2011年03期目次

  • Prokaryotic Expression,Purification and Immunogenicity of Fusion Protein of Hepatitis B Virus Core Antigen and PreS1 Antigen

    WU Gang,LI Ji-lai,WANG Juan,ZHAO Li,XU Jing(National Vaccine and Serum Institute,Beijing 100024,China)

    Objective To express the fusion protein of hepatitis B virus core antigen(HBcAg)(1 ～ 155) and preS1 antigen(PreS1)(3 ～ 55),purify the expressed product and analyze its immunogenicity.Methods HBV DNA was extracted from the sera of HBeAg-positive patients with hepatitis B and used as templates for amplification of HBcAg and PreS1 genes by PCR.The fusion gene CS1 was subcloned into prokaryotic expression vector pET-32a(+).The constructed recombinant plasmid pET-CS1 was transformed to E.coli BL21(DE3) for expression under induction of IPTG.The expressed product was purified then identified by SDS-PAGE,HPLC and Western blot.BALB / c mice were immunized with the purified fusion protein and determined for subclasses of total Ab and titers of anti-PreS1 by indirect ELISA,and for titers of anti-HBc by competitive inhibition method,then evaluated for cellular immune effect by ELISPOT.Results Both restriction analysis and sequencing proved that recombinant plasmid pET-CS1 was constructed correctly.Electron microscopy showed that the preliminarily purified CS1 was assembled to virus-like particles at diameters of about 30 nm automatically.SDS-PAGE and HPLC showed that the purities of CS1 protein were 98.2% and 93% respectively.Purified CS1 protein was bound to anti-HBc and anti-PreS1 specifically,and induced anti-HBc and anti-PreS1 in mice,most of which were of subclass IgG2a.In addition,purified CS1 protein induced the secretion of HBcAg-specific IFNγ by murine splenic cells.Conclusion Fusion protein CS1 was successfully expressed in prokaryotic cells and purified,and the purified CS1 reached a high purity and induced high humoral and cellular immune responses.

    2011 03 v.24 [Abstract][OnlineView][Download 375K]

  • Construction of Eukaryotic Expression Vector for Fusion Gene RV-G/LTB and Its Expression in Vero Cells

    LIU Juan,LIU Ming-yuan,YU Lu,GUO Heng,LI Yang,ZHAO Li-jing,SUN Shu-min,LI Hui-ping,LIU Xue,WANG Xue-lin(Key Laboratory of Zoonosis Research,Ministry of Education,Institute of Zoonosis,Jilin University,Changchun 130062,China)

    Objective To construct a eukaryotic expression vector for fusion gene RV-G / LTB and express in Vero cells.Methods The cloned gene fragments encoding rabies virus(RV) glycoprotein(G) and E.coli heat-labile enterotoxin subunit B(LTB) were fused by SOE-PCR using a linker sequence(Gly4Ser)2 and inserted into eukaryotic expression vector pVAX1.The constructed recombinant plasmid pVAX1-G-linker-LTB was transfected to Vero cells,and the expressed fusion protein was identified by indirect IFA and Western blot.Results Both restriction analysis and sequencing proved that recombinant plasmid pVAX1-G-linker-LTB was constructed correctly,and the expression of fusion protein was proved in transfected Vero cells.Conclusion A eukaryotic expression vector for fusion gene RV-G / LTB was successfully constructed and expressed in Vero cells.

    2011 03 v.24 [Abstract][OnlineView][Download 271K]

  • Construction and Identification of Recombinant Adenovirus Vector Expressing Human S100A8

    GUO Yuan-yuan,YOU Li,XU Lan-lan,ZOU Zheng-yu,LI Yu-ye,SUN Shuang-shuang,LUO Jin-yong,ZHOU Lan(Key Laboratory of Laboratory Medical Diagnostics,Department of Medical Laboratory,Ministry of Education,Chongqing Medical University,Chongqing 400016,China)

    Objective To construct recombinant adenovirus vector expressing human S100A8(hS100A8) and lay a foundation of further study on hS100A8.Methods From plasmid pGST-hS100A8,hS100A8 gene fragment was amplified and subcloned into shuttle plasmid pADTrack-TOX.The constructed recombinant shuttle plasmid pADTrack-TOX-hS100A8 was identified by restriction analysis,PCR and sequencing,then linearilized with Pme Ⅰ and transformed to competent AdEasier cells.The obtained recombinant adenovirus vector pAdhS100A8 was digested with Pac Ⅰ and transfected to HEK293 cells for packaging,and the prepared recombinant adenovirus was amplified for titration and identification by RT-PCR and Western blot.Results Both recombinant shuttle plasmid pADTrack-TOX-hS100A8 and recombinant adenovirus vector pAdhS100A8 were constructed correctly.Recombinant adenovirus AdhS100A8 was successfully packaged in HEK293 cells,reached a titer of 1011 IU / ml after amplification,and successfully expressed in HEK293 cells.Conclusion The recombinant adenovirus vector expressing hS100A8 was successfully constructed,which laid a foundation of further study on hS100A8.

    2011 03 v.24 [Abstract][OnlineView][Download 262K]

  • Prokaryotic Expression and Purification of Listeriolysin O from Listeria Monocytogenes

    GUO Jian-wei,MA Cong,WANG Zhen-guang,QIAN Yang-hui,ZHANG Yun,HAO Xiu-hong(Department of Clinical Laboratory,Navy General Hospital,Beijing 100037,China)

    Objective To express the listeriolysion O(LLO) from Listeria monocytogenes(LM) in prokaryotic cells and purify the expressed product.Methods LLO gene was amplified from the genomic RNA of LM by PCR using a pair of specific primers and inserted into vector pET-30a(+).The constructed recombinant plasmid pET-30a(+) / rLLO was transformed to E.coli BL21(DE3) for expression under induction of IPTG.The expressed product was purified by Ni2+-NTA chromatography and identified by SDS-PAGE and mass spectrum.Results PCR,restriction analysis and sequencing proved that recombinant plasmid pET-30a(+) / rLLO was constructed correctly.The expressed rLLO fusion protein,with a relative molecular mass of about 78 000,mainly existed in a form of inclusion body.Mass spectrum proved that the two protein fragments,with relative molecular masses of 35 000 and 45 000 respectively,were also LLO.Conclusion Recombinant LLO protein was successfully expressed in E.coli and purified,which laid a foundation of further study on biological function of LLO and preparation of LLO-based specific diagnostic kit.

    2011 03 v.24 [Abstract][OnlineView][Download 281K]

  • Replication Kinetics of Standard Human Metapneumovirus Strain in Various Cell Lines

    SHE Wei-wei,ZHAO Xiao-dong,ZHAO Yao(P2 Laboratory,Children's Hospital of Chongqing Medical University,Chongqing 400014,China)

    Objective To investigate the replication kinetics of standard human metapneumovirus(hMPV) strain in various cell lines to find a cell line suitable for the isolation and culture of the virus.Methods Standard hMPV strain hMPV / NL / 1 / 00 of subtype A and hMPV / NL / 1 / 99 of subtype B were inoculated to Vero,Vero-E6,LLC-MK2,A549 and HEp-2 cells separately for several blind passages.The CPEs were observed daily,and the RNAs were extracted from the cells on days 2,4,6,8,10,12,14,16,18 and 20 after inoculation,then reversely transcribed to cDNA and determined by real-time fluorescent quantitative PCR.Results CPEs were observed in Vero,Vero-E6,LLC-MK2 and A549 cells 3 d after inoculation.However,the CPEs caused by hMPV strains of various subtypes showed no significant difference.The hMPV could be stably replicated in Vero,Vero-E6 and LLC-MK2 cells,but could not be subcultured stably in A549 and HEp-2 cells.Conclusion Vero,Vero-E6 and LLC-MK2 cells were suitable,while A549 and HEp-2 cells were unsuitable for the culture of hMPV.

    2011 03 v.24 [Abstract][OnlineView][Download 468K]

  • In Vitro Bioactivity and Degradation Regularity of Synthetical Tachyplesin Ⅰ

    XIE Hai-wei,WANG Di,YANG Xian-song,SUN Lan-ping,ZHANG Bin,XU Hui(School of Biotechnology and Food,Bengbu College,Bengbu 233030,Anhui Province,China)

    Objective To investigate the in vitro bioactivity of synthetical tachyplesinⅠas well as its degradation regularity in mimic digestive environment.Methods TachyplesinⅠwas treated at various temperatures,pH values,cation concentrations,polarities of solvent and mimic digestive environments,then determined for bioactivity to evaluate the stability,and for hemolytic activity to mouse blood cells.The degradation of tachyplesinⅠin mimic digestive environment was evaluated by HPLC profile.Results TachyplesinⅠwas stable at temperatures less than 100℃ and pH values less than 10.3.Cation concentration and polarity of solvent showed certain effects on the antibacterial activity of tachyplesinⅠ.The tachyplesinⅠat a concentration of more than 80 mg / L showed a certain hemolytic activity after treatment for more than 30 min.TachyplesinⅠwas highly stable in mimic gastric juice,gastric mucosa homogenate and plasma,of which the chromatographic pattern showed no significant change,and little degradation was observed.The minimum bacteriostatic concentration of tachyplesinⅠwas 5.0 ～ 20.0 mg/L.However,tachyplesinⅠ was slightly sensitive in mimic intestine juice and intestine mucosa homogenate,expressed as decreased peak height,appearance of foreign protein peak and significantly decreased bioactivity,of which the minimum bacteriostatic concentration was 80～160 mg / L.Conclusion TachyplesinⅠshowed high thermotolerance,stability to acidic condition as well as a certain resistance to degradation with protease in vivo.

    2011 03 v.24 [Abstract][OnlineView][Download 547K]

  • Inhibitory Effect of Rosiglitazone on Proliferation of Human Colon Cancer Lovo Cells and Relevant Mechanism

    WU Ke,HE Bai-cheng,ZHOU Qi-xin(Department of Pharmacology,Chongqing Key Laboratory of Biochemistry and Molecular Pharmacology,Chongqing Medical University,Chongqing 400016,China)

    Objective To investigate the inhibitory effect of rosiglitazone(Ros) on proliferation of human colon cancer Lovo cells as well as the relevant mechanism.Methods Lovo cells were treated with various concentrations(10-6、10-5、10-4 and 10-3 mol / L) of Ros and Meloxicam(Mel) for 6,12 and 24 h separately,and determined for proliferative activity by MTT method.The cells 24 h after treatment with Ros were determined for transcription level of COX-2 mRNA by RT-PCR,and for expression level of cyclooxygenase-2(COX-2) protein by Western blot.Results The Ros at concentrations of more than 10-5 mol / L and the Mel at concentrations of more than 10-4 mol / L showed significantly dose and time-dependent inhibitory effect on the proliferation of Lovo cells.Ros decreased the expression levels of COX-2 mRNA and protein significantly,both in dose-dependent modes.Conclusion Ros inhibited the proliferation of Lovo cells by a mechanism associated with the inhibition of COX-2 expression.

    2011 03 v.24 [Abstract][OnlineView][Download 260K]

  • Predication of Spatial Structure and B Cell Epitope of VP1 Protein of Chinese Sacbrood Virus LN-QY Strain

    CHENG Jian,ZHANG Pei,MA Ming-xiao,LI Ming,YANG Song(Liaoning Medical University,Jinzhou 121000,Liaoning Province,China)

    Objective To predict the spatial structure and B cell epitope of VP1 protein of Chinese sacbrood virus(CSBV) LN-QY strain.Methods The gene encoding VP1 protein of CSBV was amplified by RT-PCR using the RNA of LN-QY strain as a template,and inserted into vector pMD18-T.The constructed recombinant plasmid was transformed to competent E.coli DH5α,and the positive clones identified by digestion with EcoRⅠ and Hind Ⅲ was sequenced.The nucleotide and deduced amino acid sequences were obtained by comparing the sequences with those of other reference strains,and the 3D model of structural protein of VP1 was established,based on which several parameters of the structural protein,such as flexible region,hydrophilicity,surface probability and antigenic index were analyzed by the Protean module in DNAStar software.Results The spatial confirmation of VP1 protein was relatively regular,in which the potential B cell antigenic epitopes were located at 47 ～ 53,139 ～ 145 and 273 ～ 284 aa.Conclusion The study provided a theoretical basis for design of VP1 epitope vaccine against CSBV LN-QY strain as well as the preparation of serological diagnostic kit.

    2011 03 v.24 [Abstract][OnlineView][Download 588K]

  • Expression of Activin A and Activin-binding Protein in Liver Tissues of Mice with Acute Alcohol-induced Liver Lesion

    YU Fang△,WANG Yi-nan,YANG Qing,MENG Xiao-dan,SUN Yang,TAI Gui-xiang,LIU Zhong-hui(△Department of Immunology,Norman Bethune College of Medicine,Jilin University,Changchun 130021,China)

    Objective To investigate the expression of activin A and activin-binding protein(Follistatin,FS) in liver tissues of mice with acute alcohol-induced liver lesion.Methods Mice were administered with alcohol by gavage at a dosage of 5 g / kg,once 12 h for 3 times,to copy the mouse model of acute alcohol-induced liver lesion.The transcription levels of activin A and FS mRNAs in liver tissues of mice were determined by real-time quantitative RT-PCR,while the expression levels of activin A and FS by immunohistochemical staining.Results Compared with those in control group,the mRNA and protein levels of activin A in liver tissues of model mice were significantly high(P < 0.01),while those of FS showed significant difference(P > 0.05).Conclusion The expression of activin A-FS in liver tissues of mice with acute alcohol-induced liver lesion was unbalanced,which was mainly expressed as increased activin A,indicating that the unbalance of activin A-FS system might be associated with alcohol-induced liver lesion.

    2011 03 v.24 [Abstract][OnlineView][Download 431K]

  • Effect of Tumor Suppressor in Lung Cancer 1 on Proliferation and Invasion Abilities of Nasopharyngeal Carcinoma Cell HNE-1 Strain

    WU Xiao△,RAN Yong-gang,CHEN Jiong-yu,YOU Yan-jie(△Cancer Hospital of Medical College,Shantou University,Shantou 515041,Guangdong Province,China)

    Objective To investigate the effect of tumor suppressor in lung cancer 1(TSLC1) on proliferation and invasion abilities of nasopharyngeal carcinoma cell HNE-1 strain.Methods The full-length of encoding region of TSLC1 gene was amplified from breast cancer MCF-7 cells by RT-PCR,based on which eukaryotic expression vector pcDNA3.1-TSLC1 was constructed and transfected to HNE-1 cells.The expression of TSLC1 at mRNA and protein levels were determined by RT-PCR and Western blot,and the effect of overexpression of TSLC1 gene on proliferation and invasion abilities of HNE-1 cells by MTT method and Transwell test.Results Both restriction analysis and sequencing proved that recombinant plasmid pcDNA3.1-TSLC1 was constructed correctly.TSLC1 gene was over-expressed in the HNE-1 cells stably transfected with the recombinant plasmid,which inhibited the proliferation and invasion abilities of HNE-1 cells significantly.Conclusion The overexpression of TSLC1 gene showed significantly inhibitory effect on proliferation and invasion abilities of HNE-1 cells,which provided an ideal molecular target for the gene therapy of nasopharyngeal carcinoma.

    2011 03 v.24 [Abstract][OnlineView][Download 240K]

  • Effect of Resveratrol on Focal Cerebral Ischemia Reperfusion Injury Via Ameliorating Oxidative Stress in Rats

    REN Jun-wei,YANG Qin(Department of Neurology,The First Affiliated Hospital of Chongqing Medical University,Chongqing Key Laboratory of Ophthalmology,Chongqing 400016,China)

    Objective To investigate the effect of resveratrol(Res) on focal cerebral ischemia reperfusion injury via ameliorating oxidative stress in rats.Methods SD rats were divided into sham operation(S),ischemia / reperfusion [I / R,i.e.rat model of cerebral artery occlusion(MCAO) copied by intraluminal thread] as well as Res at low(I / R + R1,15 mg / kg) and high(I / R + R2,30 mg / kg) dosage groups,of which the lesions of neurological function were scored 2 h after ischemia and 24 h after reperfusion separately,while determined for malondialdehyde(MDA) content and superoxide dismutase(SDO) activity in serum and brain tissue by chemical colorimetry,for infarct volume by TTC method,for moisture content in brain tissue by dry and wet weight method,and for pathological change of brain tissue by HE staining.Results Res improved the neurological deficit caused by cerebral ischemia reperfusion injury(P < 0.01),decreased the MDA content(P < 0.01) and increased the SDS activity in serum and brain tissue(P < 0.01),while decreased the infarct volume(P < 0.01) and the moisture content in brain tissue(P < 0.01),and improved the pathological changes of brain tissue,each in a dose-dependent pattern.Conclusion Res showed good protective effect on the cerebral ischemia reperfusion injury via ameliorating oxidative stress by a potential mechanism which might be associated with the clearance of free radicals and the relief of oxidative damage.

    2011 03 v.24 [Abstract][OnlineView][Download 260K]

  • Expression of Human Lysozyme-Xylanase Fusion Gene in Pichia pastoris

    GAO Jin-hu,TANG Cun-duo,WU Min-chen(School of Medicine and Pharmaceutics,Jiangnan University,Wuxi 214122,Jiangsu Province,China)

    Objective To express human lysozyme(hLY) and xylanase(XynⅡ) fusion protein in Pichia pastoris.Methods hLY gene and XynⅡgene were linked by PCR,in which a site recognized by enterokinase was introduced,then cloned into vector pPIC9K.The constructed recombinant plasmid pPIC9K-XynⅡ-EKsite-hLY was linearized with SacⅠ,then transformed to P.pastoris strain GS115 by electroporation.Multi-copy transformants were screened with G418 identified by PCR,and the positive clones were induced with methanol.The expressed fusion protein as well as that digested with enterokinase were determined for activities of hLY and XynⅡ by improved Shugar and DNS methods respectively.Results The sequence of the obtained fusion gene was completely consistent with theoretical sequence.The hLY and XynⅡactivities of the fusion protein were 170 and 158 U / ml respectively.However,after digestion with enterokinase,the activities of hLY and XynⅡwere 520 and 244 U / ml respectively.Conclusion XynⅡ-EKsite-hLY fusion gene was successfully constructed and expressed in P.pastoris,and the activity of target protein increased significantly after digestion with enterokinase.

    2011 03 v.24 [Abstract][OnlineView][Download 307K]

  • Effect of Angiopoietin-2 on Proliferation of Human Colon Cancer SW1116 Cells and Relevant Mechanism

    ZHANG Ji-hong△,WEN Chun-yang,YIN Li,LI Xiang-jun,LI Xi-ning,REN Li-qun(△School of Pharmacy,Jilin University,Changchun 130021,China)

    Objective To investigate the effect of angiopoietin-2(Ang-2) on proliferation of human colon cancer SW1116 cells as well as the relevant mechanism.Methods SW1116 cells were treated with Ang-2 at various concentrations and determined for proliferation level by MTT method.The cells were divided into normal control,serum-free DMEM,Ang-2(1.2 mg / L) and PI3K / Akt inhibitor LY294002(10 μmol / L) + Ang-2 groups,then determined for proliferation level by MTT method,and for expressions of Tie-2,PI3K and Akt proteins by Western blot 24 h after treatment.Results Compared with that at other concentrations,1.2 mg / L Ang-2 influenced the proliferation of SW1116 cells significantly.LY294002 inhibited the proliferation of SW1116 cells caused by Ang-2 effectively.Compared with those in serum-free DMEM group,the expression level of Tie-2 increased slightly(P > 0.05),and that of Akt increased significantly(P < 0.01),while that of PI3K decreased significantly(P < 0.01).However,all the expression levels of the proteins were significantly lower in LY294002 + Ang-2 group than in Ang-2 group(P < 0.01).Conclusion Ang-2 promoted the proliferation of SW1116 cells,which can be inhibited by LY294002 by a possible mechanism associated with the signal pathway regulated by Tie-2 / PI3′-kinase / Akt.

    2011 03 v.24 [Abstract][OnlineView][Download 217K]

  • Effect of Panax Ginsenoside on Immunological Function of Immuno-suppressive Mice

    ZHAO Yun-li△,WU Hua-zhang,YANG Jing,ZHANG Jie,ZHOU Chun-xian(△Department of Prevention,BengBu Medical Collage,BengBu 233030,Anhui Province,China)

    Objective To investigate the effect of Panax Ginsenoside on immunological function of immune-suppressive mice.Methods Mice were administrated with Panax Ginsenoside at various concentrations [0.12,1.20 and 12 g /(kg·d)] by gavage respectively,once a day for 21 d,and injected i.p.with cyclophosphamide at a dosage of 0.1 g /(kg·d) on days 10 ～ 14,then counted for platelet(PLT),white blood cell(WBC) and red blood cell(RBC) in sera,determined for the content of hemoglobin(Hb) and the weights of immune organs.The mice were also determined for phagocytic function of celiac macrophages by chick erythrocyte phagocytosis test and subjected to DTH skin test with 2,4-D.The proliferative activities of celiac macrophages and splenic lymphocytes,the nitric oxide(NO) content in celiac macrophages and the splenic lymphocyte transformation function of the mice were determined in vitro.Results Panax Ginsenoside recovered the decreases of PLT,WBC,RBC and Hb in sera as well as the weights of immune organs,relieved the DTH caused by 2,4-D,promoted the metabolism,enhanced the phagocytic function and induced the NO production of celiac macrophages,and increased the splenic lymphocyte transformation rate and index.Conclusion Panax Ginsenoside enhanced the phagocytic function of macrophages as well as cellular and humoral immunities of immune-suppressive mice.

    2011 03 v.24 [Abstract][OnlineView][Download 234K]

  • Effect of RNA PolⅡ Driven ALU Transcripts on Apoptosis of HEK293 Cells

    LI Xuan△,GAO Jian,YANG Mei,HU Wen-yan,TIAN Lv,PENG Pai-lan,WANG Feng,GAO Chang-yi,REN Hong,TANG Kai-fu(△Department of Digestive Medicine,The Second Affiliated Hospital of Chongqing Medical University,Chongqing 400010,China)

    Objective To investigate the effect of RNA PolⅡ driven ALU transcripts on the apoptosis of human embryonic kidney 293(HEK293) cells as well as the role of IFN in this mechanism.Methods The HEK293 cells at logarithmic growth phase were divided into six groups.The cells in ALU-293,pcDNA3.1-293,Poly I ∶ C-293 and HBs-293 groups were transiently transfected with recombinant plasmid pcDNA3.1-ALU,empty vector pcDNA3.1(-)(negative control),Poly I ∶ C(positive control),recombinant plasmid pcDNA3.1-HBs respectively,while those in IFNβ-293 group was added with 1.65 × 104 U IFNβ(positive control).However,the cells in blank control group were untreated.The cells in various groups 48 h after transfection were determined for proliferative activity by MTT method,for apoptosis by Cellular DNA Fragmentation ELISA and DNA Ladder,and for IFNβ mRNA level by real-time PCR.Results Transient transfection with recombinant plasmid pcDNA3.1-ALU inhibited the proliferation and promoted the apoptosis of HEK293 cells,and up-regulated the mRNA level in the cells significantly.Conclusion PolⅡ driven ALU transcripts induced the apoptosis of HEK293 cells by activating interferon system.

    2011 03 v.24 [Abstract][OnlineView][Download 206K]

  • Influence of Neoadjuvant Chemoradiotherapy Combined with Biological Therapy on Immunological Function of Patients with Low Rectal Cancer

    WANG Min△,YANG Yan-ming,LIU Lin-lin,ZHANG Yu,JI Fu-jian,YU Jie,FNAG Xue-dong(△General Surgery Hospital,Second Hospital of Jilin University,General Surgery Center of Jilin University,Changchun 130041,China)

    Objective To study the influence of neoadjuvant chemoradiotherapy combined with biological therapy on the cellular immunological function of patients with low rectal cancer before surgery.Methods Fifteen patients with rectal cancer at Dukes C stage were treated by two curse of XELOX protocol then by biological therapy before surgery,and monitored for the percentages of T lymphocyte subgroups,NKT cells and NK cells by flow cytometry before neoadjuvant chemoradiotherapy as well as before and after biological therapy to evaluate the short-term curative effect.Results The patients were immunosuppressive before neoadjuvant chemoradiotherapy,of whom the percentages of T lymphocyte subgroups,NKT cells and NK cells decreased significantly after neoadjuvant chemoradiotherapy(P < 0.05).However,after biological therapy,the above-mentioned percentages increased significantly(P < 0.05),while the clinical symptoms were improved,and the effective rate of therapy and remission rate of tumor were 20.0% and 66.7% respectively.Conclusion Neoadjuvant chemoradiotherapy decreased the immunologic function of patients with rectal cancer.However,the biological therapy before surgery enhanced the cellular immune function.Neoadjuvant chemoradiotherapy combined with biological therapy is of an important significance in improving the immune status of patients,shortening the time for hospitalization and decreasing the relapse rate.

    2011 03 v.24 [Abstract][OnlineView][Download 181K]

  • Construction and Immune Effect of DNA Vaccines Encoding Various Forms of Hepatitis B Virus Core Antigen

    SHEN Lin,CHEN Dan,ZHANG Xiao-xi,SUN Zhi-dan,YUAN Jing-yun,WANG Wei-long,LIU Xin-ying,LI Ding-feng,LIU Yong(National Engineering Research Center for Viral Biotechnology,Beijing 100176,China)

    Objective To compare the humoral and cellular immune effects of three kinds of DNA vaccines encoding various forms(truncated,full-length and full-length fusion) of hepatitis B virus core antigen(HBcAg) in mice to optimize the protocol for design of immunogen.Methods Three forms of HBcAg DNA vaccines with optimized codons,i.e.pRec2.0-Ct expressing truncated core protein consisting of 144 aa,pRec2.0-C expressing full-length core protein consisting of 183 aa and pRec2.0-C-preS1 expressing full-length core and preS1 fusion protein,were constructed and transfected to 293T cells,and the expression of target antigen was determined by Western blot.BALB/c mice were immunized with the three forms of DNA vaccines respectively and determined for serum anti-HBc by ELISA,and for HBcAg-specific T cell response by IFNγ ELISPOT method.Results The three forms of DNA vaccines were constructed correctly as proved by restriction analysis and sequencing,and the corresponding proteins were expressed in 293T cells.All the vaccines induced HBcAg-specific antibody and T cell immune response in mice.However,the anti-HBc and T cell immune response levels induced by pRec2.0-C were significantly higher than those by pRec2.0-C-preS1 and pRec2.0-Ct(P < 0.05).Conclusion Truncation of HBcAg to a length of 144 aa or fusion with preS1 decreased the induced humoral and cellular immune response levels.

    2011 03 v.24 [Abstract][OnlineView][Download 227K]

  • Development of A Method for Quality Control of PEGylated Recombinant Human Endostatin

    LI Yong-hong,RAO Chun-ming,WANG Lan,HAN Chun-mei,TAO Lei,WANG Jun-zhi(National Institute for the Control of Pharmaceutical and Biological Products,Beijing 100050,China)

    Objective To develop a method for quality control of PEGylated recombinant human endostatin(PEG-ES).Methods Test samples were determined for biological activity by fluorimetric endothelial cell migration assay and for purity and content by RP-HPLC.Peptide mapping was carried out by trypsin digestion,and other tests according to the requirements in Chinese Pharmacopoeia(Volumes Ⅱ and Ⅲ,2005 edition).Results The specific activities of three batches of bulks of PEG-ES determined by the developed method were 88.4,56.5 and 89.6 U / mg,while the activities of final products were 81%,92% and 151% of stated amount,respectively.The protein contents of three batches of final products determined by RP-HPLC were 101.0%,97.0% and 98.1% of stated amount respectively.All the purities of three batches of bulks determined by RP-HPLC and SDS-PAGE were more than 99.9%.All the results of peptide mapping were consistent with those of control,and all the other quality indexes met the requirements in Chinese Pharmacopoeia.Conclusion The developed method laid a foundation of effective quality control of PEG-ES.

    2011 03 v.24 [Abstract][OnlineView][Download 324K]

  • Inhibitory Effect of Amlodipine on Spontaneous Pulmonary Metastasis of Murine Melanoma B16 Cells and Relevant Mechanism

    ZHANG Xu△,LIAO Yi-lan,SUN Yang,SUN Wen-juan(△Key Laboratory of Biochemistry and Molecular Pharmacollage of Chongqing,Department of Pharmaceutical College,Chongqing Medial University,Chongqing 400016,China)

    Objective To investigate the inhibitory effect of amlodipine on spontaneous pulmonary metastasis of murine melanoma B16 cells as well as the relevant mechanism.Methods C57BL / 6J mice were inoculated s.c.with murine melanoma B16 cells in right inguen,2 × 106 cells for each,and divided into negative control,positive control as well as amlodipine high,moderate and low dosage groups the next day.The mice in negative control group were inoculated with physiological saline by oral route,0.2 ml for each,once a day for 21 d,while those in amlodipine low,moderate and high dosage groups with amlodipine at dosages of 1,3 and 10 mg / kg respectively.However,the mice in positive control group were injected i.p.with 20 mg / kg cyclophosphamide,once 2 days for 7 times.The tumor formations as well as activities of mice in various groups were observed.The mice were killed on the second day after the last inoculation,and observed for pulmonary metastasis of B16 cells,based on which the tumor inhibition rate was calculated.The platelet aggregation and adherence abilities before and after inoculation were compared by platelet aggregation test and adherence test in vitro respectively.Results Amlodipine showed dose-dependent inhibitory effect on the in vivo proliferation and spontaneous pulmonary metastasis of B16 cells,the platelet aggregation induced by B16 cells as well as the adherence of platelet to B16 cells.Conclusion Amlodipine inhibited the in vivo proliferation and spontaneous pulmonary metastasis of B16 cells by a potential mechanism which might be associated with inhibiting the tumor cells-induced platelet aggregation and the adherence of tumor cells to platelet.

    2011 03 v.24 [Abstract][OnlineView][Download 504K]

  • Effect of Indomethacin on Expression of Aquaporin 8 in Human Amniotic Epithelial Cells

    DUAN Zhao-ning,QI Hong-bo,Luo Xin(The First Affiliated Hospital of Chongqing Medical University,Chongqing 400016,China)

    Objective To investigate the effect of indomethacin on expression of aquaporin 8(AQP 8) in human amniotic epithelial cells.Methods The primary culture of human amniotic epithelial cells were identified by immuocytochemistry,then treated with indomethacin at various concentrations(10,20,50,100 and 200 μmol / L)for 24 h respectively,using the cells in normal culture as control.On the basis of this,another group of human amniotic membrane cells were treated with 200 μmol / L indomethacin for 6,12,24,36 and 48 h respectively.The expressions of AQP 8 mRNA and protein in amniotic membrane cells of various groups were determined by RT-PCR and Western blot respectively.Results Except those at a concentration of 10 μmol / L,the indomethacin at other concentrations down-regulated the expressions of AQP 8 mRNA and protein significantly(P < 0.01),while the expression levels in the cells treated with 200 μmol / L indomethacin was the lowest.The expression levels of AQP 8 mRNA and protein in the cells treated with 200 μmol / L indomethacin for more than 12 h were significantly lower than those in control groups(P <0.01),which decreased in a time-dependent mode and reached the minimum 48 h after treatment.Conclusion Indomethacin down-regulated the expression of AQP 8 mRNA and protein in human amniotic epithelial cells.

    2011 03 v.24 [Abstract][OnlineView][Download 256K]

  • Effect of Ulinastatin and Taxotere on Proliferation and Invasion of Human Breast Cancer Line MDA-MB-231 Cells and Relevant Mechanism

    ZHAO Xiao-liang,SUN Zhi-jun,LUO jie,GAO Feng(Department of General Surgery,The Second Affiliated Hospital of Chongqing Medical University,Chongqing 400016,China)

    Objective To observe the effect of ulinastatin(ULI)and taxotere(TXT) on proliferation and invasion of human breast cancer MDA-MB-231 cells as well as expressions of IL-6,IL-8 and TNF-α.Methods The estrogen receptor-negative MDA-MB-23l cells cultured in vitro were randomly divided into blank control,ULI(800 U / ml),TXT(3.7 μg / ml) and ULI + TXT groups,and determined for the transcription levels of IL-6,IL-8 and TNF-α mRNAs by fluorescent quantitative RT-PCR,for proliferation ability by MTT method,and for invasion ability by Transwell chamber assay.Results Both TXT and ULI inhibited the expressions of IL-6,IL-8 and TNF-α genes as well as the proliferation and invasion of MDA-MB-231 cells,while the inhibition ability of ULI was lower than that of TXT.However,TXT combined with ULI showed the strongest inhibitory effect in all the four groups.Conclusion ULI inhibited the proliferation and invasion of human breast cancer MDA-MB-231 cells by a mechanism which might be associated with the down-regulation of expressions of IL-6,IL-8 and TNF-α genes.

    2011 03 v.24 [Abstract][OnlineView][Download 202K]

  • Preparation of Monoclonal Antibodies against Equine Influenza Virus Subtype H7N7

    XIAO Cheng-rui,SONG Zhan-yun,LIU Yang,WANG Wei-li,MENG Qing-feng,MENG Ri-zeng(Jilin Entry-Exit Inspection and Quarantine Bureau,Changchun 130062,China)

    Objective To prepare the monoclonal antibodies(mAbs) against equine influenza virus(EIV) subtype H7N7 and develop a specific,sensitive and simple method for determination of the virus.Methods BALB / c mice were immunized with EIV subtype H7N7,of which splenocytes were fused with myeloma SP2 / 0 cells.The hybridoma cell strains stably secreting the mAbs against EIV subtype H7N7 were screened by hemagglutination inhibition(HI) test and indirect ELISA,and the biological characteristics of the secreted mAbs were identified.Results Three hybridoma cell strains stably secreting mAbs against EIV subtype H7N7 were screened,named as 5B2,1C10 and 2B7 respectively.The mAbs secreted by 5B2 and 1C10 cell strains were IgG2a,while that by 2B7 cell strain was IgG M,of which all the light chains were κ chains.All the mAbs reacted specifically with EIV subtype H7N7,while showed no cross reaction with EIV subtype H3N8,equine arteritis virus(EAV),equine infectious anemia virus(EIAV) or equine encephalitis virus(JEV),indicating high specificity.Conclusion Three kinds of mAbs specific to EIV subtype H7N7 were prepared,which provided good materials for rapid diagnosis and epidemiological investigation of EIV subtype H7N7.

    2011 03 v.24 [Abstract][OnlineView][Download 163K]

  • Analysis on The First Hepatitis B Vaccination for Newborns in Maternity Wards in Hongkou District of Shanghai City,China during 2001～2008

    WU Zhi-fang,QIAN Xiao-hua,ZHAO Dai-jun,LIN Ke(The Center for Disease Control and Prevention in Houkou District,Shanghai City,Shanghai 200082,China)

    Objective To analyze the first hepatitis B(HB) vaccination for newborns in maternity wards in Hongkou District of Shanghai City during 2001 ～ 2008,and provide a reference for further improvement of immune strategy of HB in newborns.Methods The HB vaccination rate,in time vaccination rate and cause for delayed vaccination,as well as the status of vaccination of newborns from HBsAg-positive mothers all the maternity wards in Hongkou District of Shanghai City during 2001 ～ 2008 were investigated and analyzed.Results Both the first vaccination rate and in time vaccination rate of HB vaccine in each year were more than 96%,which showed significant difference in domestic newborns and those from other places(P < 0.05).However,either vaccination rate or in time vaccination rate of newborns from HBsAg-positive and negative mothers showed no significant difference(P > 0.05).The first cause(84.49%) for delayed vaccination was the bodyweight(less than 2 500 g) of newborn,of which 46.69% were not less than 2 300 g.Conclusion We suggest that the bodyweight of not less than 2 300 g of a newborn should be included in the indication of HB vaccination so as to increase the vaccination rate and in time vaccination rate.

    2011 03 v.24 [Abstract][OnlineView][Download 169K]

  • Establishment of Guinea Pig Model of Influenza Virus Infection

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

    Objective To preliminarily establish the guinea pig model of influenza virus infection.Methods Guinea pigs were infected i.p.with influenza virus strains A / Wisconsin / 67 / 2005(H3N2),A / Hiroshima / 52 / 2005(H1N1) and B / Malaysia / 2506 / 2004 and observed for clinical symptoms.Nasal washes and lung tissues were collected on days 1,3,5,7,9,11 and 13 after infection and determined for virus titer by cell culture method.Results Guinea pigs were infected with all the three types of influenza virus.The virus titers in nasal cavity and lung tissue reached peak values 3 d after infection,then decreased gradually until disappeared.Conclusion Guinea pigs were susceptible to influenza virus types H3N2,H1N1 and B.

    2011 03 v.24 [Abstract][OnlineView][Download 179K]

  • Improvement of Production Procedure for Inactivated Vaccine against Aeromonas hydrophila J-1 Strain

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

    Objective To improve the production procedure for inactivated vaccine against Aeromonas hydrophila J-1 strain and increase the culture efficiency.Methods J-1 strain was cultured in modified and toxin-producing media by mechanical stirring under ventilating condition,based on which the time for fermentation was determined,and the culture effets of the two media were compared.The culture liquid of J-1 strain was inactivated in shake-flask and in inactivation tank respectively,and the inactivation effects by formaldehyde at various concentrations were compared.Results The growth level of J-1 strain reached a peak value 12 ～ 24 h after culture in fermentation tank by mechanical stirring.The mean viable count in culture liquid in modified medium was 216 × 108 CFU / ml,which increased by 111.7% as compared with that in toxin-producing medium(102 × 108 CFU / ml),and reached 300 × 108 CFU / ml at most.However,the virulence of culture liquid in modified medium was slightly higher than that in toxin-producing medium.The treatment with 0.3% formalin at 37℃ for 24 h inactivated the culture liquid completely,while the residual formalin content in prepared inactivated vaccine met the relevant quality standard.Conclusion Modified medium was superior to toxin-producing medium in culture of J-1 strain,and the time for culture of A.hydrophia by mechanical stirring in fermentation tank was only a half of that by original procedure.

    2011 03 v.24 [Abstract][OnlineView][Download 165K]

  • Role of Hepatitis C Virus F Protein in Virus Replication and Pathogenesis

    WANG Wen-bo,REN Hao(Department of Microbiology,The Second Military Medical University,Key Laboratory of Biodetection and Biodefense of PLA,Shanghai Key Laboratory of Medical Biodefense,Shanghai 200433,China)

    Hepatitis C virus(HCV) is an enveloped positive-strand RNA virus of the Flaviviridae family,of which the genome consists of about 9 600 nucleotides encoding a polyprotein(about 3 000 amino acids) that is processed by cellular and viral proteases into at least ten structural and nonstructural viral proteins.In recent years,a novel HCV protein has been identified by several laboratories,known as the reading frame shift protein(alternative reading frame protein,ARFP) or F protein(frameshift protein),also called as Core+1 protein.F protein gene is generated from the core reading frame shift.Although F protein has been discovered for more than 10 years,its function remains unclear,and its role in genesis and progress of liver diseases and hepatocellular carcinoma(HCC) as well as its effect on replication and infection of HCV are still in dispute.This review aims to summary the discovery,mechanism,antigenicity,biochemical characteristics and biological function of F protein.

    2011 03 v.24 [Abstract][OnlineView][Download 194K]

  • Progress in Research on Construction Strategy of Virosomal Gene Transfer Vector

    TONG Jia-bei,LU Yan-qin,HAN Jin-xiang(Laboratory of Shandong Medicinal Biotechnology Centre,Key Laboratory for Biotech Drugs Ministry of Health,Key Laboratory for Modern Medicine and Technology of Shandong Province,Jinan 250062,China)

    Virosomes are unilamellar phospholipid bilayer vesicles with viral envelope protein in them,which can be used as vectors for gene transfer and antigen-presenting.Since they are free from virus genome,and their membrane protein fractions are variable,virosmoes as gene vectors have no potential pathogenicity,and their targeting can be changed manually.At present,influenza and Sendai virosomes are paid more attention.This paper reviews the progress in research on construction strategy of virosomal gene transfer vector and provides a basis for further development and improvement of virosomes for gene transfer.

    2011 03 v.24 [Abstract][OnlineView][Download 160K]

  • Progress in Research on 10-23 DNAzyme

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

    10-23 DNAzyme(10-23 DRz) are a set of single stranded DNA segments which derived from a combinatorial library of sequences by molecular technique in vitro.With high catalytical activity and sequence specificity against the target RNA,10-23 DRz can be designed to cleave any target RNA in a sequence specific manner,and inhibit the expression of corresponding proteins.In this paper,the progress in research on 10-23 DRz,including the structure,influencing factors of activity,mechanism,biological characters and application,are reviewed.

    2011 03 v.24 [Abstract][OnlineView][Download 228K]

  • Progress in Research on Novel Virulence-related Gene of Streptococcus suis Serotype 2

    LI Peng,HE Yong-ju,FENG Shu-zhang(Institute of Military Veterinary Medicine,Academy of Military Medical Sciences,Changchun 130062,China)

    Streptococcus suis serotype 2(S.suis 2) is an important pathogen of zoonoses.However,the role of virulence factor in pathogenesis of S.suis 2 is still uncertain.More and more attentions are paid in investigating other virulence-related genes in recent years.This paper reviews the progress in research on novel virulence-related gene of S.suis 2.

    2011 03 v.24 [Abstract][OnlineView][Download 219K]

  • Progress in Research on Proteomics of Campylobacter jejuni

    ZHU Ling-wei,GUO Xue-jun(Key Laboratory of Jilin Province for Zoonosis Prevention and Control,Institute of Military Veterinary,Academy of Military Medical Science,Changchun 130062,China)

    Campylobacter jejuni is popular in the world,which is the leading cause of bacterial gastroenteritis and acute diarrhea in humans and animals.Proteomics encompasses the global analysis of proteins at the organism level.The technologies included under this term have now started to be utilized for understanding how Campylobacter species respond to changes in the environment,with an emphasis on the human host,as well as to map subcellular locations of proteins.This paper reviews the progress in research on proteomics of Campylobacter jejuni.

    2011 03 v.24 [Abstract][OnlineView][Download 160K]


@2012 Editorial Department of "Chinese Journal of Biologicals"