2010年11期目次

  • Prokaryotic Expression and Biological Activity of Recombinant Mycobacterium tuberculosis Ag85a-ESAT6 Fusion Protein

    ZHANG Qun,ZHU Lin,LOU Jue-ren(Shanghai Institute of Biological Products,Shanghai 200052,China)

    Objective To express recombinant Mycobacterium tuberculosis Ag85a-ESAT6 fusion protein in prokaryotic cells,purify the expressed product and determine its biological activity.Methods Ag85a-ESAT6 fusion gene fragment was inserted into vector pET-28a(+),and the constructed recombinant plasmid pET-28a-Ag85a-ESAT6 was transformed to E.coli BL21(DE3)for expression under induction of IPTG.The denaturalized expressed product was purified by Ni-agarose gel chromatography and renaturalized,then used for in vitro stimulation of spleno-lymphocytes of mice immunized with five kinds of recombinant vaccinia viruses expressing Ag85a and ESAT6 of M.tuberculosis.The proliferation of splenic lymphocytes was determined by MTT method.Results Both restriction analysis and sequencing proved that recombinant plasmid pET-28a-Ag85a-ESAT6 was constructed correctly.The expressed recombinant fusion protein,with a relative molecular mass of about 41 000,mainly existed in a form of inclusion body and contained 55.65% of total somatic protein.After purification and renaturation,the protein reached a purity of more than 95% and an expression level of about 1.2 g/L fermentation liquid,and showed good reactogenicity.As compared with those immunized with empty vaccinia virus or physiological saline,the proliferation activities of spleno-lymphocytes of mice immunized with the five kinds of recombinant vaccinia viruses increased significantly after co-culture with Ag85a-ESAT6 fusion protein(P < 0.01).Conclusion Ag85a-ESAT6 fusion protein was successfully expressed by using prokaryotic system and showed biological activity after purification and renaturation.

    2010 11 v.23 [Abstract][OnlineView][Download 317K]

  • Polymorphism of ptxS1 and prn Genes of Bordetella pertussis in China

    ZHANG Liu△,XU Ying-hua,ZHAO Jian-hong,XU Yun-qiang,ZHANG Shu-min(△National Institute for the Control of Pharmaceutical and Biological Products,Beijing 100050,China)

    Objective To analyze the polymorphism of pertussis toxin(PT)S1 subunit(ptxS1)and pertactin(prn)genes of Bordetella pertussis in China in the past 50 years.Methods A total of 85 B.pertussis strains were collected,from which ptxS1 and prn genes were amplified by PCR and sequenced,and the sequencing results were compared.Results The B.pertussis of four ptxS1 genotypes and six prn genotypes were found.Since 1960s,non-vaccine-derived ptxS1A strains have gradually substituted to the vaccine-derived ones.The strains containing novel prn2 and prn3 genotypes appeared after 2000.Phylogenetic tree showed that both the homologies of nucleotide and amino acid sequences of strains of ptxS1 and prn genotypes were more than 97%.Conclusion Antigen drift was observed in the ptxS1 and prn genes of clinical isolates of B.pertussis in China during the past 50 years.It laid a foundation of strengthening the epidemiological monitoring of pertussis in China and developing novel acellular pertussis vaccine.

    2010 11 v.23 [Abstract][OnlineView][Download 491K]

  • Screening of High Immunogenic Membrane Proteins from Brucella melitensis by Immunoproteomics

    YANG Yan-ling△,FU Xiao-xia,SHENG Xue-ling,WANG Xing-long(△Institute of Military Veterinary Medicine,Academy of Military Medical Sciences,Changchun 130062,China)

    Objective To develop a immunoproteomical method for screening of candidate antigen for immunization from membrane protein of Brucella melitensis.Methods The membrane protein of Brucella melitensis 16M was extracted,then separated by two-dimensional electrophoresis(2-DE)and subjected to Western blot with human,bovine and sheep sera infected with brucella.The screened immunodominant protein dots were identified by LC-MS/MS and analyzed for bioinformatics.Results A total of 56 immunodominant protein dots representing 35 open reading frames(ORFs)were screened,of which 12 were co-recognized by bovine and sheep sera.Bioinformatics retrieval showed that the proteins were mainly associated with the energy metabolism,protein and amino acid syntheses,fatty acid metabolism as well as saccharide and coenzyme syntheses of brucella.Conclusion High immunogenic membrane protein was successfully screened from Brucella melitensis by immunoproteomics,which provided a large quantity of candidate antigens for preparation of brucella subunit vaccine.

    2010 11 v.23 [Abstract][OnlineView][Download 419K]

  • Effect of shRNA Lentiviral Vector Targeting Human Growth Arrest and DNA Damage 45α Gene on Disfunction of Human Umbilical Vein Endothelial Cells Due to Hypoxia Stress

    LUO Xin,LIU Dan-dan,QI Hong-bo,YAO Zhen-wei,CHEN Guo-qing(Department of Obstetrics and Gynecology,First Affiliated Hospital,Chongqing Medical University,Chongqing 400016,China)

    Objective To study the silencing effect of shRNA lentiviral vactor,targeting human growth arrest and DNA damage 45α(Gadd45α)gene and expressing green fluorescent protein(GPF),on the up-regulation of Gadd45α expression in human umbilical vein endothelial cells(HUVECs)due to hypoxia stress,and preliminarily investigate the role of Gadd45α in disfunction of HUVECs due to hypoxia stress.Methods HUVECs were infected with the lentiviral vector after package,and the MOI and time for infection were optimized.The HUVECs were determined for expressions of Gadd45α mRNA and protein by real-time PCR and Western blot 72 h after infection,while for apoptosis rate by flow cytometry,for migration rate by Transwell test,and for secretion levels of sFlt-1 and sEng by ELISA.Results The final titer of lentiviral vector after package was 1 × 108 TU/ml,and the optimal MOI and time for infection were 20 and 72 h respectively.The infection efficacy of HUVECs was about 80%.The silencing efficacy of Gadd45α gene with Gadd45α shRNA lentiviral vector reached 80%,while the lentiviral vector as negative control showed no inhibitory effect on Gadd45α expression.Hypoxia stress caused the up-regulation of Gadd45α expression in HUVECs,however,the shRNA lentiviral vector targeting Gadd45α gene inhibited the apoptosis of HUVECs due to hypoxia stress,decreased the releases of sFlt-1 and sEng,and enhanced the migration in vitro of HUVECs(P < 0.05)vs the HUVECs cultured under hypoxia stress and untreated with the shRNA lentiviral vector targeting Gadd45α gene.The expression level of Gadd45α protein was positively related to the secretion levels of sFlt-1 and sEng(r1 = 0.89,r2 = 0.77,both P < 0.05).Conclusion Silencing Gadd45α gene showed protective effect on the biological function of HUVECs under hypoxia stress,and Gadd45α might be a key upstream activation site involved in regulating the releases of sFlt-1 and sEng in preeclampsia.

    2010 11 v.23 [Abstract][OnlineView][Download 454K]

  • Antibody Blockade of Surface Adhesion Molecules DNAM-1 and LFA-1 Inhibits Killing Activity of Natural Killer Cells to Tumor Cells

    SHANG Ying-hui△,ZHANG Li,LAO Feng-xue(△Beijing University of Traditional Chinese Medicine,Beijing 100029,China)

    Objective To investigate the effect of antibody blockage of surface adhesion molecules DNAM-1 and LFA-1 on the killing activity to tumor cells of natural killer(NK)cells amplified in vitro.Methods PBMCs-derived NK cells were activated in vitro and determined for the expression levels of surface adhesion molecules DNAM-1 and LFA-1,of which the contact to target cells were blocked by Trans-well test,and the effect of direct cell-cell contact on killing activity of NK cells was investigated.The role of relevant adhesion molecules in killing of tumor cells by NK cells was evaluated by NK cell cytotoxicity test in which the McAbs against DNAM-1 and LFA-1 were introduced to block the corresponding signal pathway.Results DNAM-1 and LFA-1 were highly expressed in the NK cells activated in vitro.The blockage of contact to target cells remarkably decreased,and the blockage of signal pathway of DNAM-1 or LFA-1 significantly decreased,while the combined blockage of signal pathways of DNAM-1 and LFA-1 further decreased the killing activity of NK cells to tumor cells.Conclusion Direct cell-cell contact is an important mechanism of killing activity of NK cells to tumor cells.The signal pathway mediated by surface adhesion molecules DNAM-1 and LFA-1 plays an important role in maintaining the killing activity of NK cells to tumor cells.

    2010 11 v.23 [Abstract][OnlineView][Download 338K]

  • Negative Regulatory Effect of Pleiotrophin on Expression of Schalfen3 and Schlafen4 Genes

    ZHOU Qiao-dan△,MI Can,HU Ying-chun,LI Gang,WU De-chang,HUO Yan-ying(△Department of Pathology,College of Basic Medical Sciences,Chongqing Medical University,Chongqing 400016,China)

    Objective To investigate the negative regulatory effect of pleiotrophin(PTN) on the expressionof Schlafen3(Slfn3) and Schlafen4(Slfn4)genes.Methods The expressions of Slfn3/Slfn4 genes in control cells(Pten-/-mouse embryo fibroblasts expressing Ptn gene)and Ptn-/-cells(Pten-/-mouse embryo fibroblasts with Ptn gene knocked down)were determined by RT-PCR and Northern blot.The relationship between the Ptn in human malignant glioma U87MG cells as well as human breast cancer MDA-MB231 cells and expression of Slfn3/Slfn4 genes were analyzed.The expression of Slfn3/Slfn4 genes in the Ptn-/-cells after 50 ng/ml of PTN was added into the medium and in the control cells cultured in the medium for Ptn-/-cells were determined.Results Slfn3/Slfn4 genes were highly expressed in Ptn-/-cells.In U87MG and MDA-MB-231 cells,Ptn was negatively related to the expression of Slfn3/Slfn4 genes.However,after PTN was added into the medium of Ptn-/-cells,the expression of Slfn3/Slfn4 genes was inhibited.Ptn-/-cell medium induced the expression of Slfn3/Slfn4 genes.Conclusion PTN showed negative regulatory effect on the expression of Slfn3/Slfn4 genes.

    2010 11 v.23 [Abstract][OnlineView][Download 287K]

  • Expression,Purification and Amplification Performance of Tgo DNA Polymerase

    SHENG Yan-min△,YU Wei-lai,WU Yang-yu,ZHAO Hua(△Department of Biology,Changchun Normal University,Changchun 130032,China)

    Objective To express and purify Tgo DNA polymerase and study its amplificatioin performance.Methods The gene encoding Tgo DNA polymerase was amplified from Thermococcus gorgonarius by PCR and cloned into vector pET101 containing His-Tag target sequence.The constructed recombinant plasmid pET101-Tgo was transformed to E.coli BL21 Star(DE3)for expression under induction of IPTG.The expressed protein was purified,analyzed by SDS-PAGE,and determined for enzyme activity using standard pfu as control.Tgo and pfu were amplified by PCR using plasmid pUC19 as template,and their proliferation efficacies were compared.The fidelity of amplification of Tgo was verified by modified blue-white selection.Results The Tgo DNA polymerase gene at a length of about 2 300 bp was amplified by PCR,of which the amino acid sequence deduced based on the nucleotide sequence was consistent with that reported in GenBank.The expressed protein was in a soluble form with a relative molecular mass of about 89 000,of which the expression level was 350 μg/g E.coli,and the enzyme activity after storage in 50% glycerin was about 5 U/μl.Both the yield and fidelity of amplification of Tgo were equivalent to those of pfu.Conclusions Tgo DNA polymerase was successfully expressed and purified,of which the amplification performance was equivalent to that of pfu.

    2010 11 v.23 [Abstract][OnlineView][Download 686K]

  • High Expression of Alkaline Lipase from Penicillium cyclopium in Pichia pastoris

    TAN Zhong-biao△,WU Min-chen,ZHANG Hui-min,LI Jian-fang(△School of Biotechnology,Jiangnan University,Wuxi 214122,Jiangsu Province,China)

    Objective To highly express the alkaline lipase(LipⅠ)from Penicillium cyclopium in Pichia pastoris.Methods The cDNA fragment of lipⅠgene was amplified from Penicillium cyclopium PG37 by RT-PCR and cloned into expression vector pPIC9K.The constructed recombinant plasmid pPIC9K-lip Ⅰ was transformed to Pichia pastoris GS115.High copy recombinant GS115/lipⅠwas screened for expression under induction of methanol,based on which the condition for expression was preliminarily optimized.Results After GS115/lipⅠ was cultured in the BMMY medium,at an original pH of 6.0,containing 1.0% methanol at 30℃ for 96 h,the LipⅠactivity in fermentation supernatant was 10.5 U/ml.However,after culture in the BMMY medium containing 1.0% methanol at 24℃ for 120 h,at an original pH of 9.0,the LipⅠactivity reached 407 U/ml.Conclusion The LipⅠ from Penicillium cyclopium was highly expressed in Pichia pastoris.

    2010 11 v.23 [Abstract][OnlineView][Download 351K]

  • Effect of Neuropilin1 Gene Specific RNA Interfering Plasmid on Proliferation and Apoptosis of Gastric Carcinoma Cells

    LIAN Hai-feng△,WU Xiao-ling(△Department of Gastrointestinalogy,The Second Affiliated Hospital of Chongqing Medical University,Chongqing 400010,China)

    Objective To investigate the effect of neuropilin1(NRP1)gene specific RNA interfering plasmid on the proliferation and apoptosis of gastric carcinoma cells.Methods NRP1 gene specific shRNA expression plasmid was constructed and transfected to human gastric carcinoma SGC7901 cells in mediation of liposome.The expression level of NRP1 in the cells was determined by Western blot,and the proliferation level and apoptosis rate of the cells by flow cytometry and Annexin-V-FITC/PI method.Results Both restriction analysis and sequencing proved that plasmid shRNA-NRP1 was constructed correctly.The expression of NRP1 in the SGC7901 cells 48 h after transfection was inhibited effectively.The percentage of cells at G0/G1 phase increased significantly,while those at S phase decreased significantly,and the apoptosis rate of cells increased.Conclusion Plasmid shRNA-NRP1 inhibited the expression of NRP1 in SGC7901 cells effectively and arrested the cell cycle at G0/G1 phase,thereby decreased the proliferation level and induced the apoptosis of the cells.

    2010 11 v.23 [Abstract][OnlineView][Download 317K]

  • Prokaryotic Expression of Recombinant Iron Siderophore Receptor IroN and Immune Protective Effect of Expressed Product against Extraintestinal Pathogenic E.coli Infection in Mice

    TONG Chun-yu△,YU Yong-zhong,CAO Hong-wei,ZHNAG Xu,CHEN Long-xin(△College of Life Sciences,Heilongjiang Bayi Agriculture University,Daqing 163319,Heilongjiang Province,China)

    Objective To express recombinant iron siderophore receptor IroN in prokaryotic cells and investigate its protective effect against extraintestinal pathogenic Escherichia coli(ExPEC)infection in mice.Methods IroN gene was amplified from the genomic DNA of E.coli J96 and cloned into prokaryotic expression vector pET-32a(+).The constructed recombinant plasmid pET-32a-IroN was transformed to E.coli BL21(DE3)for expression under induction of IPTG.The expressed recombinant protein was purified and used for immunization of BALB/c mice by subcutaneous route.The antibody levels in sera and urine of immunized mice were determined by ELISA.The immunized mice were challenged with E.coli J96 strain by intraperitoneal and intraurethral routes respectively,based on which the protective rate was calculated,and the bacteria were counted.Results The recombinant IroN protein,at an expression level of 32%,reached a purity of more than 85% after purification and showed good reactogenicity.Subcutaneous immunization with the recombinant IroN protein induced high titer specific IgG in sera of mice,while no specific IgA was detected in urine.The protective rate of recombinant IroN protein against intraperitoneal challenge with ExPEC was 81.8%(P < 0.05).After intraurethral challenge with ExPEC,the number of bacteria isolated from the kidneys of mice immunized with recombinant IroN protein decreased by more than 100 folds(P < 0.05),while those in bladder and urine showed no significant difference(P > 0.05)as compared with those in control groups(immunized with PBS).Conclusions IroN protein was expressed in prokaryotic cells and purified,which showed significantly protective effect against intraperitoneal and renal infection with ExPEC,but showed no protection against bladder infection.

    2010 11 v.23 [Abstract][OnlineView][Download 325K]

  • Immune Effects in Different Varieties of Horses

    LIU Gen-shun△,YAO Xiao-dong,WANG Deng-hai,WANG Hai-yan(△Jiangxi Institute of Biological Products,Ji,an 343100,Jiangxi Province,China)

    Objective To analyze the immune effects in different varieties of horses.Methods Horses were grouped according to the species and the regions from which they were purchased,and immunized with the same tetanus toxoid by the same route,and the antitoxin titers,immunization success rates,the numbers of courses when immune tolerance appeared,discard rates and mean life span for immunization of various groups were compared.Results Immune effect showed significant difference in various groups and in the horses with different physical qualities and health statuses.Conclusion The production of high titer immune serum needs the high-quality horses which may be obtained by breeding.

    2010 11 v.23 [Abstract][OnlineView][Download 126K]

  • Comparison of Protein Components of Excretory-Secretory Antigens of Adult Worm and Muscle Larvae of Trichinella spiralis

    MA Ming-wang△,ZHANG Zhi-lan,SHENG Li-jie,DONG Ming-zhi,(△Department of Biochemistry,Fenyang College,Shanxi Medical University,Fenyang 032200,Shanxi Province,China)

    Objective To compare the protein components of excretory-secretory(ES)antigens of adult worm and muscle larvae of Trichinella spiralis,analyze their reactogenicities and lay a foundation of separation and screening of Tirchinella spiralis antigen component with high immunogenicity and reactogenicity.Methods The adult worms and muscle larvae of Tirchinella spiralis were collected,with which ES antigens were prepared then analyzed for protein components by SDS-PAGE and for reactogenicities by Western blot.Results Seventeen protein bands with relative molecular masses of 120 000 ～ 14 000,including six main protein bands with relative molecular masses of 120 000,64 000,43 000,40 000,35 000 and 33 000 respectively were observed on the SDS-PAGE profile of adult worm of Trichinella spiralis.However,twenty protein bands with relative molecular masses of 112 000 ～ 10 000,including eleven main bands with relative molecular masses of 112 000,66 000,56 000,55 000,53 000,49 000,45 000,43 000,25 000,21 000 and 10 000 respectively were observed on the SDS-PAGE profile of muscle larvae.The ES antigen of adult worm showed seven reaction bands on Western blot profile,with relative molecular masses of 43 000,40 000,35 000,27 000,19 000,18 000 and 14 000 respectively,of which the ones with relative molecular masses of 43 000,40 000,27 000 and 18 000 were stained obviously.However,the ES antigen of muscle larvae showed fourteen bands on Western blot profile,with relative molecular masses of 74 000 ～ 12 000,of which the ones with relative molecular masses of 53 000,49 000,45 000,43 000,35 000,27 000,18 000 and 12 000 were stained obviously.Conclusion Both the protein components of adult worm and muscle larvae of Trichinella spiralis were complicated,including common and different components.ES antigen showed high reactogencity and might be used as an important candidate antigen for study on trichinelliasis.

    2010 11 v.23 [Abstract][OnlineView][Download 174K]

  • Cloning and Sequencing of Lactobacillus plantrum Histidine Decarboxylase Gene

    YU Chang-qing△,WANG Chang-yuan,MAN Yong-gang,WANG Ying(△College of Food Science,Heilongjiang Bayi Agricultural University,Daqing 163319,Heilongjiang Province,China)

    Objective To clone Lactobacillus plantrum histidine decarboxylase(HDC)gene and analyze its sequence.Methods Primers were designed according to the lactic acid bacteria HDC gene sequence reported in GenBank,with which the DNA of Uvs44 genome of L.plantrum was extracted,then amplified by PCR and cloned,and analyzed for the homology of nucleotide sequence to that of other lactic acid bacteria.Results The homologies of nucleotide sequence of the cloned HDC gene,at a length of about 900 bp,were 99.9% and 89.1% to those of Lactobacillus sakei and Lactobacillus buchneri respectively.The high variable region of the cloned gene was located at sites 32 ～ 489,in which T and C were often substituted.Conclusion It provided a theoretical basis for the detection of histramine-producing lactic acid bacteria and laid a foundation of construction of HDC gene-deleted recombinant bacterial strain.

    2010 11 v.23 [Abstract][OnlineView][Download 406K]

  • Effect of Expression of RNA Interference Gene BC047440 on Proliferation Ability of HCT-8 Cells

    TANG Liang△,ZHAO Quan,GONG Peng-tao,LI Jian-hua,YANG Ju,ZHANG Xi-chen(△College of Animal Science and Technology,Jilin Agricultural University,Changchun 130118,China)

    Objective To investigate the effect of colon cancer-related RNA interference gene BC047440 on the proliferation ability of colon cancer cells.Methods The shRNA interference plasmids pGPU6/GFP/Neo-BC047440-331(a1),pGPU6/GFP/Neo-BC047440-451(a2),pGPU6/GFP/Neo-BC047440-615(a3)and pGPU6/GFP/Neo-BC047440-756(a4)were transfected to colon cancer HCT-8 cells in mediation of liposome,then observed for transfection efficacy by fluorescent microscopy,determined for the expression level of BC047440 mRNA by fluorescent quantitative PCR,and analyzed for cell proliferation activity by MTT method.Results The transfection efficacy of HCT-8 cells was 53%.The expression levels of BC047440 mRNA in the HCT-8 cells transfected with RNA interference plasmids a1,a2,a3 and a4 decreased by 36.1%,47.0%,53.8% and 63.3% respectively,indicating the highest interference efficacy of a4 in the four plasmids.The proliferation ability of HCT-8 cells also decreased significantly.Conclusion The RNA interference plasmid carrying BC047440 gene inhibited the BC047440 mRNA expression as well as proliferation of HCT-8 cells,which might be used as a novel gene inhibiting the growth of colon cancer cells.

    2010 11 v.23 [Abstract][OnlineView][Download 357K]

  • Construction and Expression of Eukaryotic Expression Vector for Interferon α2b Gene

    CHEN Fen-ze△,WANG Yan,ZHU Yi-song,GAO Ren-jun(△Shenzhen Polytechnic Colleges,Shenzhen 518055,Guangdong Province,China)

    Objective To construct a eukaryotic expression vector for interferon α2b(IFNα2b)gene and express in CHOdhfr-cells.Methods IFNα2b gene fragment was amplified from recombinant E.coli DH5α-pbv220-IFNα2b and cloned into plasmid psv-dhfr.The constructed recombinant plasmid psv2-dhfr-IFNα2b was transfected to CHO-dhfr cells,and the monoclonal cell strains growing stably were obtained by MTX pressure screening.The antiviral activity of IFNα2b in transfected cells was determined by Wish cytopathic inhibition test.The genomic DNA of transfected cells was extracted and identified by PCR.Results Both PCR and restriction analysis proved that recombinant plasmid psv2-dhfr-IFNα2b was constructed correctly.The IFNα2b expressed in transfected cells showed high antiviral activity.IFNα2b gene band was amplified using the genomic DNA of transfected cells as a template.Conclusions A eukaryotic expression vector for IFNα2b gene was successfully constructed,and the IFNα2b with biological activity was expressed in CHO-dhfr-cells,which laid a foundation of further eukaryotic expression of IFNα2b.

    2010 11 v.23 [Abstract][OnlineView][Download 411K]

  • Construction and Application of Recombinant Plasmid with Tetanus Toxin Gene

    JIANG Chang-li△,TAN De-yong,TENG Yi,MA Jie,QU Liang,CHENG Han-song,WANG Hui-xuan(△Department of Clinical Laboratory,Kunming General Hospital of Chengdu Military Area Command of Chinese PLA,Kunming 650032,China)

    Objective To construct and apply a recombinant plasmid containing specific tetanus toxin(TT)gene fragment.Methods Three specific TT gene fragments were amplified by PCR using the genomic DNA of inactivated Clostridium tetani strain as a template and linked to vector pMD19-T Simple.Analog tetanus dangerous goods were identified by PCR using the constructed recombinant plasmid pMD19-TTx as a positive reference.Results Both PCR and restriction analysis proved that recombinant plasmid pMD19-TTx was constructed correctly.Using the recombinant plasmid as a positive reference,analog tetanus dangerous goods were detected rapidly.Conclusion Recombinant plasmid pMD19-TTx was successfully constructed,which provided a positive reference for detection of Clostridium tetani.

    2010 11 v.23 [Abstract][OnlineView][Download 196K]

  • Relationship of Adiponectin in Plasma to Brain Natriuretic Peptide and Left Ventricular Mass Index of Patients with Chronic Heart Failure

    LIU Hong-yan△,ZHANG Jin-guo,TAN Hong-yong(△Shandong University,Jinan 250100,China)

    Objective To investigate the relationship of adiponectin(APN)in plasma to brain natriuretic peptide(BNP)and left ventricular mass index(LVMI)of patients with chronic heart failure(CHF),and evaluate the value of APN level in predicting the process and prognosis of CHF.Methods Ninety male patients with CHF and 90 healthy men were determined for APN and BNP levels in plasma by ELISA,for IVST,PWT,LVIDd,LVEDV and LVESV by echocardiography,based on which LVMI was calculated.The APN,BNP and LVMI were compared between the patients with CHF and control,the patients with CHF due to various causes,and the APN and BNP levels between patients with various degrees of cardiac functions,based on which the relationship of APN to BNP and LVMI was analyzed.Results The APN and BNP levels in plasma as well as LVMI of patients with CHF were significantly higher than those of healthy men(P < 0.05).The APN levels of patients with CHF due to various causes showed no significant difference(P > 0.05).Both the APN and BNP levels of patients with NYHA Ⅳ were significantly higher than those with NYHA Ⅲ and with NYHA Ⅱ(P < 0.05).APN was positively related to both BNP and LVMI(r = 0.528,P < 0.001;r = 0.269,P < 0.05).Conclusion The APN level in plasma of patients with CHF increased with the deterioration of heart function,and might participate in the pathophysiological process of myocardial remodeling,which might be used as an important indicator for predicting the process and evaluating the prognosis of CHF.

    2010 11 v.23 [Abstract][OnlineView][Download 241K]

  • Expression of Multi-epitope Subunit Vaccine of Foot-and-mouth Disease Virus Type Asia 1 in Pichia pastoris and Immunogenicity of Expressed Product

    HU Bo△,LU Hui-jun,LI Xiao,YE Ming,ZHU Zhan-bo,DU Shou-wen,XI Ying,WANG Jing,HAI Jia-li,LI Yang,JIN Kuo-shi,TIAN Ming-yao,LI Chang,JIN Ning-yi(△College of Animal Science and Veterinary Medicine,Jilin University,Changchun 130062,China)

    Objective To construct a multi-epitope subunit vaccine of foot-and-mouthdisease virus(FMDV)type Asia 1,using cholera toxin B subunit(CTB)as a molecular adjuvant,express in Pichia pastoris and evaluate its immunogenicity.Methods Recombinant plasmid pPIC9K-P1/2A-CTB-TEpi was constructed and transformed to P.pastoris GS115 for expression under induction of methanol.The expressed product was purified and analyzed by SDS-PAGE and Western blot.BALB/c mice were immunized with the purified P1/2A-CTB-TEpi protein and inactivated FMDV vaccine respectively,twice on days 0 and 21,using PBS as control.The venous blood of immunized mice was collected every week,from which sera were separated and determined for antibody level by ELISA.The mice were killed 10 d after the 2nd immunization,of which splenic lymphocytes were isolated for proliferation test and determined for IFNγ by ELISPOT.Results The target protein was expressed in P.pastoris and secreted into the culture supernatant,which showed two bands with relative molecular masses of about 82 600(P1/2A)and 40 500(CTB-TEpi)respectively on SDS-PAGE profile and contained 21% of soluble protein in supernatant.The ratio of P1/2A to CTB-TEpi was about 1:2 in the purified target protein which showed high reactogenicity.P1/2A-CTB-TEpi protein induced high humoral and cellular immune responses in mice.The serum antibody level of mice immunized with the protein showed no significant difference with(P > 0.05),while the lymphocyte proliferation and IFNγ secretion levels were significantly higher than those with inactivated FMDV vaccine(P < 0.01).Conclusion The multi-epitope subunit vaccine of FMDV type Asia 1,using cholera toxin B subunit(CTB)as a molecular adjuvant,was constructed and showed high immunogenicity,which laid a foundation of study on multi-epitope and subunit vaccines of FMDV.

    2010 11 v.23 [Abstract][OnlineView][Download 447K]

  • Development of A Production Procedure for Bulk of Inactivated Mandarinfish Pathogenic Aeromonas hydrophila Vaccine

    TAO Jia-fa,LAI Ying-tiao,REN Yan,GONG Hua,KANG Guang-hui,ZHANG You,SHI Cun-bin,WU Shuqin(Pearl River Fisheries Research Institute,Chinese Academy of Fisheries Sciences,Guangdong Key Laboratory of Aquatic Animal Immune Techniques,Guangzhou 510380,China)

    Objective To develop a production procedure for the bulk of inactivated mandarinfish pathogenic Aeromonas hydrophila vaccine.Methods Aeromonas hydrophila GYK1 strain was cultured in shake-flask and 5 L fermenter for various hours and determined for A650.The bacterial culture was inactivated with formaldehyde solution at various concentrations for various hours,and the inactivation effect was evaluated.The strain was cultured in shake-flask,seed tank and fermenter in turn,then inactivated with formaldehyde to prepare the bulk of inactivated vaccine which was tested for safety and potency.Results The growth of strain reached a stable stage 18 h and a peak value 24 h after culture in shake-flask.However,in 5 L fermenter,the growth of strain reached a stable stage 8 h and a peak value about 10 h after culture.The bacterial liquid was completely inactivated with 0.30% formaldehyde at 37℃ for 24 h,and the residual formaldehyde was low.The prepared bulk of inactivated vaccine showed high safety and was qualified in potency test.Conclusion A production procedure for the bulk of inactivated mandarinfish pathogenic Aeromonas hydrophila vaccine was preliminarily developed.

    2010 11 v.23 [Abstract][OnlineView][Download 208K]

  • Effect of Berberine on Lipid Metabolism and Expressions of Low Density Lipoprotein Receptor and Scavenger Receptor Class-B Type 1 Genes in Livers of Hyperlipidemic Rabbits

    JIN Lei,HE Qi,ZHOU Qi-xin,YANG Jun-xia(Department of Pharmacology,Chongqing Key Laboratory of Biochemistry and Molecular Pharmacology,Chongqing Medical University,Chongqing 400016,China)

    Objective To investigate the effect of berberine(Ber)on lipid metabolism and expressions of low density lipoprotein receptor(LDLR)and scavenger receptor class-B type 1(SR-B1)genes in livers of hyperlipidemic rabbits.Methods Forty New Zealand rabbits were fed with basal forage for 1 week,of which 8 were selected as normal diet(ND)group,and the other 32 were fed with high-fat diet(HFD)to copy hyperlipidemic rabbit model.Eight weeks later,the model rabbits were randomly divided into HFD,high-fat diet + fenofibrate(FD),high-fat diet + Ber at low dosage(BLD)and high-fat diet + Ber at high dosage(BHD)groups and further fed for 8 weeks.The total TC,TG,LDL-C and HDL-C contents in sera of rabbits were determined by full automatic biochemical analyzer,and the histopathological changes of livers were observed.The expression levels of LDLR and SR-B1 genes in livers were determined by real-time fluorescent quantitative PCR.Results Compared with those in ND group,the TC,TG and LDL-C levels in sera of rabbits in HFD group increased significantly(P < 0.01),while moderate fatty and watery degenerations were observed in liver tissue.However,the serum TC,TG and LDL-C levels in BLD group decreased significantly(P < 0.01),while the moderate fatty and watery degenerations were relieved as compared with those in HFD group.The expression levels of LDLR and SR-B1 mRNAs in HFD group were significantly lower than those in ND group(P < 0.01).However,both the expressions of LDLR and SR-B1 mRNAs in BLD and BHD groups were up-regulated significantly as compared with those in HFD group(P < 0.01).Conclusion Ber showed significantly regulatory effect on blood fat by a potential mechanism of up-regulating the expressions of LDLR and SR-B1 genes therefore inhibiting the cholesterol synthesis in liver.

    2010 11 v.23 [Abstract][OnlineView][Download 371K]

  • Inhibitory Effect of Endostar on Growth of Rhadomyosarcoma PLA-802 cells and The Relevant Mechanism

    YANG Qing-ping△,Zhou Zhi,YIN Qing-hua,LUO Xiao-hong,LI Sheng-nan,XIANG Mei-huan,ZHANG Xiong(△Department of Pathology,People's Hospital of Changshou District,Chongqing City,Chongqing 401220,China)

    Objective To investigate the inhibitory effect of Endostar,an inhibitor of human vascular endothelium,on the growth of rhadomyosarcoma(RMS) PLA-802 cells and the relevant mechanisms.Methods PLA-802 cells cultured by routine method were divided into control and test groups.The cells in control group was untreated,while those in test groups were treated with Endostar at various groups respectively.The effect of Endostar on the growth of PLA-802 cells was determined by MTT method,the change of cell cycle by flow cytometry,the effect of Endostar on expressions of vascular endothelial growth factor(VEGF)mRNA and protein by RT-PCR and Western blot respectively,and the VEGF level in cell culture supernatant by ELISA.Results Endostar inhibited the proliferation of PLA-802 cells as well as expression and production of VEGF,each in a concentration-and time-dependent pattern.Conclusion Endostar can inhibit the growth of PLA-802 cells by a potential mechanism which might be associated with blocking the expression of VEGF.

    2010 11 v.23 [Abstract][OnlineView][Download 342K]

  • Effect of Amlodipine on Cell Cycle of Murine Hepatocarcinoma H22 Cells and Expression of Relevant Protein

    LI Wei-ping,HUANG Wen-jing,SUN Wen-juan(Key Laboratory of Biochemistory and Molecular Pharmacology of Chongqing,Department of Pharmacology,Pharmaceutical College,Chongqing Medical University,Chongqing 400016,China)

    Objective To investigate the effect of amlodipine on cell cycle of murine hepatocarcinoma H22 cells and the expression of relevant protein.Methods H22 cells were treated with amlodipine at various concentrations,then determined for proliferation level by MTT method,for cell cycle and apoptosis by flow cytometry,and the expressions of Cyclin B1 and tumor-inhibiting gene p53 at protein and gene levels by immunohistochemical assay and RT-PCR.Results The treatment with amlodipine at various concentrations(1.75 × 10-3,3.5 × 10-3,7 × 10-3,14 × 10-3 and 28 × 10-3 mg/ml)for 24,36 and 48 h inhibited the cell proliferation.The IC50 of amlodipine for treatment for 48 h was 13.4 μmol/L.Amlodipine arrested H22 cells at G2 phase and induced the cell apoptosis.The expression level of Cyclin B1 in the H22 cells after treatment with amlodipine decreased significantly,while that of p53 increased significantly,which showed significant difference with those of blank control(P < 0.01).Conclusions Amlodipine inhibited the proliferation of H22 cells significantly,and showed anti-tumor effect through down-regulating the expression of Cyclin B1 and up-regulating the expression of p53 gene.

    2010 11 v.23 [Abstract][OnlineView][Download 429K]

  • Application of Polymer Conjugate Using Polylysine as A Carrier in Detection of Hepatitis C Virus Core Antigen

    MAI Zhi-gang,LEI Ming-jun,YI Jun-bo,CHEN Shao-juan(Biotechnology Research Center,Shenzhen University,Shenzhen 518058,Guangdong Province,China)

    Objective To investigate the application of polymer conjugate using polylysine as a carrier in the detection of hepatitis C virus(HCV)core antigen.Methods The McAb against HCV core antigen was conjugated with HRP using polylysine as a carrier.Meanwhile,the conjugate with sodium periodate was prepared,and the relative contents and reactivities of antibodies in the two conjugates were compared.Both the conjugates were used for detection of HCV core antigen by ELISA and Western blot.The detection results by the two methods were compared,both of which were compared with those by commercial kit.Results Competitive inhibition and direct binding tests showed that,when the inhibition rate reached 50%,the free antibody concentrations in polymer and sodium periodate conjugates were 0.001 9 and 0.001 1 mg/ml respectively.When the A403 was 1.0,the dilution of polymer conjugate was about 1:200 000,while that of traditional conjugate was about 1:20 000.The minimum detection limits of HCV core antigen by ELISA with polymer and sodium periodate conjugates were 2.0 and 10 pg/ml respectively.Both the intra-and inter-coefficients of variation of detection results with the two conjugates were less than 10%.Both the conjugates showed high specificities.The coincidence rates of detection results of clinical serum samples by polymer and sodium periodate conjugates with that by commercial kit were 97.0% and 98.6% respectively.The sensitivity of Western blot using polymer conjugate increased by at least 10 folds as compared with that using sodium periodate conjugate.Conclusion The polymer conjugate using polylysine as a carrier may be used for the detection of HCV core antigen by ELISA and Western blot,and may increase the ratio of enzyme to antibody thus increase the sensitivity of detection and prevent miss detection.

    2010 11 v.23 [Abstract][OnlineView][Download 324K]

  • Optimization of Condition for Opsonophagocytic Assay for Evaluation of Potency of Pneumococcus Polysaccharide Conjugate Vaccine

    LIU Qian,CHEN Xiao-hang,REN Ke-ming,ZHANG Yi,WANG Xi,SHEN Rong(Lanzhou Institute of Biological Products,Lanzhou 730046,China)

    Objective To optimize the condition for opsonophagocytic assay(OPA)for evaluation of potency of pneumococcus polysaccharide conjugate vaccine.Methods The experimental condition for OPA was optimized according to the protocol for opsonophagocytic assay against streptococcus pneumoniae(version A.02,January 2007)published by WHO reference laboratory in US University of Alabama.The opsonophagocytic titers of drug-resistant and drug-sensitive pneumococcus strains against the sera of mice immunized with pneumococcus polysaccharide conjugates of the corresponding types were determined and compared with the corresponding ELISA titers,between which the relationship was analyzed.Results After HL-60 cells were differentiated for 4 d under induction of 0.8% DMF,both the expression of cell surface markers CD11b and CD35 were not less than 55%,and that of CD71 was not more than 15%,and the proportion of live cells was not less than 65%.The recovery rate of working seed lot of pneumococcus of various types were not more than 80%,while the non-specific killing rate of complement was 18%.All the serotypes showed high bactericidal activity,and similar correlation coefficients and slopes were observed between OPA and ELISA titers.Conclusions The optimized condition met the requirements for OPA and might be used for the evaluation of potency of pneumococcus polysaccharide conjugate vaccine.

    2010 11 v.23 [Abstract][OnlineView][Download 482K]

  • Development of Oligonucleotide Microarray Assay for Differentiation of Nsp2-deleted Mutant of Highly Pathogenic PRRSV and Classical American PRRSV Strain

    GUO Huan-cheng△,XI Jin,LI Jiang-nan,TU Chang-chun(△Institute of Veterinary Sciences,Academy of Military Medical Sciences,Changchun 130062,China)

    Objective To develop an oligonucleotide microarray assay for differentiation of Nsp2-deletedmutant of highly pathogenic PRRSV and classical American PRRSV strain.Methods Based on the nucleotide sequences of Nsp2-deleted mutant of highly pathogenic PRRSV and classical PRRSV strain,a set of duplex PCR primers were designed to amplify the conservative region of American PRRSV genome and partial Nsp2 gene sequence covering the two non-continued deletions.The 31～ 35 mer oligonuleotide probes were designed to bind the multiple target sites within the conservative region of American PRRSV genome and the sequence corresponding to the deletions of Nsp2 gene.On the basis of this,an oligonucleotide microarray assay was developed,verified for specificity and sensitivity and preliminarily applied.Results Classical and mutant PRRSV strains were differentiated obviously by hybridization patterns.The microarray probe showed no specific hybridization with classical swine fever virus(CSFV),porcine circovirus type 2(PCV2)or porcine pseudorabies virus(PRV).The sensitivity of the developed microarray method was similar to that of conventional PCR method.The detection results of 12 samples from suspected PRRS by microarray were consistent with those by conventional PCR.Conclusion The developed microarray method may be used for accurate differentiation of Nsp2-deleted mutant highly pathogenic PRRSV as well as clinical diagnosis and epidemiological investigation of highly pathogenic PRRSV infection.

    2010 11 v.23 [Abstract][OnlineView][Download 475K]

  • Detection of Rotavirus Serotype G4 by TaqMan Probe-based Real-time Quantitative RT-PCR

    CHEN Ji-jun△,HAN Yue-wu,HU Guang-hong,BAI Mu-qun,ZHOU Xu(△Department of Biochemistry and Molecular Biology,Lanzhou Medical College,Lanzhou University,Lanzhou 730000,China)

    Objective To detectthe VP7 gene of rotavirus serotype G4 by Taq Manprobe-basedreal-timequantitative RT-PCR.Methods Total RNAs were extracted from the rotavirus serotypes G1,G2,G3 and G4 cultured in Vero cells for test for the specificity of primers and probes by RT-PCR and real-time quantitative RT-PCR respectively.The plasmid carrying the VP7 gene of rotavirus serotype G4 was constructed,and RNA reference was prepared,based on which a standard curve of real-time quantitative RTPCR was plotted,and the sensitivity and precision of the method were verified.Five cultures of rotavirus serotype G4 and three cultures of each of serotypes G1,G2 and G3 were detected by TaqMan probe-based real-time quantitative RT-PCR to evaluate the practicality.Results Only from the total RNA of rotavirus serotype G4,the target gene or the fluorescent signal was amplified by using the primers and probes for VP7 gene of rotavirus serotype G4.No target gene or fluorescent signal was amplified from the total RNA of rotavirus serotype G1,G2 or G3.Within a RNA concentration range of 2.34 × 103 ～ 2.34 × 107 copies/μl,the amplification efficacy was more than 90%,and the R2 was more than 0.98.The RNA samples at a concentration level of 100 copies/μl was detected by the method,and the intra-and inter-coefficients of variations(CVs)were less than 2.5% and less than 4% respectively.By the developed method,only the detection results of five rotavirus serotype G4 samples were positive,while those of serotype G1,G2 and G3 were negative.Conclusions TaqMan probe-based real-time quantitative RT-PCR is a sensitive,specific and precise method for detection of rotavirus serotype G4.

    2010 11 v.23 [Abstract][OnlineView][Download 233K]

  • Development of A Method for Determination of Residual DNA in CHO Host Cells

    ZHU Rong,DU Hong-qiao,TAN Hong-xing,XU Ge-lin,YAN Jia-xin(Wuhan Institute of Biological Products,Wuhan 430060,China)

    Objective To develop a method and prepare an internal reference for determination of residual DNA in CHO host cells in biologics,and provide a basis for the establishment and revision of the relevant national standards.Methods DNAs were extracted from CHO cells with phenol:chloroform:isopentanol,DNA extraction kit and high salt method respectively,and the extraction effects were compared.The DNA probe extracted by the optimal method was labeled with digoxin,based on which the volumes for labeling were optimized,and the labeling efficacy was evaluated by direct determination.The internal references of DNA were divided into three groups and treated by various methods.The reference in group 1 was untreated,while that in group 2 was treated with bovine serum albumin and protease K.However,the reference in group 3 was treated by the method in group 2 and an additional procedure of DNA extraction.The sensitivities of above-mentioned samples were determined by dot blot with the labeled probe.Results The extraction effect of DNA from CHO cells by high salt method was satisfactory.The volumes for labeling was optimized as DNA 10 μg,Vial 4 16 μl and total volume 64 μl.The sensitivities of internal references in groups 1,2 and 3 were 1 pg,10 pg and 1 ng respectively.Conclusion The developed dot blot method with digoxin-labeled CHO cell DNA probe is stable and reliable,which may be used for determination of residual DNA content in CHO host cells in biologics.

    2010 11 v.23 [Abstract][OnlineView][Download 206K]

  • Influenza Vaccine and Guillan-Barré Syndrome

    LIU Dong-hui△,WU Xing,WANG Jun-zhi,WANG Xing-tai(△Bureau of Laboratory Science,Department of Public Health,Massachusett,USA)

    Guillan-Barre syndrome(GBS)is referred to as acute inflammatory demyelinating polyneuropathy,which is usually caused by microbial,bacteria,viruses or other factors.The adverse effects including GBS emerging from the wide inoculation of H1N1 influenza vaccines have been receiving wide concerns.This paper reviews the history,clinical features,research status of GBS and its relationship to influenza vaccine inoculation,as well as the significance and risk management of influenza vaccination.

    2010 11 v.23 [Abstract][OnlineView][Download 162K]

  • Progress in Research on Staphylococcus aureus Vaccine

    CHEN Yi-guo,YANG Da-qing,FU Ning(Department of Immunology,Southern Medical University,Guangzhou 510515,China)

    Staphylococcus aureus(SA)remain an important cause of infectious diseases in both the community and the clinic,and has developed resistance to multiple drugs since the wide application of antibiotics,bring a severe challenge for the clinics to combat againist the pathogen.Several approaches to prevent SA infection have undertaken since 1960s.Though there is no currently available approach in clinic,immunoprophylaxis is found to be of an excellent prospect.This paper reviews the progress in research of SA vaccine in recent years.

    2010 11 v.23 [Abstract][OnlineView][Download 220K]


@2012 Editorial Department of "Chinese Journal of Biologicals"