OnlineView][Download 281K]

  • Enzymatic Property and Immunogenicity of Uricase Liposome of Candida utilis

    WANG Na△,TAN Qun-you,XU Mei-ling,LI Yi,ZHAO Chun-jing,ZHANG Jing-qing(△Medicine Engineering Research Center in University,Chongqing Key Laboratory of Biochemical & Molecular Pharmacology,Chongqing Medical University,Chongqing 400016,China)

    Objective To investigate the enzymatic property and immunogenicity of liposome-mediated uricase(UC) of Candida utilis.Methods UC liposome was prepared by reverse-phase evaporation method,based on which the optimal temperatures,pH values,acid-and basic-stabilities of UC liposome and UC were determined,and the effects of partial metal ions and organic compounds on enzymatic activity were evaluated.SD rats were immunized with the UC liposome as an antigen,and their serum antibody titers were determined.Results Both the optimal temperatures for UC liposome and UC were 40℃,while the optimal pH values were 8.0 and 8.5 respectively.The stability of UC liposome was significantly higher than that of UC.The serum antibody titers of rats immunized with UC liposome and UC were 1 ∶ 500 and 1 ∶ 8 000 respectively.Conclusion UC liposome was significantly superior in enzymatic property and lower in immunogenicity to UC,which provided en experimental basis for clinical application of UC.

    2011 05 v.24 [Abstract][OnlineView][Download 287K]

  • Construction and Expression of Prokaryotic Expression Vector for Canine MC4R Gene

    HE Bao-jun,BA Cai-feng,WANG Guang-chuan,ZHAO Wei,SONG Hui-juan,LV Jin-gang(Experimental Animal Center,Liaoning Medical University,Jinzhou 121001,Liaoning Province,China)

    Objective To construct a prokaryotic expression vector for canine MC4R(cMC4R) gene and express recombinant MC4R protein.Methods MC4R gene was amplified by PCR using recombinant plasmid pcDNA3.1-myc-his / A-cMC4R as a template and cloned into prokaryotic expression vector pET-30a(+).The constructed recombinant plasmid pET-30a(+)-MC4R was transformed to E.coli Transetta(DE3) for expression under induction of IPTG.The expressed product was identified by SDS-PAGE and Western blot.Results Restriction analysis proved that recombinant plasmid pET-30a(+)-MC4R was constructed correctly.DNA sequencing proved that the homology of inserted sequence to that reported in GenBank was 99%,of which only the base T at site 777 of encoding region changed into C.The relative molecular mass of expressed protein was about 37 000.The expression level reached the maximum after induction for 4 h,which accounted for 8% of total somatic protein.The recombinant protein showed specific binding to anti-His monoclonal antibody.Conclusion The prokaryotic expression vector for cMC4R gene was successfully constructed,and recombinant cMC4R protein was expressed,which provided an antigen for preparation of polyclonal and monoclonal antibodies against cMC4R protein.

    2011 05 v.24 [Abstract][OnlineView][Download 289K]

  • Effects of Betaine and Methionine Chelated Chromium (Ⅲ) on Serum Biochemical Indicators and Subcutaneous Fatty Acid Synthase mRNA Expression in Finishing Pigs

    CUI Bo,GENG Zhong-cheng,PAN Xing-ling,SUN Hai-tao,LIU Sheng-jun(College of Animal Science and Technology,Heilongjiang Bayi Agriculture University,Daqing 163319,Heilongjiang Provinec,China)

    Objective To investigate the effects of betaine and methionine chelated chromium(Ⅲ) on serum biochemical indicators and subcutaneous fatty acid synthase(FAS) mRNA expression in finishing pigs.Methods Eighty-one finishing pigs with a mean bodyweight of about 52 kg were divided into nine groups according to a 3 × 3 [methionine chelated chromium(Ⅲ) × betaine] test design with replicates at two factors and three levels.The methionine chelated chromium(Ⅲ)(0,200 and 400 μg / kg) and betaine(0,1 000 and 1 250 mg / kg) at various chromium levels were added into basal diet consisting of corn and bean dregs,and their effects on serum biochemical indicators and subcutaneous FAS mRNA expression in finishing pigs were evaluated.Results Both betaine and methionine chelated chromium(Ⅲ) decreased the serum triglyceride(TG),total cholesterin(CHO) and insulin contents as well as expression level of FAS mRNA,and increased growth hormone(GH) level in finishing pigs.The TG content in group 9,CHO,GH and INS contents in group 6 as well as FAS mRNA level in group 8 showed the most significant differences with those in control group(each P < 0.05),and reciprocal effects were observed.Conclusion Both betaine and methionine chelated chromium(Ⅲ) decreased the fat deposition.However,the complex addition of betaine and methionine chelated chromium(Ⅲ) showed satisfactory decreasing effect and a certain reciprocal effect.

    2011 05 v.24 [Abstract][OnlineView][Download 206K]

  • Expression of SALL4 Gene during Matrine-induced Apoptosis of Leukemia THP-1 Cells

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

    Objective To investigate the expression of oncogene SALL4 in martine-induced apoptosis of human acute leukemia THP-1 cells as well as its significance.Methods THP-1 cells were treated with 1.0,1.5 and 2.0 g / L matrine respectively and observed for morphological change by electron microscopy 24,48 and 72 h later,while determined for proliferative activity by MTT method,for apoptosis rate by flow cytometry,and for expression of SALL4 mRNA by RT-PCR.Results The THP-1 cells 24 h after treatment with matrine showed apoptotic morphology at various degrees.The matrine at various concentrations inhibited the proliferation and promoted the apoptosis of THP-1 cells significantly,both in dose-and time-dependent modes(P < 0.05).However,the expression levels of SALL4 mRNAs in THP-1 cells treated with martine at various concentrations decreased significantly as compared with those in control group,in dose-and time-dependent mode(P < 0.05).Conclusion The down-regulation of SALL4 gene expression might play an important role in martine-induced apoptosis of THP-1 cells.

    2011 05 v.24 [Abstract][OnlineView][Download 361K]

  • In Vivo Induction of Differentiation of Rat Bone Marrow Mesenchymal Stem Cells into Myofibroblasts

    ZHOU Wei△,XU Yan-hua,WU Xiao-ling,JIANG Rong,CHEN Peng-fei(△Department of Gastroenterology,The Second Affiliated Hospital of Chongqing Medical University,Chongqing 400010,China)

    Objective To investigate the possibilities of differentiation of rat bone marrow mesenchymal stem cells(BMSCs) into myofibroblasts in various disease models.Methods BMSCs were isolated and purified from the marrows in femora and tibiae of rats by whole bone marrow adherent culture method and subcultured in vitro.The BMSCs of passage 3 were identified for surface antigen and induced to evaluate the abilities of adipogenesis and osteogenesis.Thirty-five rats were divided into normal control(A),liver fibrosis model(B),ulcerative colitis model(C),liver fibrosis model transplanted with BMSCs(D) and ulcerative colitis model transplanted with BMSCs(E) groups,seven for each.The rats in groups B and D were injected s.c.with 40% carbon tetrachloride to copy liver fibrosis model,and those in group D were transplanted with 1 × 106 DAPI-labeled BMSCs through caudal vein 8 weeks later.However,the rats in groups C and E were treated with ethanol solution containing trinitrobenzenesulfonate(TNBS) by enema to copy ulcerative colitis model,and those in group E were transplanted with 1 × 106 DAPI-labeled BMSCs through caudal vein 24 h later.All the rats were killed 2 weeks after transplantation with BMSCs,of which the liver tissues in groups B and D were observed for pathological change by HE and VG staining,while the intestinal tissues in groups C and E by HE staining.The expression of α-smooth muscle actin(α-SMA) in all the tissue samples were determined by IFA.The distribution of BMSCs in liver and intestinal tract and α-SMA in liver were observed by laser confocal microscopy.Results BMSCs were positive for CD29,CD166 and CD90 but negative for CD45,and were differentiated into osteoblasts and adipocytes.Ten weeks after copy of disease models,the hepatic cords of rats were arranged irregularly,with the proliferation of a large quantity of collage fibers in portal region and formation of pseudolobules.However,in groups C and E,the mucosal thickening of colon,ulcer and infiltration of a large quantity of inflammatory cells into the mucosa and sub mucosxa of colon were observed.DAPI-labeled BMSCs were mainly distributed in the fibrous cords of livers of rats in group D,and in the mucosa and sub mucosa of colons in group E,both of which were positive for α-SMA.Conclusion BMSCs may be differentiated into myofibroblasts in both the liver of rats with liver fibrosis and the colon of rats with ulcerative colitis.

    2011 05 v.24 [Abstract][OnlineView][Download 446K]

  • Cloning and Prokaryotic Expression of VEGF-SLC Fusion Gene

    JIANG Pan,CHEN Quan,ZHENG Yi,LIU Ge-li,ZHANG Lu-yu(Department of Immunology,College of Basic Medicine,Chongqing Medical University,Chongqing 400016,China)

    Objective To construct a prokaryotic expression vector for vascular endothelial growth factor(VEGF)-secondary lymphoid-tissue chemokine(SLC) fusion gene and purify the expressed fusion protein.Methods VEGF-SLC gene was amplified by Gene SOEing and inserted into vector pQE30.The constructed recombinant plasmid pQE30-VEGF-SLC was transformed to E.coli M15 for expression under induction of IPTG.The expressed product was identified by SDS-PAGE and Western blot,then purified by Ni-Agarose His-tagged protein purification kit.Results Both restriction analysis and sequencing proved that recombinant plasmid pQE30-VEGF-SLC was constructed correctly.The relative molecular mass of expressed recombinant fusion protein was about 28 000.The expression level reached a peak value 5 h after induction,which accounted for about 19% of total somatic protein.The expressed product mainly existed in a form of inclusion body,showed specific binding to mouse anti-human VEGF monoclonal antibody,and reached a purity of more than 90% after purification.Conclusion Recombinant VEGF-SLC fusion protein was successfully expressed in E.coli,which laid a foundation of further study on biological activity and targeting anti-tumor effect of the fusion protein as well as development of biologic agents targeting to tumors such as lung cancer.

    2011 05 v.24 [Abstract][OnlineView][Download 447K]

  • Screening of Differential Mitochondrial Protein in Breast Cancer Cells Cultured In Vitro

    YUN Xue-xue△,YANG Yong-chang,JIANG Wei,XIAO Dai-wen,YAN Hui,HUANG Wen-fang,LUO Chun-li(△Key Laboratory of Laboratory Medical Diagnostics of Ministry of Education,Department of Laboratory Medicine,Chongqing Medical University,Chongqing 400016,China)

    Objective To screen differential mitochondrial protein by comparing the protein fingerprint of breast cancer cells cultured in vitro with that of normal breast cells,and lay a foundation of study on subcellular protein biomarker of breast cancer.Methods Mitochondrial proteins were extracted from breast cancer cell MDA-MB-231 and MCF-7 strains and normal breast cell HBL-100 strain,and analyzed for fingerprints by surface enhanced laser desorption & ionization time-of-flight mass spectrometry(SELDI-TOF-MS).The results were analyzed by Biomarker Wizard Software,based on which differential mitochondrial proteins were screened,and the effect of dissolvent and long-term storage in frozen on the result analysis was evaluated.Results Nearly 200 protein peaks were observed in a relative molecular mass range of 2 000 to 100 000.Compared with that of HBL-100 cells,45 and 36 differential protein peaks were observed in MDA-MB-231 and MCF-7 cells(P < 0.05) respectively.The expression levels of proteins with relative molecular masses of 9 200,13 800,14 000 and 22 500 in the two breast cancer cell strains decreased,while those with relative molecular masses of 10 000 and 11 600 increased.Triton X-114 at a concentration of 4‰ and storage in frozen for 3 ～ 6 months showed no effect on result analysis.Conclusion SELDI-TOF-MS may be used for rapid and sensitive detection of differential mitochondrial protein in breast cancer cells,which provided a novel route for study on subcellular protein biomarker of breast cancer.

    2011 05 v.24 [Abstract][OnlineView][Download 245K]

  • Isolation,Purification and Enzymatic Activity of Phospholipase A2 from Venom of Guangdong Cobra(Naja naja atra)

    HAO Cai,HAN Li-ping,JIANG Lin-lan(Department of Pharmacy,Guangzhou General Hospital of Guangzhou Military Command,Guangzhou 510010,China)

    Objective To isolate and purify phospholipase A2(PLA2) from the venom of Guangdong cobra(Naja naja atra) and analyze its physic-chemical property,enzymatic activity as well as influencing factors of the activity.Methods PLA2 was isolated and purified by chemical precipitation,Sephadex G-50 gel filtration and UND Sphere S cation exchange chromatography,and determined for protein concentration by BCA method,for purity by HPLC,for relative molecular mass by SDS-PAGE,for enzymatic activity by automatic titration.The influences of temperature,pH value and metal ion on enzymatic activity as well as the heat stability of PLA2 were evaluated.Results The protein concentration,recovery rate,HPLC purity,relative molecular mass,specific activity,optimal temperature and pH value of PLA2 isolated from the venom of Guangdong cobra were 0.6 mg / ml,9.5%,99%,14 300,133 μmol /(mg·min),about 60℃ and about 8.5 respectively.Ca2+ and Mg2+ increased,while K+,Ni2+,Fe2+,Cu2+,Zn2+,Co2+ and Cr2+ inhibited the enzymatic activity of PLA2.PLA2 showed high heat stability.Conclusion The PLA2 with high purity,high optimal temperature and high heat stability was purified from the venom of Guangdong cobra.

    2011 05 v.24 [Abstract][OnlineView][Download 329K]

  • Protective Effect of Mucosal Immunization with ΔA146Ply against Streptococcus pneumoniae

    YAO Run,CUI Ya-li,YUAN Jun,WANG Hong,GONG Yi,YIN Yi-bing,ZHANG Xue-mei(Key Laboratory of Medical Diagnostics,Ministry of Education,Department of Laboratory Medicine,Chongqing Medical University,Chongqing 400016,China)

    Objective To investigate the protective effect of ΔA146Ply protein(hemolysin of Streptococcus pneumonia with a single deletion of A146) and the feasibility of the said protein as a candidate S.pneumonia vaccine.Methods BALB / c mice were divided into test and control groups,and immunized i.n.with 30 μl of mixture of 15 μg ΔA146Ply with 1 mg / ml CT adjuvant and 30 μl of mixture of PBS with CT adjuvant respectively,once a week for 4 weeks.The specific antibody levels in sera and saliva of mice were determined by ELISA 1 week after the last immunization.Meanwhile,active protection test against S.pneumonia of several serotypes was performed on ΔA146Ply,while passive protection test on antisera of ΔA146Ply.The conservation of Ply protein in S.pneumonia was analyzed by Western blot.The IgG subtype and IL-17A level in sera of mice were determined by indirect ELISA.Results The IgG titer in sera of mice in test group was 5.12 × 105,while IgG and IgA titers in saliva were 4.0 × 103 and 4.8 × 103 respectively.Mucosal immunization with ΔA146Ply prolonged the survival time and increased the survival rate of mice effectively.The protective rates to S.pneumonia NCTC7466,CMCC(B) 31436,CMCC(B) 31614 and CMCC(B) 31207 were 50.0%,41.7%,50.0% and 41.7% respectively.In passive protection test,the survival rate of mice in test group was 70%.The subtypes of antibodies induced by mucosal immunization with ΔA146Ply were mainly IgG1 and IgG2b.IL-17A level reached a peak value of(678.55 ± 189.00) pg / ml 48 h after treatment.Ply was highly conserved in all the 4 S.pneumonia strains.Conclusion Mucosal immunization with ΔA146Ply induced both humoral and cellular immune responses against infections with several S.pneumonia strains effectively,indicating that ΔA146Ply might be a good candidate protein of S.pneumonia vaccine.

    2011 05 v.24 [Abstract][OnlineView][Download 270K]

  • Preparation of Rabies Vaccine for Human Use with Vero Cells in Basket Bioreactor

    YANG Yi△,RU Dong-yu,GUO Xiu-xia,CUI Wen-guang,MIAO Li,LIU Yan,WANG Ya-jun,CAI Yue-hong,YUAN Zhi-gang(△Changchun Institute of Biological Products,Changchun 130062,China)

    Objective To prepare rabies vaccine for human use with Vero cells in basket bioreactor.Methods Rabies vaccine virus strain aGV was cultured in Vero cells in 14 L bioreactor(500 g of disk carriers in basket) by perfused culture.The continuously harvested virus liquid was concentrated by ultrafiltration,inactivated,and purified by column chromatography,then added with stabilizer and prepared into freeze-dried vaccine.The prepared vaccine was determined for titers before and after lyophilization and after storage at 37℃ for 28 d.Results The cell density reached 1.0 × 107 cells / ml at most,and the titer of harvested virus liquid was 7.5 × 106 ～ 3.4 × 108 FFU / ml.After purification,the total protein content,residual Vero cell protein content,residual Vero cell DNA content and vaccine titer were less than 80 μg / 0.5 ml,less than 3.5 μg / 0.5 ml,less than 100 pg / 0.5 ml and more than 5.0 IU / 0.5 ml.Conclusion Basket bioreactor may be used for preparation of rabies vaccine for human use with high quality.

    2011 05 v.24 [Abstract][OnlineView][Download 240K]

  • Biosafety of Surface Modified Polyethylene Terephthalate Fiber

    HUANG Zhao-song,BI Long,ZHANG Zhen-yu,ZHANG Dong-xian,SUN Peng-xiao,HAN Yi-sheng(Department of Orthopedics,Xijing Hospital,The Fourth Military Medical University,Xi'an 710032,China)

    Objective To evaluate the biosafety of surface modified polyethylene terephthalate(PET) fiber.Methods Surface modified PET fiber was subjected to cytotoxicity test according to the relevant national requirements,and the cytotoxicities before and after modification were compared.The extract of surface modified PET fiber was subjected to acute toxicity,haemolysis,pyrogen and sensitization tests,based on which its biosafety was evaluated.Results Compared with those in negative control group,the cytotoxicity of PET fiber before modification was judged as Ⅲ ～ Ⅳ(P < 0.05),while the PET after modification showed no cytotoxicity(P > 0.05).Surface modified PET fiber showed no systemic acute toxicity,and was qualified in pyrogen test as well as skin sensitization test in guinea pigs,of which the haemolysis rate was 0.07%(< 5%,P < 0.05).Conclusion Surface modified PET showed no toxicity,and was biocompatible,which met the relevant national standard for biosafety and provided a basis for further study on artificial ligament tissue engineering.

    2011 05 v.24 [Abstract][OnlineView][Download 178K]

  • Preparation of Monoclonal Antibody and Development of Quantitative ELISA for Pertussis Filamentous Hemagglutinin

    PENG Xiang-bing,CHEN Wen,ZHAN Qian,XIANG Mei-juan,ZHANG Ai-hua,YANG Xiao-ming(Wuhan Institute of Biological Products,Wuhan 430060,China)

    Objective To prepare monoclonal antibody(McAb) and develop an ELISA method for quantitative determination of pertussis filamentous hemagglutinin(FHA).Methods The McAbs against FHA was prepared by hybridoma technique and identified.The reaction condition of ELISA system was optimized,based on which double antibody sandwich ELISA for quantitative determination of FHA was developed and verified for precision and accuracy.The FHA contents in 12 batches of intermediate products of acellular pertussis vaccine were determined by the developed ELISA method.Results Four hybridoma cell strains stably secreting McAb against FHA were obtained,and the secreted McAbs were of IgG1 and IgG2b subtypes,with an ELISA titer of 1 ∶ 105 ～ 1 ∶ 106 in ascites,and showed specific reaction with FHA,while showed no cross reaction with pertussis toxin or influenza virus hemagglutinin.The linear detection range of developed ELISA method was 1.56 ～ 100.00 ng / ml,while both the intra-and inter-coefficients of variation were less than 10%.The recovery rates of FHA at high,moderate and low concentrations were 94.73%,108.67% and 116.37% respectively.The FHA contents(X ± 2SD) in 12 batches of intermediate products of acellular pertussis vaccine were stable.Conclusion The McAbs against FHA of pertussis were successfully prepared,and a double antibody sandwich ELISA method with high precision and accuracy was developed,which might be used for the determination of FHA content in intermediate products of acellular pertussis vaccine.

    2011 05 v.24 [Abstract][OnlineView][Download 298K]

  • Clinical Significance of Screening of Rh-negative Blood Group and Detection of Irregular Antibody

    LI Lan,WU Chang-lin,DANG Xin-tang,DONG Hong-qiang,ZHU Yi(Department of Blood Transfusion,The Second People's Hospital of Shenzhen,Shenzhen 518035,Guangdong Province,China)

    Objective To choose the matching blood to Rh-negative patients who were positive for irregular antibody by the screening of Rh-negative blood group and the detection of irregular antibody,and analyze its clinical significance.Methods A total of 363 RhD-negative patients were subjected to antibody screening and Rh blood grouping by micro-column agglutination method,based on which the Rh factor-matched blood was selected for transfusion.For lack of matched blood source,the RhD-negative patients with anti-c and anti-e antibodies were transfused with erythrocytes group O of ccdEE and CCdee phenotypes.Results Of the 363 RhD-negative patients,21 were positive for irregular antibody,including 5 for anti-D,8 for anti-E,3 for anti-c,2 for anti-c and E,2 for anti-C and 1 for anti-e.The patients with irregular antibodies were transfused with selected Rh factor-matched blood,and satisfactory clinical efficacy was observed.Conclusion By transfusion with Rh factor-matched blood selected based on antibody screening and Rh blood grouping,the production of irregular antibodies was avoided,the efficacy of clinical blood transfusion was improved,and the adverse reactions of blood transfusion was reduced.

    2011 05 v.24 [Abstract][OnlineView][Download 134K]

  • Cloning of Long Tandem Repeat Sequence of Vero Cells and Its Application in Quantitative PCR for DNA

    CAO Shou-chun,DONG Guan-mu,LI Jia,TANG Jian-rong,WU Xiao-hong,LIU Jing-hua,SHI Lei-tai(National Institute for the Control of Pharmaceutical and Biological Products,Beijing 100050,China)

    Objective To clone the long tandem repeat sequence(LTR) in genomic DNA of Vero cells and develop a quantitative PCR method for Vero cell DNA.Methods To clone the long tandem repeat sequence(LTR) in genomic DNA of Vero cells and develop a quantitative PCR method for Vero cell DNA.Results Eight positive clones of pET-30a-172 were obtained by restriction analysis and sequencing,with homologies of 98.3% ～ 99.4% to the gene sequence reported in GenBank.The sensitivity and liner range of the developed quantitative PCR method were 1 × 10-6 ng / μl and 1 ng / μl ～ 1 × 10-6 ng / μl respectively.By the developed PCR method,amplification curve was only obtained from the DNA of human diploid MRC-5 cells but not from that of chick embryo cells,CHO cells or hamster kidney cells,indicating high specificity.Conclusion A 172 bp LTR-based quantitative PCR method for Vero cell DNA was successfully developed,which might be used for residual Vero cell DNA content in vaccines.

    2011 05 v.24 [Abstract][OnlineView][Download 280K]

  • Validation of Inactivation of Parvovirus in Human Prothrombin Complex Concentrate by Short-wave Ultraviolet C and Impaction on Product Quality

    WANG Min,YUE Guang-zhi,YANG Li-hong,JIA Li-li,YANG Jing-qing,ZHOU Qian,HAO Jie,WANG Qing-zhou,HOU Ji-feng(National Institutes for Food and Drug Control,Beijing 100050,China)

    Objective To inactivate the parvovirus in human prothrombin complex concentrate(PCC) by using short-wave ultraviolet(UVC),validate the inactivation effect as well as the impaction on product quality.Methods The porcine parvovirus(PPV) as an indicator virus in PPC was inactivated with short-wave UVC at dosages of 200,250 and 300 J / m2 respectively.The virus titer was determined by micro-cell culture method.The samples showing no CPE were subjected to 3 blind passages and validated for inactivation effect.The PPC samples were evaluated for structure and function by 2-DE and HPLC,and determined for activity.Results The titer of PPV inactivated with short-wave UVC decreased by more than 4.0 log.CPEs were observed in all the samples after 3 blind passages.The recovery rate of coagulation factor activity in absence of stabilizer was more than 70%.HPLC proved that the coagulation factor protein polymer content and chromatographic behavior of the samples after UVC irradiation showed no significant change as compared with that before irradiation.2-DE showed that the number of protein spots after irradiation was basically consistent with that before irradiation.However,changes were observed in positions and gray values of two protein spot groups in the samples treated with 300 J / m2 UVC.Conclusion Short-wave UVC showed good effectiveness in inactivation of non-enveloped viruses,with little impaction on protein activity of PCC.

    2011 05 v.24 [Abstract][OnlineView][Download 361K]

  • Optimization of Medium for Fermentation of Fuscoporia obliqua by Response Surface Method

    HE Chang-chuan△,ZHANG Zong-ju,ZOU Wei-sheng,CHU Qing-huan,ZHU Hu [△Center for Bioengineering and Biotechnology,China University of Petroleum(East China),Qingdao 266555,Shandong Province,China]

    Objective To optimize the medium for fermentation of Fuscoporia obliqua by response surface method and increase the yield of polysaccharide.Methods The component of medium for fermentation of Fuscoporia obliqua was optimized by Plackett-Burman design and response surface method.Polynomial regression analysis was performed on the experimental data by using Design-Expert software,based on which the functional relationship between polysaccharide yield and three main factors(starch,yeast extract and sodium dihydrogen phosphate) were explored.The finally optimized formula of medium was subjected to three verification test.Results The optimal concentrations of starch,yeast extract and sodium dihydrogen phosphate were 7.5,1.71 and 1.35 g / L respectively,and the extracellular polysaccharide yield reached 9.12 g / L at most.The polysaccharide yields obtained by three verification tests were 9.08,8.96 and 9.12 g / L respectively,with a mean of 9.05 g / L,which was basically consistent with that expected.Conclusion By using the optimized medium,the extracellular polysaccharide yield increased significantly.

    2011 05 v.24 [Abstract][OnlineView][Download 353K]

  • Application of Mixed Mode Chromatography to Polishing Purification of Monoclonal Antibody

    LV Ruo-yun,CHENG Li-jun(NCPC New Drug Research and Development Co.Ltd.,Hebei Iindustry Microbial Metabolic Engineering & Technology Research Center,State Key Laboratory of Antibody Drug Development,Shijiazhuang 050015,China)

    Highly effective polishing purification shall be introduced to purification procedure of monoclonal antibody(McAb),and each step of the procedure shall be verified for the ability of removing foreign matters.Polishing purification techniques,such as ion exchange chromatography and hydrophobic chromatography,may be used for removing special matters,which are of important significance in the qualities of final products.However,a novel mixed mode chromatography is increasingly used for the polishing purification of McAb.The principles,key influencing factors and the abilities in removing foreign matters of various polishing purification methods suitable for industrial production,including hydrophobic charge induction chromatography,multi-modal anion exchange and hydroxyapatite chromatography,are reviewed in this paper.

    2011 05 v.24 [Abstract][OnlineView][Download 155K]

  • Progress in Research on Evaluation of Safety of Products for Gene Therapy

    ZHOU Xiao-bing,LI Bo(National Institute for the Control of Pharmaceutical and Biological Products,National Center for Safety Evaluation of Drugs,Beijing 100176,China)

    As a novel method for treatment of disease,gene therapy has been paid more and more attentions.The safety of products for gene therapy,as novel drugs,is one of the most important indexes for evaluation of the novel method for treatment.Proceeding from vector and expressed product,this paper reviews the progress in research on safety and its evaluation of products for gene therapy.

    2011 05 v.24 [Abstract][OnlineView][Download 184K]

  • Progress in Research on Pandemic Influenza H5N1 Vaccine

    ZHANG Yan-yu,LIAO Guo-yang,LI Wei-dong(Institute of Medical Biology,Chinese Academy of Medical Science and Peking Union Medical College,Kunming 650118,China)

    Highly pathogenic avian influenza H5N1 virus may cause serious fatal diseases and constitute a grave threat to human health.Although the current H5N1 influenza strains appear not to be transmissible from human to human,it is of major concern that mixing with human influenza strains could convert H5N1 to a strain that would spread that would spread from human to human and cause a serious pandemic.In addition,avian influenza H5N1 virus after continuous variation may break through the species barrier and spread to mammals and humans,thereby cause disease even death under the influence of various virus and host factors.This paper reviews the progress in research on pandemic influenza vaccine.

    2011 05 v.24 [Abstract][OnlineView][Download 155K]


@2012 Editorial Department of "Chinese Journal of Biologicals"