2011年04期目次

  • Effect of Hepatitis C Virus JFH1 NS5A Gene on Replication and Infectivity of HC-J4 Virus

    WANG Yong-zhi,WANG Wen-bo,XU Gang,CAO Ming-mei,REN Hao,QI Zhong-tian(Department of Microbiology,Shanghai Key Laboratory of Medical Biodefense,PLA Key Laboratory of Biodetection and Defense,Second Military Medical University,Shanghai 200433,China)

    Objective To investigate the effect of hepatitis C virus(HCV) 2a FL-J6JFH NS5A gene substitution on replication and infectivity of HC-J4 virus genotype 1b and lay a foundation of establishment of HCV 1b cell model.Methods JFH1 NS5A was introduced into HC-J4 genome by gene substitution to construct chimeric full-length genome HC-J4/JFHNS5A.The RNA transcripts of wild HC-J4,chimaera and FL-J6JFH were prepared in vitro and transfected to Huh-7.5 cells in mediation of liposome.The protein expression in transfected cells was determined by IFA,and the gene copy by HCV negative-strand RNA-specific RT-PCR and fluorescent quantitative PCR(FQ-PCR).The supernatant of cells were collected on various days after transfection and used for infection of naive Huh-7.5 cells to observe the infectivity.Results No expression of HCV protein was observed by IFA in the cells transfected with wild HC-J4 and with the chimaera.However,the negative-strand RNAs of HCV were detected at all the time points within 18 d after transfection,indicating low replication levels of chimaera and wild HC-J4 in transfected cells.FQ-PCR showed that,on days 9 and 12 after transfection,the HCV RNA level in the cells transfected with chimaera was significantly higher than that with wild HC-J4(P < 0.05).No expression of HCV protein was observed by IFA in naive Huh-7.5 cells at any time point after infection with supernatant of transfected cells.Conclusion Though JFH1 NS5A protein enhanced the replication ability of HC-J4 strain of genotype 1b in the cells cultured in vitro at a certain degree,no detectable infectious virus particles were produced.The establishment of HCV 1b cell model was influenced by other factors.

    2011 04 v.24 [Abstract][OnlineView][Download 934K]

  • Establishment of Mammalian Cell Line for Stable Expression of Hemagglutinin Protein of Influenza Virus Type A

    GUO Jian-qiang,CHEN Ai-jun,YAO Li-hong,LIU Xiao-yu,FU Jin-qi,XU Peng-wei,ZHANG Zhi-qing(State Key Laboratory for Molecular Virology and Genetic Engineering,Institute for Viral Disease Control and Prevention,Chinese Center for Disease Control and Prevention,Beijing 100052,China)

    Objective To establish a mammalian cell line for stable expression of hemagglutinin(HA) of influenza virus type A.Methods Full-length HA gene of influenza virus(A/PR/8/34) was amplified by PCR and cloned into eukaryotic expression vector pcDNA5/FRT(pDF).Flp-In-CHO cells were co-transfected with the constructed recombinant plasmid pDF-HA and plasmid pOG44 expressing Flp recombinase,and the target gene was integrated into chromosome of CHO cells by homologous recombination in vivo.Recombinant CHO-HA cell line was screened by continuous pressure screening with hygromycin B,and the expression of HA protein was determined by IFA and Western blot.The recombinant cell line was subcultured for 10 passages and tested for the stability of genetic and expression of HA by PCR and IFA respectively.Results Both restriction analysis and sequencing proved that recombinant plasmid pDF-HA was constructed correctly.Twenty recombinant cell strains highly expressing HA protein were screened with hygromycin B.Western blot proved that HA protein was expressed in the recombinant cells and were cleaved into HA1 and HA2 proteins.The expressions of HA at gene and protein levels were proved by PCR and IFA in the recombinant cells after subculture for 10 passages.Conclusion The mammalian cell line for stable expression of HA of influenza virus type A was successfully established,which provided target cells for immunological determination of HA protein and influenza virus as well as study on function of HA protein.

    2011 04 v.24 [Abstract][OnlineView][Download 1064K]

  • Expression of Excellular Domain of Glycoprotein D of Herpes Simplex Virus Type 2 in Mammalian Cells and Immunologic Activity of Expressed Product

    GUAN Wen-yan,WANG Zheng-mao,LI Lin,LI Yue-xi(Department of Biochemistry & Molecular Biology,School of Preclinical Medicine,Nanjing Medical University,Nanjing 210029,China)

    Objective To express the excellular domain of glycoprotein D(gD) of herpes simplex virus type 2(HSV-2) in mammalian cells and analyze the immunologic activity of expressed product.Methods The gDt gene sequence encoding excellular domain of gD of HSV-2 G strain was chemically synthesized and inserted into vector pCEP4.The constructed recombinant plasmid pCEP4-gDt,with a His label at N-terminus,was transfected to HEK293 cells for expression.The expressed protein was purified by nickel ion column affinity chromatography and tested for antigenicity by ELISA.Polyclonal antisera were prepared by immunizing mice with the purified recombinant protein and determined for titer by ELISA.Results PCR,restriction analysis and DNA sequencing proved that recombinant plasmid pCEP4-gDt was constructed correctly.Western blot showed a target protein band with relative molecular mass of about 46 000.The purified recombinant protein,at a concentration of about 45 μg/ml,showed good antigenicity as proved by ELISA,which induced specific antibody titer of 5 × 103 in the sera of mice 5 weeks after immunization.Conclusion The excellular domain of gD of HSV-2 was expressed in mammalian cells,and showed good antigenicity and immunogenicity,which laid a foundation of preparation of recombinant subunit vaccine against HSV.

    2011 04 v.24 [Abstract][OnlineView][Download 1047K]

  • Expression of Recombinant Fusion Protein BoNT-LH(N)-Elafin in Pichia pastoris and Activity of Expressed Product

    YU Hong-mei,LI Qi,ZHOU Xiang-dong,Perelman Juliy M,Kolosov Victor P(Department of Respiratory Medicine,The Second Affiliated Hospital,Chongqing Medical University,Chongqing 400010,China)

    Objective To express BoNT-LH(N)-Elafin fusion protein in Pichia pastoris and determine its bioactivity.Methods Recombinant eukaryotic expression vector pPIC9K-BoNT-LH(N)-Elafin was constructed and transformed to P.pastoris GS115 strain for expression under induction of methanol.The culture supernatant was collected,from which the fusion protein was purified by cation exchange chromatography and determined for reactogenicity,anti-elastase activity and cleaving effect on SNARE protein complex.Results Both restriction analysis and sequencing proved that recombinant plasmid pPIC9K-BoNT-LH(N)-Elafin was constructed correctly.The expressed BoNT-LH(N)-Elafin fusion protein,with a relative molecular mass of about 110 000,reached a purity of 92% and a concentration of 54 mg/L after purification,which showed good reactogenicity,anti-elastase activity as well as cleaving effect on SNARE protein complex.Conclusion Recombinant fusion protein BoNT-LH(N)-Elafin was successfully expressed in P.pastoris GS115 strain and showed high bioactivity,which laid a foundation of further study on the mechanism of its inhibitory effect on airway mucus hypersecretion.

    2011 04 v.24 [Abstract][OnlineView][Download 1081K]

  • Effect of Heparan Sulfate Combined with Zinc Hydroxide on Immune Response Induced by HBsAg in Mice

    WU Mei-ni,WANG Hai-xuan,LAN Yun,HU Ning-zhu,HU Yun-zhang(Institute of Medical Biology,Chinese Academy of Medical Sciences and Peking Union Medical College,Kunming 650118,China)

    Objective To investigate the effect of heparan sulfate(HS) combined with zinc hydroxide,as an immunological adjuvant,on immune response induced by HBsAg in mice.Methods ICR mice were divided into twenty-three groups,including test groups as well as negative control,antigen control,aluminum adjuvant control,HS control and zinc hydroxide groups.The HS and zinc hydroxide,at various dosages,were combined and further mixed with 2 μg of HBsAg,then immunized to the mice in various test groups by different schedules.Blood samples of the mice were collected 4,8,12,16,20 and 24 weeks after the last immunization,from which sera were separated and determined for anti-HBs level by ELISA.BALB/c mice were immunized at the same dosage by the same schedule as those in the group which showed the most satisfactory humoral immune effect and determined for cytotoxic activity by lactate dehydrogenase method 8 weeks later.Results No IgG was detected in blank control group at any time points.However,the IgG titers in all the rest groups reached peak values 8 weeks after the last immunization and showed a decreasing tendency as time went on.The IgG level in group 23(100 μg HS + 1.0 mg zinc hydroxide,immunized twice) was the highest and lasted for a long time.Eight weeks after the last immunization,the humoral immune response levels of mice immunized with HS combined with zinc hydroxide were significantly higher than those with aluminum adjuvant and with HS adjuvant alone(P < 0.05),and insignificantly higher than that with zinc hydroxide adjuvant alone(P > 0.05).HS combined with zinc hydroxide induced specific CTL cellular immune response.Conclusion HS combined with zinc hydroxide enhanced the specific humoral immune response induced by HBsAg and induced CTL cellular immune response effectively.

    2011 04 v.24 [Abstract][OnlineView][Download 1005K]

  • Prokaryotic Expression and Purification of Minor Capsid Protein L2 Derived from Human Papillomavirus 58 and Preparation of Monoclonal Antibody

    LIN Jie,LV Qi,TONG Liang,LIU Fang,MA Lan(Life Science Division,Graduate School at Shenzhen,Tsinghua University,Shenzhen 518055,Guangdong Province,China)

    Objective To express the minor capsid protein L2 derived from human papillomavirus(HPV) 58 which caused majority of cervical cancer in China in prokaryotic cells,purify the expressed product and prepare its monoclonal antibody(McAb).Methods L2 gene was amplified from plasmid pHPV58 containing the genome of HPV-58 and inserted into modified prokaryotic expression vector pGEX-KGV.The constructed recombinant plasmid pGEX-KGV-HPV58 L2 was transformed to E.coli BL21(DE3) for expression under induction of lactose.The expressed protein was re-naturalized by dialysis and purified by affinity chromatography,based on which McAb was prepared by hybridoma technique and identified.Results Restriction analysis and sequencing proved that recombinant plasmid pGEX-KGV-HPV58 L2 was constructed correctly.The expressed protein,mainly existing in a form of inclusion body,contained 15.5% of total somatic protein and reached a purity of 95% after purification.A total of cell strains secreting high titer McAbs were obtained.Conclusion HPV-58 L2 protein was successfully expressed in prokaryotic cells and purified,and its McAb was prepared,which laid a foundation of further study on the antigenic epitope of L2 protein as well as development of relevant prophylactic antibody drug and broad spectrum recombinant vaccine.

    2011 04 v.24 [Abstract][OnlineView][Download 1192K]

  • Inhibitory Effect of P115 Gene Silencing by shRNA on Expression of Macroph-ge Migration Inhibitory Factor in Gastric Carcinoma Cells

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

    Objective To construct the shRNA expression vector for Golgi-vesicular transport protein P115 and investigate the regulatory effect of P115 gene silencing on expression of macrophage migration inhibitory factor(MIF) in gastric carcinoma cell BGC-823 strain.Methods Four shRNA sequencing specific to P115 gene were designed,based on which recombinant expression vectors were constructed and transfected to BGC-823 cell strain for high expression of P115.The expressions of P115 and MIF mRNAs were determined by RT-PCR,while those of proteins by Western blot.Results All the four shRNA plasmids for P115 gene were constructed correctly as proved by restriction analysis and sequencing,and inhibited the expression of P115 gene in transfected BGC-823 cells.The silencing effect of pGPU6/GFP/Neo-shP115-2 was satisfactory,of which the inhibiting rate to expressions of P115 mRNA and protein were 75.07% and 70.97% respectively.Both the expression levels of MIF mRNA and protein in cells transfected with pGPU6/GFP/Neo-shP115-2 decreased significantly(P < 0.05).Conclusion The expression level of MIF in BGC-823 cells after P115 gene silencing decreased significantly,indicating that P115 might involve in regulating the expression of MIF in gastric carcinoma cells and P115 gene might be used as a novel target for study on the molecular mechanism of onset and progress of gastric carcinoma.

    2011 04 v.24 [Abstract][OnlineView][Download 1267K]

  • Construction and Identification of Eukaryotic Coexpression Vector for PPE68/Ipr1

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

    Objective To construct a eukaryotic vector for coexpression of PPE68 gene of Mycobacterium tuberculosis and intracellular pathogen resistance 1(Ipr1) gene of mice,and express in murine macrophage RAW264.7 cells.Methods PPE68 and Ipr1 genes were subcloned into coexpression vector pBudCE4.1 containing multiple promoters,and the constructed recombinant plasmid pBud68-Ipr1 was transfected to RAW264.7 cells.The transcriptions of PPE68 and Ipr1 genes were determined by RT-PCR,and their expressions by Western blot.Results PCR,restriction analysis and sequencing proved that both PPE68 and Ipr1 genes with correct sequences were inserted into recombinant plasmid pBud68-Ipr1.Both PPE68 and Ipr1 genes were transcribed and expressed in transfected RAW264.7 cells,and the relative molecular masses of expressed protein were 37 000 and 50 000 respectively.Conclusion Eukaryotic coexpression vector pBud68-Ipr1 was successfully constructed and expressed in RAW264.7 cells,which laid a foundation of construction and further study on the immune protection of recombinant BCG with PPE68/Ipr1.

    2011 04 v.24 [Abstract][OnlineView][Download 1151K]

  • Construction and Identification of shRNA Expression Vector for Human Rac1 Gene

    WANG Hong,ZHANG Ju-qing,CHEN Yu-bing,ZHANG Hong-mei,CUI Man-hua(Second Hospital of Jilin University,Changchun 130041,China)

    Objective To construct the shRNA expression vector for human Rac1 gene and lay a foundation of increasing the sensitivity of ovarian cancer to radiotherapy and chemotherapy.Methods The oligo DNA for construction of shRNA expression vector was designed and synthesized by using shRNA design software according to the sequence of human Rac1 gene in GenBank,based on which recombinant shRNA vector with human Rac1 gene was constructed and tranfected to ovarian cancer Skov3 cells,and effective recombinant shRNA plasmid was screened by RT-PCR 48 h later.Results Recombinant shRNA plasmid with Rac1 gene was identified by digestion with BamHⅠ and PstⅠ,of which the sequence was completely consistent with theoretical sequence.RT-PCR showed that the transcription level of Rac1 mRNA in Skov3 cells transfected with plasmid pGPU6/GFP/Rac1-524 decreased.Conclusion The shRNA expression vector for human Rac1 gene was successfully constructed,and effective interfering plasmid pGFU6/GFP/Rac1-524 was screened.

    2011 04 v.24 [Abstract][OnlineView][Download 1265K]

  • Prokaryotic Expression and Purification of Protein D of Haemophilus influenza Type b

    DU Qian,ZHANG Hua-jie,WANG Yi-fei,ZHANG Shu-min(Biomedicine Research and Development Center,Jinan University,Guangzhou 510632,China)

    Objective To clone the hpd gene encoding protein D of Haemophilus influenza type b(Hib),express in proka-ryotic cells,purify the expressed product and lay a foundation of further development of protein D-based conjugate vaccine.Methods The hpd gene fragment was amplified from genomic DNA of Hib CMCC strain by PCR and cloned into vector pET-30a(+).The constructed recombinant plasmid pET-30a-hpd was transformed to competent E.coli BL21(DE3) for expression under induction of IPTG.The expressed protein was de-naturalized with 6 mol / L urea,purified by DEAE anion exchange column chromatography,re-naturalized by dialysis and identified for reactogenicity by Western blot.Results PCR and sequencing proved that the recombinant plasmid pET-30a-hpd was constructed correctly.The expressed protein,in a form of inclusion body,contained about 40% of total somatic protein and reached a purity of about 95% after purification by one-step column chromatography.The purified recombinant protein showed specific reaction with the antisera prepared by immunizing mice with Hib.Conclusion The hpd gene of Hib was successfully cloned and expressed in E.coli,which laid a foundation of further development of protein D-based conjugate vaccine.

    2011 04 v.24 [Abstract][OnlineView][Download 1148K]

  • Effect of Carbohydrate Response Element Binding Protein on Glucose and Lipid Metabolism in L02 Cells

    XIONG Ling,SHEN Wei(Department of Gastroenterology,Second Affiliated Hospital,Chongqing University of Medical Sciences,Chongqing 400010,China)

    Objective To investigate the role of carbohydrate response element binding protein(ChREBP) in hepatocyte fatty degeneration induced by high glucose concentration.Methods L02 cells were cultured in 18 and 25 mmol/L glucose,using those in 11 mmol/L glucose as control,then determined for triglyceride(TG) content,observed for fatty degeneration by oil red staining and for nuclear translocation of ChREBP by IFA.The expression level of liver pyruvate kinase(LPK) mRNA in the cells was determined by RT-PCR,while that of fatty acid synthase(FAS) protein by Western blot.Results Compared with low glucose concentration,high glucose concentration increased the TG and fatty drop contents in L02 cells,stimulated the nuclear translocation of ChREBP and up-regulated the expressions of LPK mRNA and FAS protein.Conclusion Glucose might induce hepatocyte fatty degeneration by its metabolic product through ChREBP-LPK-FAS pathway.

    2011 04 v.24 [Abstract][OnlineView][Download 1359K]

  • Effect of YKL-40 Transfection on Proliferation and Invasion Activities of Prostate Cancer LNcap Cells

    XIA Yan,ZHA Cheng-xi,YANG Hai-jing,WEN Yu-ting,YANG Hao,CHEN Wei(The Institute of Biochemistry and Molecular Biology,Basic Medical College,Lanzhou University,Lanzhou 730000,China)

    Objective To investigate exogenous YKL-40 gene transfection on the proliferation,invasion,migration and adhesion activities of prostate cancer LNcap cells.Methods The expressions of endogenous YKL-40 gene in prostate cancer DU-145,PC-3 and LNcap cells were determined by RT-PCR.LNcap cells were transfected with plasmid pcDNA3.1-YKL-40 and determined for proliferation,invasion,migration and adhesion activities before and after transfection by MTT method,Boyden chamber method and adhesion test,based on which in vitro drug-sensitivity test was performed.Results Endogenous YKL-40 gene was only expressed in DU-145 cells.On days 2 ~ 5 after transfection with plasmid pcDNA3.1-YKL-40,the A490 values of LNcap cells were significantly higher than those of control(P < 0.05).The numbers of cells with invasion and transmembrane migration were(75.11 ± 4.40) and(133.00 ± 5.07) respectively,and the adhesion rate was 107.57%,which were significantly higher than those of control(P < 0.05).The IC50 of transfected LNcap cells to 5-FU,cisplatin and etoposide were(31.15 ± 0.43),(4.15 ± 0.13) and(55.22 ± 0.57) μmol / L respectively,which were significantly higher than those of control(P < 0.05).Conclusion YKL-40 gene promoted the proliferation and increased the invasion,migration and adhesion activities as well as the resistance to 5-FU,cisplatin and etoposide of LNcap cells.

    2011 04 v.24 [Abstract][OnlineView][Download 1249K]

  • Construction and Identification of Recombinant Adenovirus with Rat Protein Inhibitor of Activated STAT1 Gene

    CHEN Ping,HUANG Li-ya,YUAN Yao-zong(Department of Gastroenterology,Ruijin Hospital,Shanghai Jiaotong University School of Medicine,Shanghai 200025,China)

    Objective To construct and identify a recombinant adenovirus with rat protein inhibitor of activated STAT1(PIAS1) gene.Methods Full-length PIAS1 gene was amplified from rat pancreatic acinar AR42J cell strain by RT-PCR and,after T-A cloning,subcloned into shuttle plasmid pDC316.Adenovirus skeleton plasmid Nad5/F35 and shuttle plasmid pDC316-PIAS1 were co-transfected to 293 cells by homologous recombination,and the obtained recombinant adenovirus plasmid Ad5/F35-PIAS1 was packaged and amplified to obtain recombinant adenovirus.The virus infection was observed by fluorescent microscopy,and the expressions of PIAS1 gene and protein by RT-PCR and Western blot respectively,based on which the titer of recombinant adenovirus was calculated.Results PIAS1 gene at a length of 1 956 bp was amplified from AR42J cell strain.Restriction analysis proved that recombinant adenovirus plasmid Ad5/F35-PIAS1 was constructed correctly.RT-PCR and Western blot showed that PIAS1 gene and protein were expressed in 293 cells.Fluorescent microscopy showed that the infection rate of recombinant adenovirus reached 90%,and the virus titer was 4.45 × 1010 PFU/ml.Conclusion The recombinant adenovirus with rat PIAS1 gene was successfully constructed,which laid a foundation of further study on the role of PIAS1 gene in the relevant diseases as well as its application in clinic.

    2011 04 v.24 [Abstract][OnlineView][Download 1266K]

  • Relationship of IL-27p28 Gene Polymorphism and Asthma in Chinese Korean Nationality Population

    SHAN Yuan-chun,CUI Shan,TIAN Guo-he,ZENG Wo-tan,ZHANG Qing-gao(The Sci-tech Mis of Yanbian University,Yanji 133000,Jilin Province,China)

    Objective To investigate the relationship of polymorphisms at g.-964T > C site in promoter region,g.2905T > G site in the second exon region and g.4730T > C site in the fourth exon region of IL-27p28 gene to the asthma in Chinese Korean Nationality population.Methods The g.-964 T > C,g.2905T > G and g.4730T > C sites of IL-27p28 gene were amplified from the blood of 32 Chinese Korean Nationality patients with asthma by PCR for genotyping by single nucleotide extension method,and analyzed for genotype and allele frequencies,using those from 93 healthy persons of the same nationality as control.Results Both the distributions of genotypes in trial and control groups were fitted to Hardy-weinberg Law(P > 0.05).However,the genotype and allele frequencies at g.-964T > C site in trial group showed significant difference(P < 0.001),while those at g.2905 and g.4730 sites showed no significant difference with those in control group(P > 0.05).Conclusion The polymorphism at g.-964T > C site of IL-27p28 gene might be related to the susceptibility to asthma in Chinese Korean Nationality population.However,no relationship was observed between the polymorphism at g.2905T > G or g.4730T > C sites and asthma in the said population.

    2011 04 v.24 [Abstract][OnlineView][Download 1218K]

  • Physiological Property of Pichia pastoris X-33 Glycosylation Mutant

    XU Yong-li,ZHANG Da-cheng,ZHU Rui-yu,CHEN Yun,JIN Jian(School of Medicine and Pharmaceutics,Jiangnan University,Wuxi 214122,Jiangsu Province,China)

    Objective To investigate the physiological property of wild Pichia pastoris X-33 strain(wX-33) and X-33α-1,6 glycosyltransferase(Och1p) mutant strain(och1 mX-33) and lay a foundation of study on further glycosylation of och1 mX-33 strain.Methods The growth curve of wX-33 and och1 mX-33 strains were plotted,based on which the strains were tested for sensitivities to temperature and Congo red,observed for morphological change by methylene blue dyeing and determined for cell survival rate.Results Compared with wX-33 strain,och1 mX-33 strain grew slowly,of which the biomass decreased,the sensitivities to temperature and Congo red increased.The deficiency in cell wall division was observed and,at unsuitable temperature,the cell survival rate decreased.Conclusion Glycosylation mutation influenced the sensitivities of P.pastoris X-33 to temperature and Congo red,decreased the mannose content in cell wall and caused significant change of physiological property of och1 mX-33 strain.

    2011 04 v.24 [Abstract][OnlineView][Download 1301K]

  • Action Mechanism of Phospholipase C Epsilon in Regulating Angiogenesis of Renal Cell Carcinoma

    WANG Chun-yuan,LUO Chun-li,YANG Shu-zhe,PAN Cui-cui,YAN Ling,ZHANG Yan-yi(Department of Laboratory Medicine,Key Laboratory of Clinical Laboratory and Diagnostics of Ministry of Education,Chongqing Medical University,Chongqing 400016,China)

    Objective To investigate the mechanism of vascular endothelial growth factor(VEGF) in influencing the angiogenesis of tumors through phospholipase C epsilon(PLCε)-NF-κB signal pathway.Methods Plasmid pGenesil-PLC for expression of PLC ε-shRNA was transfected to human renal cell carcinoma 786-0 cell strain to silence the expression of PLCε gene.The expressions of PLC ε,NF-κB and VEGF in transfected cells at mRNA level were determined by RT-PCR,while those at protein level by Western blot.The cells were treated with BAY11-7082,a specific inhibitor of NF-κB,and determined for inhibiting effect on cell proliferation by MTT method,then for expressions of VEGF mRNA and protein by RT-PCR and Western blot respectively.Results Recombinant plasmid pGenesil-PLCε inhibited the expressions of PLCε at mRNA and protein levels significantly,of which the inhibiting rates were 71.43% and 50.01% respectively.The plasmid also down-regulated the expressions of NF-κB and VEGF mRNAs and proteins significantly.BAY11-7082 showed significantly dose-and time-dependent inhibitory effect on the proliferation of 786-0 cells.Both the expressions of VEGF mRNA and protein in the cells after treatment with BAY11-7082 were inhibited significantly.Conclusion PLCε might inhibited the expression of NF-κB-dependent VEGF gene by inhibiting the expression of NF-κB gene and influence the angiogenesis of renal cell carcinoma.

    2011 04 v.24 [Abstract][OnlineView][Download 1476K]

  • Prokaryotic Expression and Purification of Minor Capsid Protein L2 Derived from Human Papillomavirus 58 and Preparation of Monoclonal Antibody

    LIN Jie,LV Qi,TONG Liang,LIU Fang,MA Lan(Life Science Division,Graduate School at Shenzhen,Tsinghua University,Shenzhen 518055,Guangdong Province,China)

    Objective To express the minor capsid protein L2 derived from human papillomavirus(HPV) 58 which caused majority of cervical cancer in China in prokaryotic cells,purify the expressed product and prepare its monoclonal antibody(McAb).Methods L2 gene was amplified from plasmid pHPV58 containing the genome of HPV-58 and inserted into modified prokaryotic expression vector pGEX-KGV.The constructed recombinant plasmid pGEX-KGV-HPV58 L2 was transformed to E.coli BL21(DE3) for expression under induction of lactose.The expressed protein was re-naturalized by dialysis and purified by affinity chromatography,based on which McAb was prepared by hybridoma technique and identified.Results Restriction analysis and sequencing proved that recombinant plasmid pGEX-KGV-HPV58 L2 was constructed correctly.The expressed protein,mainly existing in a form of inclusion body,contained 15.5% of total somatic protein and reached a purity of 95% after purification.A total of cell strains secreting high titer McAbs were obtained.Conclusion HPV-58 L2 protein was successfully expressed in prokaryotic cells and purified,and its McAb was prepared,which laid a foundation of further study on the antigenic epitope of L2 protein as well as development of relevant prophylactic antibody drug and broad spectrum recombinant vaccine.

    2011 04 v.24 [Abstract][OnlineView][Download 1276K]

  • Effect of Soluble Interleukin-5 Receptor Combined with Soluble Interleukin-13 Receptor on Airway Hyperresponsiveness and Airway Inflammation of Asthmatic Mice

    WAN Qi,WU Shuo,SONG Ai-ling,LI Yun-ming,ZHANG Ying-qi,LI Zhi-kui(Department of Respiratory Medicine,Xijing Hospital,The Fourth Military Medical University,Xi'an 710032,China)

    Objective To observe the effect of soluble interleukin-5 receptor α(sIL-5Rα) combined with soluble interleukin-13 receptor α2(sIL-13Rα2) on airway hyperresponsiveness(AHR) and airway inflammation of asthmatic mice.Methods Forty BALB/c mice were randomly divided into normal control,asthmas,sIL-5Rα,sIL-13Rα2 and sIL-5Rα + sIL-13Rα2 groups.Except those in normal control group,the mice in the other four groups were sensitized and challenged with ovalbumin to establish the mouse model of asthmas.The mice in sIL-5Rα,sIL-13Rα2 and sIL-5Rα + sIL-13Rα2 groups were injected i.p.with 100 μg of sIL-5Rα,100 μg of sIL-13Rα2 and 100 μg of sIL-5Rα + 100 μg of sIL-13Rα2 respectively 30 min before each challenge,while those in normal control and asthmas groups with physiological saline.Twenty-four hours after the last challenge,the mice in various groups were challenged with acetylcholine chloride at various concentrations and determined for airway resistance,counted for cells in broncho-alveolar lavage fluid(BALF),observed for pathological change in lung tissue by HE staining and determined for IL-5 and IL-13 levels in BALF by ELISA.Results After challenge with acetylcholine chloride,the airway resistance of mice in asthmas group increased significantly as compared with that in normal control group(each P < 0.01).The airway resistance was significantly lower in sIL-13Rα2 and sIL-5Rα + sIL-13Rα2 groups than in normal control group(P < 0.01),while was significantly lower in sIL-5Rα + sIL-13Rα2 group than in sIL-13Rα2 group(P < 0.01).Compared with those in normal control group,the total cell and eosinophil(EOS) counts in BALF of mice in asthmas group increased significantly(P < 0.01),and obvious lesion was observed in lung tissue.The total leukocyte and EOS counts in BALFs of mice were significantly lower in sIL-5Rα,sIL-13Rα2 and sIL-5Rα + sIL-13Rα2 groups than in asthmas group(each P < 0.01),while significantly lower in sIL-5Rα + sIL-13Rα2 group than in sIL-5Rα and sIL-13Rα2 groups(each P < 0.01).Pathological examination showed that,compared with those in asthmas group,the inflammatory infiltrations in sIL-5Rα,sIL-13Rα2 and sIL-5Rα + sIL-13Rα2 groups decreased,while the inflammatory reactions were mild.The IL-5 and IL-13 levels in BALF of mice were significantly higher in asthmas group than in normal control group(P < 0.01),while significantly lower in sIL-5Rα + sIL-13Rα2 group than in asthmas group(P < 0.01).Conclusion The sIL-5Rαcombined with sIL-5Rα2 decreased the AHR and relieved the airway inflammation of asthmatic mice.

    2011 04 v.24 [Abstract][OnlineView][Download 1438K]

  • Effect of Total Panax Notoginseng Saponins on Proliferation and Apoptosis of Pulmonary Artery Smooth Muscle Cells in Rats and Relevant Mechanism

    LI Chang-yi,WANG Dao-xin(Department of Respiratory,The Second Affiliated Hospital of Chongqing Medical University,Chongqing 400010,China)

    Objective To investigate the effect of total panax notoginseng saponins(PNS) on proliferation and apoptosis of pulmonary artery smooth muscle cells(PASMCs) in rats as well as the relevant mechanism.Methods PASMCs were divided into control(untreated),bFGF(8 ku/L) as well as bFGF + PNS at low(300 μg/L),moderate(450 μg/L) and high(600 μg/L) dosage groups,and determined for proliferation activity by MTT method,for distribution of cell cycle and apoptosis rate by flow cytometry and for expression of caspase-3 by Western blot 24 h later.Results PNS intervention inhibited the proliferation of PASMCs,arrested the cell cycle of PASMCs induced by bFGF into S stage,and increased the proporation of PASMCs at G0/G1 stages,the apoptosis rate of PASMCs as well as the expression level of caspase-3.The apoptosis rate of PASMCs treated with PNS was(18.70% ± 0.33%) ~(28.40% ± 0.79%),which was 10 ~ 14 times higher than those treated with bFGF.Conclusion PNS significantly inhibited the bFGF-induced proliferation and promoted the apoptosis of PASMCs by a potential mechanism which might be associated with regulating the conversion of cell cycle and activating the caspase-3 expression.

    2011 04 v.24 [Abstract][OnlineView][Download 1357K]

  • Prokaryotic Expression of Lipoprotein-associated Phospholipase A2 and Preparation of Its Polyclonal Antibody

    HUANG Mei-rong,HUANG Jian,WANG Hong,XU Xiu-yu,WANG Peng,YIN Yi-bing,XU Wen-chun(Key Laboratory Medical Diagnostics,Ministry of Education,Department of Laboratory Medicine,Chongqing Medical University,Chongqing 400016,China)

    Objective To construct a prokaryotic expression vector for lipoprotein-associated phospholipase A2(LP-PLA2) gene,express recombinant protein and prepare the polyclonal antibody against human LP-PLA2.Methods Total RNA was extracted from differentiated THP-1 cells,from which the full-length gene encoding LP-PLA2 was amplified by RT-PCR and inserted into prokaryotic expression vector pCold TF.The constructed recombinant plasmid was transformed to E.coli BL21(DE3) for expression under induction of IPTG at low temperature.The expressed protein was purified by Ni-Sepharose 6FF affinity chromatography and DEAE-Sephadex ion exchange chromatography and immunized into New Zealand rabbits.The prepared polyclonal antibody was determined for titer by indirect ELISA and for reactogenicity by Western blot.Results Restriction analysis and sequencing proved that recombinant plasmid pCold TF-LP-PLA2 was constructed correctly.The expressed TF-LP-PLA2 in a soluble form,with a relative molecular mass of about 93 000,reached a purity of 90% after purification and reacted with commercial antibody against human LP-PLA2.The prepared antiserum reached a titer of more than 1 ∶ 5.12 × 106 and showed good reactogenicity.Conclusion Recombinant LP-PLA2 protein was successfully expressed in prokaryotic cells,and high titer polyclonal antibody was prepared,which laid a foundation of further preparation of monoclonal antibody and development of immunological method for determination of LP-PLA2.

    2011 04 v.24 [Abstract][OnlineView][Download 1349K]

  • Prevalence of Human Coronavirus in Children in Chongqing Area,China

    XIE Yan-pi,CHEN Xin,DU Li-na,SHE Wei-wei,LI Rong-pei,ZHAO Xiao-dong(Children′s Hospital of Chongqing Medical University,Chongqing 400014,China)

    Objective To investigate the prevalence and epidemic features of four kinds of human coronavirus(HCoV),i.e.HCoV-HKU1,HCoV-NL63,HCoV-OC43 and HCoV-229E in hospitalized children with acute respiratory tract infection(ARTI) in Chongqing Area,China.Methods Nasopharyngeal aspirates(NPAs) were collected from 996 hospitalized children with ARTI in Children's Hospital of Chongqing Medical University from April 2007 to March 2009,of which 460 specimens positive for respiratory syncytial virus(RSV) and human metapneumovirus(hMPV) as proved by RT-PCR were excluded,while the other 536 specimens negative for RSV and hMPV were determined for HCoV.Results Ten(1.86%) of the 536 specimens were proved as HCoV-positive,of which 3(0.56%) were positive for HCoV-HKU1,4(0.74%) for HCoV-NL63,3(0.56%) for HCoV-OC43 and 0 for HCoV-229E.Of the 10 children,9 were male and 1 was female,6(60%) were at ages of 0 ~ 5 months,2(20%) at 6 ~ 11 months,1(10%) at 12 ~ 23 months,1 at 2 ~ 3 years,while none at age of more than 3 years.HCoV infection was sporadic in the whole year,of which HCoV-HKU1 infection mainly occurred in winter and spring,while HCoV-NL63 and HCoV-OC43 in summer and fall.The clinical symptoms of HCoV infection included fever,cough,sputum,pant,vomiting and diarrhea.Four of the 10 cases were diagnosed as bronchopneumonia,3 as interstitial pneumonia,1 as persistent pneumonia,1 as capillary bronchitis,and 1 as acute laryngotracheal bronchitis,of which 6 were complicated with symptomatic diarrhea.Conclusion HCoV was low prevalent in the hospitalized children with ARTI in Chongqing Area,which might cause infections in lower respiratory tract and alimentary canal.

    2011 04 v.24 [Abstract][OnlineView][Download 1357K]

  • Relationship of HBV-DNA to Age and Blood Lipid Metabolism of Patients with Chronic Hepatitis B

    PENG Pai-lan,HE Song,HU Wen-yan,LI Xuan,TIAN Lv,REN Hong,TANG Kai-fu(Department of Gastroenterology,The Second Affiliated Hospital of Chongqing Medical University,Chongqing 400010,China)

    Objective To analyze the relationship of HBV-DNA to age and blood lipid metabolism of patients with chronic hepatitis B.Methods The clinical data on 788 HBV-DNA-positive(with HBV-DNA titers of more than 1 × 103 copies/ml) patients with chronic hepatitis B,hospitalized in The Second Affiliated Hospital of Chongqing Medical University from November 2007 to October 2009,were analyzed retrospectively,based on which the relationship of HBV-DNA to age and blood lipid metabolism was analyzed by Spearman rank correlation and multiple linear regression.Results The result of Spearman rank correlation showed that HBV-DNA level was negatively related to the age of patients and positively related to the triglyceride(TG),total cholesterol(TC),high density lipoprotein(HDL),Low density Lipoprotein(LDL) and apolipoprotein A-Ⅰ(ApoA-Ⅰ) contents in blood.The result of multiple linear regression showed that HBV-DNA level was linearly dependent to the age and ApoA-Ⅰcontent of the patients.Conclusion The HBV-DNA level of patients with chronic hepatitis B was negatively related to the age and positively related to ApoA-Ⅰ content.

    2011 04 v.24 [Abstract][OnlineView][Download 1301K]

  • Clinical Curative Effect of Etanercept on Rheumatoid Arthritis Complicated with Coronary Heart Disease

    QU Bao-ze,GAO Wei(Department of Cardiology,First Affiliated Hospital of Liaoning Medical College,Jinzhou 121000,Liaoning Province,China)

    Objective To observe the clinical curative effect on Etanercept,an antagonist of TNFα,on rheumatoid arthritis(RA) complicated with coronary heart disease.Methods Sixty patients with RA complicated with coronary heart disease were divided into slow-acting drug and Etanercept groups randomly.The 30 patients in slow-acting drug group were treated with slow-acting drug during the whole observation period,while the other 30 patients in Etanercept group were treated with slow-acting drug at first then injected s.c.with Etanercept at a dosage of 25 mg twice a week for 3 months.Serum samples were collected before and 12 months after treatment and determined for homocysteine(HCY) level by full-automatic fluorescent polarization immunoassay.The clinical curative effect was evaluated.The coronary flow reserve(CFR) was determined by transthoracic echocardiography.High-resolution type B ultrasonic examination was used for scanning of arteria brachialis to measure the endothelium-dependent arterial flow-mediated dilation rate(FMD).The cardiovascular events,adverse reactions and changes in liver and kidney functions within 12 months were recorded.Results Compared with those before treatment,the serum HCY levels of patients in Etanercept decreased significantly(P < 0.05),while the CFR and FMD increased significantly(P < 0.05).However,in slow-acting drug group,the HCY,CFR and FMD levels after treatment decreased insignificantly as compared with those before treatment(P > 0.05).Compared with those after treatment with slow-acting drug,the serum HCY level of patients after treatment with Etanercept decreased significantly(P < 0.05),while CFR and FMD increased significantly(P < 0.05).The effective remission rate of clinical symptoms of patients in Etanercept group was significantly higher,while the occurrence rate of major cardiovascular events within 12 months was significantly lower than those in slow-acting drug group(P < 0.05).However,the adverse reaction rates in the two groups showed no significant difference(P > 0.05).Conclusion Etanercept relieved the clinical symptoms of patients with RA significantly,delayed the progress of coronary heart disease and decrease the occurrence of cardiovascular events.

    2011 04 v.24 [Abstract][OnlineView][Download 1388K]

  • Preparation and Identification of Complete Sulfadimidine Antigen

    WANG Ying,YAO Di,WANG Yue,AN Yu,ZHANG Dong-jie,LIU Zeng-shan(Food College of Heilongjiang Bayi Agriculture University,Daqing 163319,Heilongjiang Province,China)

    Objective To prepare and identify complete sulfadimidine(SM2) antigen.Methods SM2-BSA as antigen for immunization and SM2-OVA as antigen for detection were prepared by coupling with diazotization,and analyzed qualitatively and quantitatively.Results Both 1% agarose gel electrophoresis and 10% SDS-PAGE showed that complete antigens SM2-BSA and SM2-OVA were coupled successfully.The molecule conjugate ratios of SM2 to BSA and to OVA were 23 ∶ 1 and 17 ∶ 1 respectively.The protein concentrations of SM2-BSA and SM2-OVA were 3.24 and 2.29 mg/ml respectively.Complete SM2-BSA antigen stimulated high titer specific antibody against SM2 in mice.The monoclonal antibody against SM2 showed no cross reaction with OVA or BSA.Conclusion Complete SM2-BSA and SM2-OVA antigens were successfully prepared,which laid a foundation of further developing more sensitive,specific amd simple method for immunological test.

    2011 04 v.24 [Abstract][OnlineView][Download 1413K]

  • Development of Loop-mediated Isothermal Amplification Method for Detection of Swine Mycoplasma hyopneumoniae

    LI Peng,MA Yan-jiao,YU Jian,XUE Xiao-meng,LIU Cong,XIA Wei(College of Animal Science and Technology,Heilongjiang Bayi Agriculture University,Daqing 163319,Heilongjiang Province,China)

    Objective To develop a loop-mediated isothermal amplification(LAMP) method for detection of swine Mycoplasma hyopneumoniae.Methods Four specific primers were designed according to the nucleotide sequence of swine M.hyopneumoniae reported in GenBank.The external primers were used for PCR,and the concentrations of internal and external primers,dNTP+ and magnesium sulfate as well as the units of Bst DNA polymerase added were optimized,based on which a LAMP method for swine M.hyopneumoniae was developed and verified for specificity and sensitivity.Results When the concentrations of internal and external primers were 0.5 and 0.20 pmol/L,while those of dNTP+ and magnesium sulfate were 0.5 and 3.75 mmol/L respectively,and 8.0 or 9.6 U of Bst DNA polymerase was added,the reaction effect was satisfactory.No Pasteurella multocida,Actinobacillus,Streptococcus pneumonia,Bordetella bronchi,Haemophilus parasuis or Streptococcus were detected by the developed LAMP.The M.hyopneumoniae with a copy number of less than 3 could not be detected by the method.Conclusion A LAMP method for detection of swine M.hyopneumoniae was developed,which showed high specificity and sensitivity.

    2011 04 v.24 [Abstract][OnlineView][Download 1459K]

  • Preparation and Quality Control of High Titer Antiserum against Measles Virus

    SUN Lu,MA Yu,ZHNAG Guo-qiang,ZHANG Li-jun(Beijing Tiantan Biological Products Co.Ltd.,Beijing 100024,China)

    Objective To prepare high titer antiserum against measles virus and use for the virus identification,mycoplasma examination in control tests on vaccine as well as combined titration test on MM and MMR vaccines.Methods Measles virus L4 strain was subcultured in Vero cells to obtain high titer virus bulk and prepare the antigen for immunization.Rabbits were injected with the prepared antigen by i.p.,i.v.and s.c.routes respectively,and the obtained antisera were subjected to control tests according to the requirements in Chinese Pharmacopoeia.Results The neutralizing titers of antisera prepared by immunization of rabbits by i.p.,i.v.and s.c.routes were 1 ∶ 960,1 ∶ 2 560 and 1 ∶ 3 840 respectively.All the antisera met the requirements for control tests on vaccine.Conclusion The preparation of antiserum against measles virus by s.c.injection in several sites on back of rabbits was simple,and the prepared serum reached the highest titer,which might be an optimal method for preparation of high titer antiserum against measles virus.

    2011 04 v.24 [Abstract][OnlineView][Download 1346K]

  • Advance in Research on Therapeutic Vaccine

    LU Xiao-wu,ZHANG Ai-hua(Wuhan Institute of Biological Products,Wuhan 430060,China)

    Therapeutic vaccine has not only the indirect targeting specificity and long-action of prophylactic vaccine,but also the curative effect of therapeutic drug,especially the curative effect on some infectious diseases without specifically effective drugs at present and tumors.In recent years,the research on therapeutic vaccine has made progress extensively.This paper reviews the significance,action mechanism and progress in research of therapeutic vaccine.

    2011 04 v.24 [Abstract][OnlineView][Download 1412K]

  • Progress in Research on Oral Live Attenuated Rotavirus Vaccine Rotarix

    LI Xiao-feng,GUO Tai(National Institute for the Food and Drug Control,Beijing 100050,China)

    Rotavirus(RV) is an important pathogen of severe diarrhea in infants worldwide.Since there is no specific drug for RV infection,the development of safe and effective vaccine is of important significance.In the three kinds of domestic and imported commercial RV vaccines,Rotarix is of type G1P[8].Since RV type G1 is epidemic widely,this paper reviews the status of Rotarix.

    2011 04 v.24 [Abstract][OnlineView][Download 1354K]

  • Research Status of Cryopreservation of Parasites

    WANG Ai-min,ZHOU Guo-yan(Institute of Thermal Science,School of Medical Instrument and Food Engineering,Shanghai University of Science and Technology,Shanghai 200093,China)

    Traditional methods for storage of parasites,including in vitro culture and subcultivation in animals,are complicated to handle and of high costs,in which the problems such as genetic drift as well as artificial error or confusion may occur.Cryopreservation overcomes the problems while maintains the original biological characters of parasites.This paper reviews the kinds of parasites for as well as influencing factors of cryopreservation,and provides a reference for study on cryopreservation of parasites.

    2011 04 v.24 [Abstract][OnlineView][Download 1348K]
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