2011年06期目次

  • Expression of Structural Protein VP1 of Poliovirus Sabin2 Strain in Insect Baculovirus Expression System

    LIU Jin-hua,DONG Guan-mu,AN Qi,CAO Shou-chun,KONG Yan(National Institutes for Food and Drug Control,Beijing 100050,China)

    Objective To express the structural protein VP1 of poliovirus(PV) Sabin2 strain in insect baculovirus expression system.Methods VP1 gene was amplified by RT-PCR from the bulk of PV vaccine prepared with Sabin2 strain,and cloned into vector pFastBac1.The constructed recombinant plasmid pFast-VP1 was transformed to E.coli DH10Bac carrying baculovirus shuttle plasmid Bacmid,and the obtained recombinant plasmid Bacmid-VP1 was transfected to sf9 insect cells in mediation of liposome to prepare recombinant baculovirus rBac-VP1.The sf9 cells were infected with rBac-VP1 of passage 2 and determined for expression of VP1 by IFA,based on which the condition for infection was optimized.Results The target gene fragment at a length of 3 203 bp was amplified from the transformants of Bacmid-VP1.The rBac-VP1 of passage 2 reached a titer of 6 × 107 pfu/ml,with which the infected sf9 cells showed green fluorescence under fluorescent microscope.The expression levels of VP1 in sf9 cells infected with rBac-VP1 at various MOIs showed no significant difference,which increased gradually with the increasing time for infection within 96 h.Conclusion The structural protein VP1 of PV Sabin2 strain was successfully expressed in insect baculovirus expression system,which laid a foundation of development of poliovirus subunit vaccine.

    2011 06 v.24 [Abstract][OnlineView][Download 268K]

  • Expression of Viral Macrophage Inflammatory Protein-Ⅱin Pichia pastoris and Its Anti-HIV-1 Activity

    MO Xue-mei,SUN Han-xiao,JIA Zhong-wei,LI Xiu-ying,ZHANG Guang(Institute of Genome Medicine,Pharmacy College,Jinan University,Guangzhou 510632,China)

    Objective To express viral macrophage inflammatory protein-Ⅱ(vMIP-Ⅱ) in Pichia pastoris and determine its anti-HIV-1 activity.Methods The vMIP-Ⅱ gene was amplified from plasmid pQE-vMIP-Ⅱ by PCR and inserted into expression vector pPICZaA.The constructed recombinant plasmid pPICZaA-vMIP-Ⅱ was transformed to P.pastoris X33 for expression under induction of methanol.The expressed product was purified by nickel ion affinity chromatography and size-exclusion chromatography,identified by Western blot and determined for inhibitory effect on cell syncytium formation induced by HIV-1.Results Both restriction analysis and sequencing proved that recombinant plasmid pPICZaA-vMIP-Ⅱ was constructed correctly.The expressed recombinant vMIP-Ⅱ protein,with a relative molecular mass of about 8 500,existed in a secretory form in fermentation supernatant,reached a purity of 97.3% after purification and showed specific reaction with HRP-labeled polyclonal antibody against vMIP-Ⅱ.Western blot showed that recombinant vMIP-Ⅱ was expressed in secretory form in culture supernatant.The number of syncytia in MT4 cells treated with recombinant vMIP-Ⅱ was significantly smaller than that in positive control group(P < 0.05).The IC50 of recombinant vMIP-Ⅱ to syncytium formation was 1.35 ng/ml.Conclusion Recombinant vMIP-Ⅱ protein was successfully expressed in P.pastoris,which showed high activity in inhibiting HIV-1.

    2011 06 v.24 [Abstract][OnlineView][Download 409K]

  • Effect of JTV1 Gene Expression Inhibition on Emodin-induced Apoptosis of K562 cells and Relevant Mechanism

    WU Yan,LIU Bei-zhong,WANG Chong,ZHU Dan,WANG Chun-guang,JIN Dan-ting,GAO Yan-jun,LI Liang(Key Laboratory of Laboratory Medical Diagnostics of Ministry of Education,Faculty of Laboratory Medicine in Chongqing Medical University,Chongqing 400016,China)

    Objective To investigate the effect of inhibiting expression of tumor-suppressing gene JTV1 on the emodin-induced apoptosis of human chronic myeloid 1eukemia(CML) K562 cell line as well as the relevant mechanism.Methods K562 cells were transfected with recombinant plasmids pGeneSil-1-JTV1-1.1 siRNA,pGeneSil-1-N.1 as negative control and empty vector pGeneSil-1 respectively and determined for proliferation level by colony formation test.The transfected K562 cells in various groups were treated with 80 μmol/L emodin for 72 h,then analyzed for cell cycle and apoptosis rate by flow cytometry,for transcription levels of apoptosis-related Bax,Bcl-2 and C-myc genes by RT-PCR,and for translation levels of the genes by Western blot.Results After the expression of JTV1 gene was inhibited,the proliferation level of K562 cells increased significantly,the percentage of K562 cells treated with 80 μmol/L emodin at G1 phase decreased,while the emodin-induced apoptosis was relived.Meanwhile,both the mRNA transcription and protein expression levels of Bax gene decreased significantly,while those of Bcl-2 and C-myc genes increased significantly.Conclusion The inhibition of JTV1 gene expression might promoted the proliferation and arrest the apoptosis of K562 cells by up-regulating the expressions of Bcl-2 and C-myc genes and down-regulating the expression of Bax gene.

    2011 06 v.24 [Abstract][OnlineView][Download 548K]

  • Construction and Identification of Recombinant Saccharomyces cerevisiae Strain for Superoxide Dismutase Gene of Brucella melitensis

    WANG Jing-long,QU Hai-long,YANG Yan-ling,SUN Chun-hui,WANG Xiu-ran,LANG Xu-long,LI Xiao-yan,BU Zhao-yang,WANG Xing-long(College of Animal Science and Veterinary Medicine,Jilin University,Changchun 130062,China)

    Objective To construct and identify a recombinant Saccharomyces cerevisiae strain for superoxide dismutase(SOD) gene of Brucella melitensis.Methods The SOD gene of B.melitensis 16M strain was amplified by PCR and inserted into vector psos.The constructed recombinant plasmid was transformed to S.cerevisiae cdc25H,and the recombinants were identified by PCR,verified for phenotype,and tested for auto-activation and location.Results Both restriction analysis and sequencing proved that recombinant plasmid psos-SOD was constructed correctly.The SOD gene at a length of 525 bp was amplified by PCR from the recombinant S.cerevisiae which showed normal phenotype.The bait plasmid showed no auto-activation to host S.cerevisiae,and the expressed fusion protein was located correctly in the cytoplasma.Conclusion A recombinant S.cerevisiae strain for SOD gene of B.melitensis was successfully constructed,which laid a foundation of study on molecular mechanism of pathogenesis of brucella.

    2011 06 v.24 [Abstract][OnlineView][Download 236K]

  • Effect of Recombinant Adenovirus pAd-sTGF-β RⅡ on Ventricular Remodeling after Myocardial Infarction in Rats

    ZHNAG Ling-ling,LIU Zeng-zhang,XIAO Pei-lin,SU Li,YIN Yue-hui(Department of Cardiology,Second Affiliated Hospital,Chongqing Medical University,Chongqing 400010,China)

    Objective To investigate the effect of recombinant adenovirus carrying the extracellular domain gene of transforming growth factor-β typeⅡreceptor(pAd-sTGF-βRⅡ) on the ventricular remodeling after myocardial infarction(MI) in rats as well as the potential mechanism.Methods Rat model of MI was established by ligating the anterior descending of left coronary artery,then divided into MI,pAd-sTGF-β RⅡand empty vector groups and treated with physiological saline,pAd-sTGF-β RⅡand pAdTrack respectively,using those receiving sham operation and treated with physiological saline as control.Four weeks after treatment,the expression of sTGF-β RⅡwas observed under fluorescent microscope by frozen sections of myocardium tissue,structure of myocardium tissue by HE staining,expressions of typesⅠandⅢcollagens by Sirius red-saturated picric acid staining,expressions of α-smooth muscle actin(α-SMA) and matrix metalloproteinase-9(MMP-9) by immunohistochemical method,and expressions of bax and bcl-2 mRNAs by RT-PCR.Results Compared with those in MI group,the expression levels of total typesⅠand Ⅲcollagens,MMP-9 and bax mRNA in myocardium tissue of rats in pAd-sTGF-β RⅡgroup decreased significantly(P < 0.01),while the number of cells positive for α-SMA decreased significantly,and the expression level of bcl-2 mRNA increased(P < 0.01).However,compared with those in sham operation group,the expression levels of total typesⅠandⅢcollagens,MMP-9 and bax mRNA in myocardium tissue of rats in pAd-sTGF-β RⅡgroup increased significantly(P < 0.01),while that of bcl-2 mRNA showed no significant difference(P > 0.05).Conclusion The intervention with pAd-sTGF-β RⅡ may mitigate ventricular remodeling after MI by a potential mechanism of inhibiting myocardial fibrosis and cardiomyocyte apoptosis mediated by TGF-β.

    2011 06 v.24 [Abstract][OnlineView][Download 499K]

  • Construction of Eukaryotic Expression Vectors for Tissue Inhibitor of Metalloproteinase 1 Gene and Their Expressions in MCF-7 Cells

    LI Peng,MA Yan-jiao,ZHAO Na,YUAN Ting,GONG Peng-tao,LI Jian-hua,OUYANG Hong-sheng,ZHANG Xi-chen(Institute of Animal Science and Technology,Heilongjiang Bayi Agriculture University,Daqing 163319,Heilongjiang Province,China)

    Objective To construct three eukaryotic expression vectors for tissue inhibitor of metalloproteinase 1(TIMP1) and express in MCF-7 cells.Methods TIMP1 gene was inserted into plasmids pcDNA3.1(+),pVAXⅠand pEGFP-C1,and the constructed recombinant plasmids pcDNA3.1(+)-TIMP1,pVAXⅠ-TIMP1 and pEGFP-C1-TIMP1 were transfected to MCF-7 cells respectively.The expressed TIMP1 was identified by real-time PCR and Western blot.Results Restriction analysis and sequencing proved that the three recombinant plasmids were constructed correctly.Green fluorescence was observed in the MCF-7 cells 48 h after transfection with recombinant plasmid pEGFP-C1-TIMP1,while was not observed in those with pcDNA3.1(+)-TIMP1 or pVAXⅠ-TIMP1.In the turn of TIMP1 mRNA transcription and protein expression levels,the MCF-7 cells were those transfected with pcDNA3.1(+)-TIMP1,pVAXⅠ-TIMP1 and pEGFP-C1-TIMP1.Conclusion Three eukaryotic expression vectors for TIMP1 were successfully constructed,of which the expression levels in MCF-7 cells were different.

    2011 06 v.24 [Abstract][OnlineView][Download 242K]

  • Construction and Identification of Eukaryotic Expression Vector for GalR2 Gene

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

    Objective To construct a eukaryotic expression vector for GalR2 gene and express in HeLa cells.Methods Total RNA was extracted from the hippocampus of C57BL/6J mice,with which GalR2 gene was amplified by RT-PCR and cloned into pEGFP-C1.The constructed recombinant plasmid pEGFP-GalR2 was transfected to HeLa cells.The intracellular location of fusion protein was observed by fluorescent microscopy.The transcription and expression of GalR2 gene were determined by RT-PCR and Western blot.Results GalR2 gene at a length of 1 116 bp was amplified from the hippocampus of C57BL/6J mice.PCR,restriction analysis and sequencing proved that recombinant plasmid pEGFP-GalR2 was constructed correctly.The expressed fusion protein was located in nuclei of transfected cells.GalR2 gene was expressed at both mRNA and protein levels.Conclusion The eukaryotic expression vector pEGFP-GalR2 was successfully constructed,which laid a foundation of further study on biological activity of GalR2 and provided a novel route for development of drugs for depression.

    2011 06 v.24 [Abstract][OnlineView][Download 270K]

  • Inhibitory Effect of Spiegelmer NOX 1255 on Binding of Luteinizing Hormone-releasing Hormone(LHRH)-Pseudomonas aeruginosa Exotoxin 40 to LHRH Re-ceptor on HeLa Cells

    DENG Xin,Sven Klussmann,LI Gen-song,NIE Zhi-wei,SONG Yang,ZHANG Guo-li,ZHU Ping(The Experimental Center of Functional Subjects,China Medical University,Shenyang 110001,China)

    Objective To investigate the inhibitory effect of Spiegelmer NOX 1255 on the binding of luteinizing hormone-releasing hormone(LHRH)-Pseudomonas aeruginosa exotoxin(PE) 40 to LHRH receptor on HeLa cells.Methods HeLa cells were co-incubated with 25 μmol/L LHRH-PE40,PEA and PE40 for 24 h respectively and observed for morphological change.The inhibitory effect of Spiegelmer NOX 1255 on LHRH-PE40 was observed by optical microscopy,while that on binding of LHRH-PE40 to LHRH receptor by ELISA.Results LHRH-PE40 and PEA killed HeLa cells directly,while PE40 showed no toxic effect on HeLa cells.Spiegelmer NOX 1255 could bind to the LHRH in LHRH-PE40 and inhibit the binding of LHRH-PE40 to LHRH receptor as well as the toxic effect of PE40.Conclusion The toxic effect of LHRH-PE40 on HeLa cells was associated with LHRH receptor.Spiegelmer NOX 1255 may block the binding of LHRH-PE40 to LHRH receptor on the surface of HeLa cells.

    2011 06 v.24 [Abstract][OnlineView][Download 375K]

  • Effect of Hedgehog Signaling Pathway on Invasion and Metastasis of Human Breast Cancer MDA-MB-231 Cells and Relevant Mechanism

    ONG Ling,DENG Hua-yu,HAN Ming,CHEN Li,JIANG Rong(Department of Pathophysiology,Laboratory for Stem Cell and Tissue Engineering,Chongqing Medical University,Chongqing 400016,China)

    Objective To investigate the effect of Hedgehog signaling pathway on invasion and metastasis of human breast cancer MDA-MB-231 cells as well as the relevant mechanism.Methods MDA-MB-231 cells were treated with cyclopamine,an inhibitor of Hedgehog signaling pathway,and evaluated for invasion and metastasis abilities in vitro by Transwell test,for matrix metal protease-9(MMP-9) and MMP-2 activities by gelatin zymography,for expressions of P-c-Jun and MMP-9 proteins by Western blot,and for expressions of Glil,c-jun and MMP-9 mRNAs by RT-PCR.Results The invasion and metastasis abilities of MDA-MB-231 cells after treatment with cyclopamine decreased significantly,while MMP-9 activity,expression levels of P-c-Jun and MMP-9 proteins as well as Glil,c-jun and MMP-9 mRNAs decreased.Conclusion Hedgehog signaling pathway was activated in MDA-MB-231 cells,which promoted the invasion and metastasis of tumor cells by up-regulating the expressions of c-jun and MMP-9.

    2011 06 v.24 [Abstract][OnlineView][Download 496K]

  • Cloning and Prokaryotic Expression of Nonstructural Protein 1 Gene of a H3N2 SIV Isolate from Henan Province,China

    ZHAO Pu,ZHAO Kun,JIA Bei-bei,WANG San-hu,LIU Xing-you,YAO Si-xin,ZHENG Yu-shu(Department of Animal Science and Technology,Henan Institute of Science and Technology,Xinxiang 453003,Henan Province,China)

    Objective To clone the nonstructural protein 1(NS1) gene of a swine influenza virus isolate of subtype H3N2(H3N2 SIV) from Henan Province,China,express in prokaryotic cells,and provide a basis for development of a method for differentiation of immunized and naturally infected pigs.Methods Viral RNA was extracted from the Henan isolate of H3N2 SIV,with which NS1 gene was amplified by RT-PCR and cloned into prokaryotic expression vector pET-28a(+).The constructed recombinant plasmid was transformed to E.coli BL21(DE3) for expression under induction of IPTG.The expressed product was identified by SDS-PAGE and Western blot.Results Sequencing result proved the homologies of more than 98% of amplified NS1 gene to several domestic and abroad SIV strains.The expressed recombinant NS1 protein,with a relative molecular mass of about 27 000,showed specific reaction with the sera of pigs infected with SIV.Conclusion The NS1 gene of Henan isolate of H3N2 SIV was successfully cloned,and recombinant NS1 protein was expressed in E.coli BL21(DE3),which laid a foundation of development of a method for differentiation of immunized and naturally infected pigs.

    2011 06 v.24 [Abstract][OnlineView][Download 184K]

  • Inhibitory Effect of siRNA-NRP1 on Growth of Transplanted Gastric Carcinoma in Nude Mice

    PENG Yang,WU Xiao-ling,LIAN Hai-feng,CHEN Peng-fei,LU Xi(Department of Gastrointestinalogy,The Second Affiliated Hospital of Chongqing Medical University,Chongqing 400010,China)

    Objective To investigate the inhibitory effect of small interference RNA(siRNA) plasmid carrying neuropilin 1(NRP1) gene on growth of transplanted gastric carcinoma in nude mice.Methods The siRNA sequence targeting to human NRP1 gene was designed,based on which a pair of complementary oligonucleotides were synthesized,annealed and cloned into plasmid pGenesil-1.1 to construct recombinant plasmid siRNA-NRP1.Transplanted gastric carcinoma model was established by injecting s.c.nude mice with human gastric carcinoma SGC7901 cells,and siRNA-NRP1 was injected into the tumors formed.The nude mice were observed for clinical manifestation,and determined for size and weight of transplanted tumors.The tumor tissue and angiogenesis were observed by examination on pathological sections,and the expression of NRP1 and microvessel density in transplanted tumors by immunohistochemical assay.Results Restriction analysis and sequencing proved that target oligonucleotide fragment was cloned into plasmid pGenesil-1.1.Both the size and weight of transplanted tumors of nude mice injected with siRNA-NRP1 were less than those of control(P < 0.01).Pathological examination of pathological sections showed that,compared with that in control group,obvious necrosis of tumor cells was observed siRNA-NRP1 group.Immunohistochemical assay showed the both the expression levels of NRP1 and microvessel density in transplanted tumors of nude mice in siRNA-NRP1 were significantly lower than those in control group(P < 0.01).Conclusion The siRNA-NRP1 inhibited the NRP1 expression in transplanted gastric carcinoma of nude mice and influenced the angiogenesis of tumors,thus inhibited the tumor growth.

    2011 06 v.24 [Abstract][OnlineView][Download 478K]

  • Prokaryotic Expression and Purification of Carboxyl Terminus of Adhesin P1 of Mycoplasma pneumoniae

    LIU Zhi-qiang,WANG Dong,CHEN Yu-bao(School of Chemistry and Chemical Engineering,Hunan University of Science and Technology,Xiangtan 411201,Hunan Province,China)

    Objective To express the carboxyl terminus of adhesion P1 of Mycoplasma pneumoniae(MP) in prokaryotic cells and purify the expressed product.Methods Genomic DNA of MP type FH was extracted and used as a template,with which the carboxyl terminal gene of MP was amplified by PCR and inserted into prokaryotic expressed vector pET-32a(+).The constructed recombinant plasmid pET-32a-MP-P1C was transformed to E.coli Rosetta for expression under induction of IPTG.The expressed product was purified by nickel ion affinity chromatography and identified by SDS-PAGE,Western blot and ELISA.Results PCR,restriction analysis and sequencing proved that recombinant plasmid pET-32a-MP-P1C was constructed correctly.The recombinant protein with a relative molecular mass of about 55 900 was expressed in a soluble form,and contained 47% of total somatic protein.The purified recombinant protein,at a concentration of 3.77 mg/ml,reached a purity of more than 94% and showed specific reaction with the sera of patients infected with MP,even at a dilution of 1 ∶ 3 000.Conclusion The carboxyl terminus of adhesion P1 of MP was expressed in prokaryotic cells and purified,which laid a foundation of further study on pathogenic mechanism of MP as well as deve-lopment of diagnostic kit and DNA vaccine.

    2011 06 v.24 [Abstract][OnlineView][Download 294K]

  • Regulatory Effect of Bletilla striata Polysaccharide on Immune Function of Mice

    QIU Hong-mei,ZHANG Ying,ZHOU Qi-xin,LAI Shu(Department of Pharmacology,Chongqing Key Laboratory of Biochemistry and Molecular Pharmacology,Chongqing Medical University,Chongqing 400016,China)

    Objective To investigate the regulatory effect of Bletilla striata polysaccharide(BSPS) on immune function of mice.Methods BSPS was extracted from Bletilla striata and determined for content.Immunosuppressive mouse model was established by intraperitopeal injection with cyclophosphamide at a dosage of 100 mg /(kg·d) for 3 d.The mice were administered intragastrically with BSPS at various dosages and observed for effect on carbon clearance.The effect of BSPS on proliferation levels of splenocytes of mice induced with either concanavalin A(Con A) or lipopolysaccharide(LPS) was determined by MTT method.Results The polysaccharide contents in three batches of extracted BSPS were 93.490%,92.280% and 92.629% respectively.In carbon clearance test,the phagocytic exponent of mice treated with BSPS at a dosage of 500 mg /(kg·d) increased significantly as compared with those in control group,which was equivalent to that treated with levamisole at a dosage of 100 mg /(kg·d).In lymphocyte proliferation test,the BSPS at dosages of 1,3 and 10μg/ml promoted the proliferation of mouse splenocytes induced by Con A.However,the proliferation level of splenocytes treated with 3μg/ml BSPS was higher than those with BSPS at the other two concentrations.The BSPS at a dosage of 3μg/ml also promoted the proliferation of mouse splenocytes induced by LPS.Conclusion BSPS enhanced both non-specific and specific immune functions of mice.

    2011 06 v.24 [Abstract][OnlineView][Download 168K]

  • Serum IgG and IgE Levels of Mice Infected with Mycoplasma suis

    BA Cai-feng,XIE Jian-zhong,SONG Li-xian(Experimental Animal Center,Liaoning Medical University,Jinzhou 121001,Liaoning Province,China)

    Objective To establish BALB/c mouse model of Mycoplasma suis infection and observe the changes of serum IgG and IgE levels.Methods BALB/c mice uninfected with M.suis were divided into groups A,B and C,and injected with M.suis-positive blood,M.suis-negative blood and physiological saline respectively.The mice in various groups were determined for M.suis infection by microscopy and PCR,and for serum IgG and IgE levels by ELISA.Results M.suis was observed in blood of mice in group A by microscopy.Specific band at a length of 602 bp was amplified from blood of mice in group A by PCR.The serum IgG levels of mice in the three groups showed no significant difference on days 7 and 19 after injection(P > 0.05),while were significantly higher in group A than in groups B and C(P < 0.01),and in group B than in group C(P < 0.01),on days 10,13 and 16.However,the serum IgE levels of mice in the three groups showed no significant difference on days 10,13 and 22(P > 0.05),while were significantly higher in group A than in groups B and C(P < 0.01),and in group B than in group C(P < 0.01) on days 16 and 19.Conclusion The mouse model of M.suis infection was successfully established.IgG and IgE played important roles in immune response against M.suis.

    2011 06 v.24 [Abstract][OnlineView][Download 265K]

  • Construction and Identification of Short Hairpin RNA Expression Vector Targeting to Human Proto-oncogene c-fos

    JING Zhi-jie,LIU Tian-fu,SHI Rui-zan(Laboratory Animal Center of Shanxi Medical University,Taiyuan 030001,China)

    Objective To construct and identify a short hairpin RNA(shRNA) expression vector targeting to human proto-oncogene c-fos.Methods Recombinant shRNA expression vector Psilencer3.1-sic-fos targeting to human c-fos gene was constructed and transfected to breast cancer MCF-7 cells.The expression of c-fos mRNA in stably transfected cell was determined by RT-PCR.Results PCR,restriction analysis and sequencing proved that recombinant plasmid Psilencer3.1-sic-fos was constructed correctly.The recombinant plasmid inhibited the expression of c-fos gene at mRNA level in MCF-7 cells significantly.Conclusion The recombinant shRNA expression vector for human c-fos gene was successfully constructed,which provided a technical tool for further study on the role of c-fos gene in genesis and progress of tumors.

    2011 06 v.24 [Abstract][OnlineView][Download 276K]

  • Role of Peroxisome Proliferator-activated Receptor-α/Nitric Oxide in Induction of Proliferation of Vascular Smooth Muscle Cells by Angiotensin Ⅳ

    WANG Quan-hua,CHEN Jia-fei,CHENG Ou-mei,WU Qin,JIANG Qing-song(Department of Pharmacology,Chongqing Medical University,Chongqing 400016,China)

    Objective To investigate the role of peroxisome proliferator-activated receptor-α(PPARα)/nitric oxide(NO) pathway in induction of proliferation of vascular smooth muscle cells(VSMCs) by angiotensin Ⅳ(AngIV).Methods Rat VSMCs were cultured in vitro and divided into six groups,then added with medium(control),Ang Ⅳ,Ang Ⅳ+ fenofibrate(FF),Ang Ⅳ + FF +MK 886,Ang Ⅳ+L-arg and AngⅣ+L-arg+L-NAME respectively and determined for proliferation level by MTT method,for total cellular protein content by BCA method,for expressions of PPARα and eNOS mRNAs by real-time PCR,for expression of PPARα protein by Western blot,for nitric oxide synthase(NOS) activity in culture supernatant by colorimetry,and for NO concentration by nitrate reduction method.Results The Ang Ⅳ at a concentration of 0.1 nmol/L induced the proliferation of VSMCs,increased the total cellular protein content,decreased the expressions of PPARα mRNA and protein and eNOS mRNA as well as the NOS activity and NO concentration in cell culture supernatant.However,FF and L-arg reversed the above-mentioned effects of Ang Ⅳ,which were inhibited by the corresponding antagonists MK 886 and L-NAME respectively.Conclusion The proliferation of VSMCs induced by Ang Ⅳ might be associated with the damage of PPARα/NO signal transduction pathway.

    2011 06 v.24 [Abstract][OnlineView][Download 254K]

  • Immune Responses Induced with Recombinant Toxoplasma gondii Heat Shock Protein 70 by Intranasal and Subcutaneous Routes in Mice

    YIN Li-tian,CAO Lei,WANG Hai-long,MENG Xiao-li,ZHANG Jian-hong,YIN Guo-rong(Department of physiology,Key Laboratory of Cellular Physiology Co-constructed by Province and Ministry of Education,Shanxi Medical University,Taiyuan 030001,China)

    Objective To compare the immune responses and their persistence induced with recombinant Toxoplasma gondii heat shock protein 70(rTgHSP70) in mice by intranasal and subcutaneous routes.Methods Ninety 5-week-old BALB/c mice were randomly divided into 3 groups.The mice in intranasal group were immunized twice with 20 μg of rTgHSP70 by intranasal drip,and those in subcutaneous group with 80 μg of rTgHSP70 by subcutaneous injection,at an internal of 2 weeks,while those in control group were unimmunized.Five mice in each group were killed respectively at weeks 1,2,3,4,5 and 6 after the last immunization and determined for IgG levels in sera and sIgA levels in nasopharynx and intestinal washes by ELISA,of which the spleen lymphocytes and intraepithelial lymphocytes(IELs) were isolated and counted.Results The IgG levels in sera of mice at weeks 1 ~ 6 after the last immunization were higher,while those at weeks 3 ~ 5 were significantly higher in intranasal group than in subcutaneous and control groups(P < 0.001),and those at weeks 3 ~ 4 were significantly higher in subcutaneous group than in control group(P < 0.001).The sIgA levels in nasopharynx washes of mice in intranasal group at weeks 3 ~ 5 as well as those in subcutaneous group at weeks 4 ~ 5 were significantly higher than those in control group(P < 0.001).The sIgA levels in intestinal washes of mice at weeks 1 ~ 6 were significantly higher in intranasal group than in subcutaneous and control groups(P < 0.001),while those at weeks 3 ~ 4 were significantly higher in subcutaneous group than in control group(P < 0.001).The spleen lymphocyte counts were significantly higher in intranasal group than in control group at weeks 2 ~ 4(P < 0.000 1),in intranasal group than in subcutaneous group at weeks 3 ~ 4,and in subcutaneous group than in control group at weeks 2 ~ 4(P < 0.001).The IEL counts were significantly higher in intranasal group than in control group at weeks 1 ~ 6(P < 0.000 1),in intranasal group than in subcutaneous group at weeks 3 ~ 4(P < 0.000 1),and in subcutaneous group than in control group at weeks 1 ~ 5(P < 0.05).Conclusion Both immunization with rTgHSP70 by intranasal and subcutaneous routes induced effective mucosal and systemic immune responses,while the response levels induced by intranasal route were higher than those by subcutaneous route,indicating intranasal drip a more effective immune route of rTgHSP70.

    2011 06 v.24 [Abstract][OnlineView][Download 283K]

  • Stability of PIKA Recombinant Hepatitis B Vaccine(Hansenula polymorpha)

    LI Xiao-yu,GAO Jun-qing,ZHANG Yan,JIN Zhen-ji,LIU Zhao-hui(Dalian Hissen Bio-Pharm Inc.,Dalian 116600,Liaoning Province,China)

    Objective To observe the stability of PIKA recombinant hepatitis B(HB) vaccine(Hansenula polymorpha).Methods PIKA recombinant HB vaccine(H.polymorpha) was stored at 37,25 and 2 ~ 8℃ respectively for various days,then determined for relative potency(RP) in vitro,pH value and purity,and observed for stability.Results The RP of vaccine decresed slightly after storage at 37℃ for 45 d but still met the relevant requirements.Neither RP nor pH value showed signficant change after storage at 25℃ for 6 months.However,after storage at 2 ~ 8℃ for 12 months,no signifcant change was observed in RP,pH value or purity of the vaccine.Conclusion PIKA recombinant HB vaccine(H.polymorpha) showed high stability.

    2011 06 v.24 [Abstract][OnlineView][Download 154K]

  • Verification of Purification Procedure of Recombinant Human Prourokinase for Virus Removal

    LI Shi-chong,YE Hua-hu,ZHAO Guo-han,ZHANG Zheng-guang,WANG Dai-ping,XU Zhao-ping,GAO Li-hua,CHEN Zhao-lie,HU Xian-wen(Beijing Institute of Biotechnology,Academy of Military Medical Sciences,Beijing 100071,China)

    Objective To verify the purification procedure of recombinant human prourokinase(Pro-uk) for virus removal.Methods The effect of viral removal by ion exchange chromatography was evaluated by scale-down model simulating the anion and cation exchange chromatography in purification procedure of Pro-uk,using esivular stomatitis virus(VSV),encephalomyocarditis virus(EMCV),herpes simplex virus type-1(HSV-1) as indicators.Results Anion exchange chromatography was effective in removal of lipid-enveloped viruses such as VSV and HSV-1,while was less effective in non-lipid-enveloped EMCV.However,cation exchange chromatography was less effective in removal of VSV and HSV-1 while was effective in EMCV.All the cumulative removal efficacies of the three kinds of indicator viruses by ion exchange chromatography were more than 99.99%(more than 4 log10).Conclusion The ion exchange chromatography step in purification procedure of Pro-uk was effective in control of safety of viruses,by which the removal efficacy of viruses met the General Requirements for Technical Evaluation on Virus Safety in Products Extracted from Organic Tissues and Expressed in Eukaryotic Cells.

    2011 06 v.24 [Abstract][OnlineView][Download 243K]

  • Preparation and Clinical Application of Anti-Human Platelet Monoclonal Antibody

    MU Zhong-mei,DUAN Sheng-bao,LIU Ying,LI Chun-ming,CHEN Xi-peng,LI Yong(Changchun Brother Biotech Co.Ltd,Changchun 130012,China)

    Objective To prepare anti-human platelet monoclonal antibody(McAb) used for clinical screening of platelet antibody and cross matching of platelet.Methods BALB/c mice were immunized with platelet group O as immunogen,of which the splenocytes were fused with NS-1 cells.Positive hybridoma cells were screened by indirect ELISA and cloned by limiting dilution method.The positive hybridoma cells and the secreted McAb identified,and the McAb was purified by affinity chromatography and used for development of solid agglutination method.Clinical blood samples were determined by the developed method,and the result was compared with that by MASPAT kit.Results A hybridoma cell strain 1C1 stably secreting anti-human platelet McAb was obtained,by which the secreted McAb was specific to human platelet glycoprotein Ib/IX.After the cells were subcultured for more than 8 months,the titer of McAb in culture supernatant was 104,which lasted for 35 d at 37℃.The titer of purified antibody reached 1 ∶ 108.The determination results of 50 blood samples by the developed solid agglutination method were consistent with that by MASPAT kit.Conclusion Anti-human platelet McAb was successfully prepared,which laid a foundation of its further clinical application.

    2011 06 v.24 [Abstract][OnlineView][Download 146K]

  • Preparation of National Reference Serum Used for Detection Kit for IgG against Toxoplasma

    ZHANG Jin,XIN Xiao-fang,BO Shu-ying,YANG Ying-chao,ZHANG Ying,WANG Guo-zhi(National Institutes for Food and Drug Control,Beijing 100050,China)

    Objective To prepare national reference serum used for detection kit for IgG against toxoplasma.Methods The sera positive for IgG against toxoplasma as well as normal human sera were collected and screened by using detection kits manufactured by six manufacturers and confirmed by dye test and direct agglutination test,then the positive serum samples were calibrated with international standard for IgG against toxoplasma.The references were calibrated coordinately,based on which the minimum detection limit was determined.Results A set of national reference consisting of 9 positive(including one for minimum detection limit) and 15 negative serum samples was prepared.Conclusion The first set of national reference serum used for detection kit for IgG against toxoplasma was prepared,and the corresponding quality standard was developed,which has been approved by the relevant national departments.

    2011 06 v.24 [Abstract][OnlineView][Download 122K]

  • Comparison of Third Generation of Domestic and The Fourth Generation of Imported Detection Kits for HIV

    CHEN Zhen-zhou,XIAO Ming-xing,TONG Xin,ZHOU Yun,LU Wei-rong,CHEN Ting(Chaozhou Blood Center,Chaozhou 521000,Guangdong Province,China)

    Objective To compare the difference in detection results by the third generation of domestic and the fourth generation of imported kits for HIV,and provide a reference for selection of reagents and establish the standard for discard of blood.Methods A total of 35 487 blood samples were tested by the third generation of domestic(A and B) and the fourth generation of imported(C) kits separately for HIV following the instructions.The kits before use were evaluated for qualities by National Institute for the Control of Pharmaceutical and Biological Products(NICPBP).Results All the three kits met the relevant requirements for quality.The detection results of s/co values of not less than 1.0 by kits A and B showed no significant difference,while those with s/co values of not less than 0.8 showed significant difference,with those by kit C.Conclusion Taking account for the safety of blood products,the standard for discard of blood is suggested as a s/co value of not less than 0.5 by the third generation of detection kit for HIV,or not less than 0.8 by the fourth generation of imported kit.

    2011 06 v.24 [Abstract][OnlineView][Download 129K]

  • Safety and Immunogenicity of Domestic Combined Live Attenuated Measles,Mumps and Rubella Vaccine

    CHU Yan,LU Yu-zhong,TAO Hong,WANG Biao,WANG Yan,WANG Shu-qiao,QIN Jie,XU Wen-qing(Shanghai Institute of Biological Products,Shanghai 200052,China)

    Objective To observe the safety and immunogenicity of domestic combined live attenuated measles,mumps and rubella(MMR) vaccine.Methods A total of 765 healthy children unimmunized with MMR previously,aged 8 ~ 15 months,were randomly divided into HG,HD and control groups and immunized with MMR vaccines with high and low mumps virus titers manufactured by Shanghai Institute of Biological Products and domestic MMR vaccine as control respectively,then observed for safety and immunogenicity.The IgG levels against measles,mumps and rubella in sera of the children were determined by ELISA.Results The systemic and local adverse reaction rates of the 765 immunized children were 18.95% and less than 0.4% respectively.The positive conversion rates of antibody against measles in HG,HD and control groups were 97.34%,97.18% and 95.04%,while those against mumps were 94.68%,89.83% and 76.60%,and those against rubella were 94.68%,94.35% and 91.49%,respectively.The geometric mean concentrations(GMCs) of antibody against measles in the three groups were 6 221.57,5 766.34 and 5 056.75 mIU/ml respectively,which increased by about 200 folds in average;while those against mumps were 381.33,308.46 and 207.01 U/ml,which increased by about 26,22 and 17 folds in average respectively;and those against rubella were 81.77,77.61 and 81.49 IU/ml respectively,which increased by about 23 folds in average.No significant differences were observed in the positive conversion rates,GMCs as well as their mean increasing folds of antibodies against measles and rubella in various groups(P > 0.05).However,the indexes were significantly higher in HG and HD groups than in control group(P < 0.05),while showed no significant differences in HG and HD groups(P > 0.05).Conclusion All the three kinds of MMR vaccines showed high safety,of which the immunogenicity to measles and rubella were similar while those to mumps showed significant difference.However,the antibody positive conversion rates induced by high and low titer MMR vaccines manufactured by Shanghai Institute of Biological Products showed no significant difference.

    2011 06 v.24 [Abstract][OnlineView][Download 192K]

  • Development of Pilot Fermentation Procedure for Recombinant Pichia pastoris for Tumor-targeting Fusion Protein EGF-E4orf4

    SUN Cheng-jin,WANG Jian,CUI Chun-qing,GUO Peng,CHEN Xiang-peng,ZHANG Zhen-long(National Vaccine & Serum Institute,Beijing 100024,China)

    Objective To develop a pilot fermentation procedure for recombinant Pichia pastoris for tumor-targeting fusion protein EGF-E4orf4(adenovirus early region 4 open reading frame 4 protein).Methods The time for culture of secondary seeds of recombinant P.pastoris for EGF-E4orf4 was optimized.The seeds were inoculated into 20 L of fermentation medium at a volume ratio of 5%,and the temperatures at culture and induction stages were set as 30 and 28℃ respectively.The DO during fermentation was maintained at 20% ~ 35% by the measures such as regulating stirring rate,aeration and pressure in fermenter.The recombinant P.pastoris was cultured by three-step fermentation,and the pH value and time for induction were optimized.Three batches of recombinant P.pastoris were fermented under the optimal condition.Results The optimal time for culture of secondary seeds was 15 h,while the optimal pH value and time for induction were 6.0 and 48 h respectively.The mean A600 value and wet weight of 3 batches of recombinant P.pastoris cultured by fermentation were(327.67 ± 17.96) and(308.33 ± 9.07) g/L respectively,while the mean expression level of EgF-E4orf4 was(200.00 ± 5.57) mg/L.Conclusion The pilot fermentation procedure for recombinant P.pastoris for EGF-E4orf4 was preliminarily developed,which laid a foundation of industrialization of EGF-E4orf4.

    2011 06 v.24 [Abstract][OnlineView][Download 269K]

  • Optimization of Condition for Culture of CHO Cells by Tubespin,a Disposable High-throughput Bioreactor

    XIE Kui,LEI Yun,HUANG Li,WANG Yi-ting,XIONG Sheng(Biomedical R&D Center,Guangdong Provincial Key Laboratory of Bioengineering Medicine,National Engineering Research Center of Genetic Medicine,Jinan University,Guangzhou 510632,China)

    Objective To optimize the condition for culture of CHO cells by Tubespin,a disposable high-throughput bioreactor and increased the yield of protein.Methods CHO cells were subjected to suspension culture in oscillation by using Tubespin at various rotation speeds(160,180 and 200 r/min) in various volumes(5,10,20 and 35 ml).The gas exchange rates,cell densities,cell viabilities and relative expression levels of protein under various conditions were compared,based on which the culture condition for cells was optimized.Under the optimal condition,the medium was added with 30,60 and 90 mmol/L sodium chloride respectively,and the effects on cell density,cell viability,oxygen uptake rate,dissolved oxygen and expression of recombinant protein were observed.Results Tubespin showed high gas exchange rate which met the necessity of high density cell culture.The optimal rotation speed shaker and culture volume of cells were 180 r/min and 10 ml respectively.Under the optimized condition,cells growth density was high,and the stable growth stage was reached rapidly,which was suitable for expression of recombinant protein.The addition of 60 mmol/L sodium chloride into medium 72 h after culture increased the expression level of recombinant protein by 1 fold.Conclusion The condition for culture of CHO cells by Tubespin as well as the concentration of sodium chloride for induction were optimized,and the yield of recombinant protein increased,which laid a foundation of large-scale production of recombinant protein by fermentation.

    2011 06 v.24 [Abstract][OnlineView][Download 475K]

  • Preparation of Monoclonal Antibody against Heavy Metal Cupper Ion and Development of Indirect Competitive ELISA Method

    GUO Chang-wei,TANG Yong,XIANG Jun-jian,ZOU Jun-hui,YANG Hong-yu(Molecular Immunology and Antibody Engineering Center,Jinan University,Guangzhou 510632,China)

    Objective To prepare monoclonal antibody(McAb) against heavy metal cupper ion and develop an indirect competitive ELISA method.Methods Copper ion was coupled to vector proteins bovine serum albumin(BSA) and ovalbumin(OVA) via a bifunctional chelating agent p-SCN-Bn-DTPA respectively.BALB/c mice were immunized with the prepared antigens Cu(Ⅱ)-DTPA-BSA for immunization and Cu(Ⅱ)-DTPA-OVA for detection respectively,and the hybridoma cell strains stably secreting McAbs against Cu(Ⅱ)-DTPA were screened.The ascites of mice were collected,in which the McAbs were determined for titer,identified for subclass and tested for specificity.An indirect competitive ELISA method was developed,by which the determination result was compared with that by ICP-AES method.Results Three hybridoma cell strains stably secreting McAbs against Cu(Ⅱ)-DTPA were screened,of which 4C2E7 strain was selected for preparation of ascites.The titer and subclass of McAb in ascites were 1 × 106 and IgG1 respectively.The McAb showed cross reaction rates of 17.5%,8.75% and 1.46% with Co(Ⅱ),Zn(Ⅱ) and Hg(Ⅱ) respectively,while showed low cross reaction rates with other seven kinds of metal ions.The minimum detection limit of the developed ELISA method was 2.0 ng/ml.The total coincidence rate of the developed ELISA method and ICP-AES method was more than 94%.Conclusion The McAb against Cu(Ⅱ) was successfully prepared,and an indirect competitive ELISA method was developed.

    2011 06 v.24 [Abstract][OnlineView][Download 218K]

  • Optimization of Medium for Fermentation of Phellinus igniarius by Response Surface Method

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

    Objective To optimize the medium for fermentation culture of Phellinus igniarius by response surface method and increase the yield of exopolysaccharide.Methods The primary components of liquid medium for P.igniarius was optimized by Plackett-Burman design and response surface method.The experimental data were subjected to polynomial regression analysis by Design-Expert software,based on which the functional relationship between the three primary components(peptone,yeast extract and potassium dihydrogen phosphate) and the yield of exopolysaccharide was explored.Three check tests were performed on the optimized medium.Results The concentrations of peptone,yeast extract and potassium dihydrogen phosphate were optimized as 14.72,11.04 and 1.472 g/L respectively by ridge analysis.By using the optimized medium,the highest yield of exopolysaccharide reached 7.04 g/L.The yields of exopolysaccharide in three check tests were 6.92,6.86 and 7.04 g/L respectively,with a mean of 6.94 g/L,which were basically consistent with those expected.Conclusion The yield of exopolysaccharide increased significantly by using the optmizied medium.

    2011 06 v.24 [Abstract][OnlineView][Download 318K]

  • Optimization of Fermentation Medium for Erythromycin Production of Streptomyces erythreus by Response Surface Methodology

    HUA Cheng-wei,YU Jiang-ao,XIE Feng-zhen(School of Life Science and Technology,Henan Institute of Science and Technology,Xinxiang 453003,Henan Province,China)

    Objective To optimize the fermentation medium for erythromycin production Streptomyces erythreus by response surface methodology.Methods Min Run Res Ⅳ design was used to evaluate the effect of compositions of organic nitrogen source and carbon source,and the steepest ascent path was employed to determine the central region of the medium composition for the further central composite design(CCD).Results Starch,dextrin,soybean cake meal and corn steep liquor showed significant effect on the yield of erythromycin,of which the optimal proportion in medium were 5.24%,1.43%,4.56% and 1.47% respectively.Conclusion The carbon and nitrogen sources of fermentation medium for erythromycin production of Streptomyces erythreus were successfully optimized by response surface methodology.The chemical titer of erthyromycin produced by fermentation in shake flask was 6 192 U/ml.

    2011 06 v.24 [Abstract][OnlineView][Download 396K]

  • Progress in Research on Antiviral Therapy Based on MicroRNAs

    ZHANG Qing-hua,LIU Zhe-wei(Capital Institute of Pediatrics,Beijing 100020,China)

    MicroRNAs represent a recently discovered group of conservative,endogenous,short noncoding regulatory RNA molecules that negatively modulate protein expression at post-transcriptional level by matching to the bases of target mRNA to inhibit the translation or degrade the mRNA.They are 22 nt in length and identified in many eukaryotic organism and some viruses.MicroRNA pathway participates in the regulation of gene expression in either physiological or pathologic state,and plays a key role in host-virus interactions.It is believed that both human and viral microRNAs will become attractive targets of antiviral therapy.The biogeny and action mechanism of miRNA,host-virus interaction at miRNA level,as well as roles of host-and virus-encoded miRNAs in antiviral therapy are reviewed in this paper.

    2011 06 v.24 [Abstract][OnlineView][Download 193K]

  • Progress in Research on Oral Vaccine against Common Diarrheal Alimentary Infectious Diseases

    LI Shi-bin,MA Yong-ping(Research Center of Medical Molecules and Cancer,Department of Biochemistry and Molecular Biology,Chongqing Medical University,Chongqing 400016,China)

    Common diarrheal alimentary infectious diseases are usually caused by pathogens including bacteria and viruses,and disseminated through water or food,which induce fulminant epidemic that cause invasion and death of infant especially.This article reviews the progress in research on oral vaccine against common diarrheal alimentary infectious diseases caused by bacteria and viruses.

    2011 06 v.24 [Abstract][OnlineView][Download 167K]

  • Progress in Research on Role of Nrf2/ARE Transduction Pathway in Central Nervous System Diseases

    ZHU Feng-chen,JIANG Dian-ming(Department of Orthopaedics,The First Affiliated Hospital,Chongqing Medical University,Chongqing 400016,China)

    Nuclear factor erythroid-2p45-related factor 2(Nrf2)is an important transcription factor for cells to mediate the resistance to oxidative stress.As a pivotal protective pathway to resist internal and external oxidative stress,it binds to antioxidant response element(ARE) and regulates the expressions of phase Ⅱ detoxification enzyme genes and antioxidant enzyme genes.With high oxygen consumption and abundant polyunsaturated fatty acids,central nervous system is sensitive to oxidative stress.The role of Nrf2/ARE transduction pathway in the central nervous system is paid more and more attentions.This article summarizes the latest progress in research on regulatory mechanisms of Nrf2/ARE transduction pathway as well as its role in central nervous system diseases.

    2011 06 v.24 [Abstract][OnlineView][Download 168K]

  • Progress in Research on Glucose-6-Phosphate Dehydrogenase

    AN Xuan,LIU Liang-zhong,HU Peng(Department of Infectious Diseases,Institute of Viral Hepatitis,The Second Affiliated Hospital of Chongqing Medical University,Chongqing 400010,China)

    Glucose-6-phosphate dehydrogenase(G6PD),the key regulatory enzyme in the hexose monophosphate shunt,plays an important role in maintenance of the cytosolic pool of NADPH and thus the cellular redox balance.G6PD deficiency,an X-linked disorder,is one of the most common enzymopathies in the world.Previous studies were focused on hemolysis and anemia.Recently,numerous studies have shown the importance of G6PD in cell growth,development and disease progression.Deficiency in G6PD activity,and hence a disturbance in redox homeostasis,can lead to dysregulation of cell growth and signaling,anomalous embryonic development,alter the susceptibility to viruses and increase the susceptibility to degenerative diseases.The present review covers recent developments in the fields of the correlation between G6PD and tumor,viral infection and cardiovascular diseases.This article focuses on the progress in research on G6PD.

    2011 06 v.24 [Abstract][OnlineView][Download 173K]
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