2010年07期目次

  • Prokaryotic Expression,Purification and Bioactivity of Recombinant Mouse Interleukin 17F/His Fusion Protein

    CHEN Ling,GUO Sheng,HAO Chun-li,WU Liang-xia,ZHANG Jian-hua(Department of Pediatrics,Shanghai Sixth People's Hospital Affiliated to Shanghai Jiaotong University,Shanghai 200233,China)

    Objective To express mouse interleukin 17F(mIL-17)/His fusion protein in E.coli,purified the expressed product and analyze its bioactivity.Methods The mIL-17f gene was amplified by RT-PCR and inserted into prokaryotic expression vector pET28a.The constructed recombinant plasmid pET28a /mIL-17f was transformed to E.coli BL21(DE3)was transformed to E.coli BL21(DE3)for expression under induction of IPTG.The expressed rmIL-17F /His fusion protein was purified by Ni2+-NTA affinity chromatography and identified for reactogenicity.BALB/c mice were immunized with the purified and re-naturalized rmIL-17 /His fusion protein by nasal drip,then determined for expression of IL-6 mRNA in lung tissue by real-time fluorescent quantitative PCR,and for IL-17F,IL-4 and IFNγ levels in peripheral blood by ELISA.Results The DNA sequence of amplified mIL-17f gene was consistent with that reported in GenBank.Recombinant plasmid pET28a /mIL-17f was constructed correctly.The expressed recombinant protein,with a relative molecular mass of about 19 000,mainly existed in a form of inclusion body and contained about 30% of total somatic protein.The purified fusion protein reached a purity of about 95% and showed good reactogenicity.The mucosal immunization with the purified fusion protein promoted the expression of IL-6 mRNA in lung tissues and increased the IL-4 and IFNγ levels in sera of mice.Conclusion The rmIL-17F with bioactivity was highly expressed in E.coli and purified,which laid a foundation of further study on the function of IL-17F.

    2010 07 v.23 [Abstract][OnlineView][Download 356K]

  • Difference in Expressions of CENP-E Variant in Various Tumor Cell Strains and Carcinoma Tissues

    YAN Chen△,LING Kang,LIU Zi-jie,WENG Ya-guang(△The Key Laboratory of Laboratory Medical Diagnostics,Department of Medical Laboratory,Chongqing Medical University,Chongqing 400016,China)

    Objective To investigate the expressions of CENP-EⅠ,a CENP-E variant,in various tumor cell strains as well as peri-cancerous and carcinoma tissues.Methods The expressions of mRNA of CENP-EⅠ,a CENP-E variant with deletion of the 38th exon,in 14 cell strains as well as 17 carcinoma tissue specimens and their corresponding peri-cancerous tissue specimens were determined by nested RT-PCR,and compared with those of wild type CENP-E(CENP-EWT).The variations of CENP-E at DNA level in the above-mentioned specimens were determined by nested PCR.The relationship of CENP-EⅠ to the malignant degree of cells were analyzed by cell proliferation test,determination of cell cycle and identification of chromosome karyotype.Results RT-PCR proved that both CENP-EWT and CENP-EⅠexisted in the same tissue or cell strain.CENP-EWT was highly expressed in normal cell strains and peri-cancerous tissues,while CENP-EⅠ in tumor cell strains and carcinoma tissues,which showed significant difference(P < 0.05).No deletion of the 38th exon at DNA level was observed in the specimens,and the expression levels of CENP-E in various cells showed no significant difference.However,CENP-EⅠ showed a certain relationship to the malignant degree of cells.Conclusion The CENP-E mRNA in not less than two forms may exist in the same cell strain,and CENP-E variant may be related to tumorigenesis.

    2010 07 v.23 [Abstract][OnlineView][Download 524K]

  • Establishment of C1R-AAD Cell Clone Stably Expressing HLA-A2 Restricted Multi-epitopes Gene of Hepatitis C Virus

    WEI San-hua,ZHANG Hai,DONG Ke,LIN Fang,WANG Xi,LI Bin,SHEN Jian-jun,ZHANG Li-jun,ZHANG Hui-zhong(Department of Clinical Laboratory and Blood Transfusion,The Fourth Military Medical University,Xi'an 710038,China)

    Objective To establish a C1R-AAD cell clone stably expressing HLA-A2 restricted multi-epitopes gene of hepatitis C virus(HCV).Methods Human ubiquitin(Ub)and HCV HLA-A2 restricted multi-epitopes(Mep)genes were synthesized and cloned into prokaryotic expression vector pRSET-A.The constructed recombinant plasmid pRSET-Ub-Mep was digested with BamHⅠ and Hind Ⅲ,and the obtained complex multi-epitopes gene Ub-Mep was subcloned into eukaryotic expression vector pcDNA 3.1()-.The constructed recombinant plasmid pcDNA 3.1(-)-Ub-Mep was transfected to C1R-AAD cells,based on which the cell clone stably expressing Ub-Mep protein were obtained by G418 pressure screening and limiting dilution method.The transcription of Ub-Mep gene in stably transfected cells was determined by RT-PCR,and the expression of Ub-Mep protein by IFA and Western blot.Results Both restriction analysis and sequencing proved that recombinant plasmidpcDNA3.1(-)-Ub-Mepwasconstructed correctly.The expressions of Ub-Mep at mRNA and protein levels were proved in the C1R-AAD cells stably transfected with the recombinant plasmid.Conclusion A C1R-AAD cell clone stably expressing HLA-A2 restricted multi-epitopes gene of HCV was established,which provided target cells for further study on cellular immune response induced by HLA-A2 restricted multi-epitopes gene.

    2010 07 v.23 [Abstract][OnlineView][Download 229K]

  • Effect of Kozak Sequence on Expression of Gene Encoding Tissue Inhibitor 1 of Metalloproteinase in Human Breast Cancer MCF-7 Cells

    MA Yan-jiao △,LI Peng,ZHOU Hai-feng,RUAN Nan,GONG Peng-tao,LI Jian-hua,OUYANG Hong-sheng,ZHANG Xi-chen(△College of Animal Science and Technology,Heilongjiang August First Land Reclamation University,Daqing 163319,Heilongjiang Province,China)

    Objective To investigate the effect of Kozak sequence on the expression of gene encoding tissue inhibitor 1 of metalloproteinase(TIMP1) in human breast cancer MCF-7 cells.Methods Recombinant plasmids pcDNA3.1-TIMP1 and pcDNA3.1-TIMP1-K containing Kozak sequence were constructed then transfected to MCF-7 cells using Fugene HD transfection agent,and their expressions were determined by fluorescent quantitative PCR and Western blot.Results PCR,restriction analysis and sequencing proved that recombinant plasmids pcDNA3.1-TIMP1 and pcDNA3.1-TIMP1-K were constructed correctly.The transcription level of TIMP1 mRNA and the expression level of TIMP1 protein in the MCF-7 cells transfected with pcDNA3.1-TIMP1-K increased by about 0.95 and 0.43 folds,while those in the MCF-7 cells transfected with pcDNA3.1-TIMP1 increased by 0.37 and 0.25 folds,respectively,as compared with those in blank control cells.Conclusion The expressions of recombinant plasmids pcDNA3.1-TIMP1 and pcDNA3.1-TIMP1-K in MCF-7 cells were significantly different at both mRNA and protein levels,indicating that Kozak sequence enhanced the expression of TIMP1 gene.

    2010 07 v.23 [Abstract][OnlineView][Download 355K]

  • Inhibitory Effect of L-Arginine on Cardiomyocyte Hypertrophy Induced by High Glucose and Insulin

    QIU Hong-mei△,WANG Ming-feng,WU Qin,JIANG Qing-song(△Department of Pharmacology,Chongqing Key Laboratory of Biochemistry and Molecular Pharmacology,Chonqing Medical University,Chongqing 400016,China)

    Objective To investigate the inhibitory effect of L-arginine(L-Arg)on the cardiomyocyte hypertrophy induced by high glucose and insulin(HGI).Methods The cardiomyocytes of suckling mice were cultured in vitro and divided into normal control,model(HGI),L-Arg at low,moderate and high dosages and L-Arg + L-NAME groups,and treated with 5.5 mmol /L glucose,25.5 mmol /L glucose + 0.1 μmol /L insulin,25.5 mmol /L glucose + 0.1 μmol /L insulin + 0.01,0.1 and 1.0 mmol /L L-Arg and 25.5 mmol /L glucose + 0.1 μmol /L insulin + 0.1 mmol /L L-Arg + L-NAME [a nitric oxide synthase(NOS)-specific inhibitor]respectively.The effect of L-Arg on cardiomyocyte hypertrophy was observed using cell surface area,protein content and atrial natriuretic factor(ANF)mRNA expression as indicators.The expressions of mRNAs of endothelial NOS(eNOS),and inducible NOS(iNOS)were determined by real-time PCR.The NOS activity and NO content in cell culture were determined by colorimetry and nitrate reduction method respectively.Results L-Arg showed a dosage-dependent inhibitory effect on the cardiomyocyte hypertrophy induced by HGI,while up-regulated the expression of eNOS mRNA and NOS activity and increased the NO content.However,L-NAME blocked all the effects of L-Arg completely.Conclusion L-Arg inhibited the HGI-induced cardiomyocyte hypertrophy by activating the expression of eNOS and promoting the release of NO.

    2010 07 v.23 [Abstract][OnlineView][Download 265K]

  • Relationship of Hypoxia-inducible Factor-1α and Invasion of Gastric Cancer Cells in Hypoxia

    DENG Ting,ZHANG Jun-wen(Department of Gastroenterology,The First Affiliated Hospital,Chongqing Medical University,Chongqing 400016,China)

    Objective To investigate the expression of hypoxia-inducible factor-1α(HIF-1α)and its relationship to the adhesion,migration and invasion abilities of human gastric cancer cells in hypoxia and HIF-1α RNA interference(RNAi).Methods Gastric cancer SGC-7901 cells were transfected with eukaryotic expression vector pGPU6 /GFP /Neo-HIF-1α-2484 to construct a stably transfected SGC-7901 clones of HIF-1α RNA.The effects of hypoxia and HIF-1α RNAi on expression of HIF-1α in SGC-7901 were determined by Western blot,on adhesion ability of SGC-7901 cells by MTT method,and on migration and invasion abilities by Boyden Chamber membrane invasion system.Results HIF-1α was expressed in SGC-7901 cells 4,8 and 16 h after culture in hypoxia,and its expression level increased significantly with the increasing hours for culture.The adhesion rate as well as the counts of migrated and transmembrane invaded SGC-7901 cells increased significantly with the increasing hours for culture,and showed significantly positive relationship to HIF-1α expression.After HIF-1α RNAi,the expression level of HIF-1α,adhesion rate as well as the counts of migrated and transmembrane invaded hypoxic SGC-7901 cells in hypoxia decreased significantly.Conclusion The overexpression of HIF-1α enhanced the adhesion,migration and invasion abilities of gastric cancer cells,which might be one of the important reasons for local invasion and distant metastasis of gastric cancer.

    2010 07 v.23 [Abstract][OnlineView][Download 243K]

  • Expression of Kidney Injury Molecule-1 in Rat Model of Acute Kidney Injury Induced by Gentamycin

    SHI Yan△,WANG Lu-fei,LUO Ping,SUN Bo,LI Xiang-jun,DONG De-lu,REN Li-qun(△Department of Experimental Pharmacology and Toxicology,School of Pharmacy,Jilin University,Changchun 130021,China)

    Objective To investigate the expression of kidney injury molecule-1(KIM-1)in rat model of acute kidney injury induced by gentamycin(GM).Methods Rat model of acute kidney injury induced by GM was established.Using those of normal rats as control,the biochemical indexes in blood and urine of model rats were determined,the kidney tissue was observed for histopathology,the secretion of KIM-1 in urine was determined by ELISA,the transcription of KIM-1 mRNA in kidney tissue by RTPCR,and the expressions of KIM-1,α-smooth muscle actin(SMA)and vimentin in kidney tissue by immunohistochemical assay with SP staining.Results Compared with those in control group,the biochemical indexes in blood and urine as well as histopathological observation of kidney tissues of model rats indicated pathological changes at various degrees;ELISA showed that the KIM-1 content in urine of model rats increased,and RT-PCR proved that the transcription level of KIM-1 mRNA in kidney tissue of model rats was upregulated(P < 0.05),both in time-dependent modes.Immunohistochemical assay showed significant time-dependent increase of expression level of KIM-1 with the increasing severity of kidney injury.Conclusion KIM-1 may be used as a sensitive and specific marker for early diagnosis of kidney injury induced by GM.

    2010 07 v.23 [Abstract][OnlineView][Download 312K]

  • Spectral Analysis on Conformational Change of Recombinant and Plasma-derived Human Serum Albumins Induced at Various pH Values

    LIU Jun-le,WEI Jing-shuang,CHENG Li-jun,DU Wei-shi,ZHANG Shi-xiong,WANG Zhi-ming,JIA Qian(New Drug R&D Center,North China Pharmaceutical Corporation,Shijiazhuang 050015,China)

    Objective To analyze the conformational change of recombinant human serum albumin(rHSA)and plasma-derived HSA(pHSA)induced at various pH values.Methods The second derivative spectrums as well as fluorescent spectrums at 275 and 295 nm of rHSA and pHSA at various pH values were analyzed by ultraviolet and fluorescent spectrometry respectively.Results Both the maximum absorption wavelengths of rHSA and pHSA were 278 nm.The second derivative and fluorescent spectrums of rHSA and pHSA changed with the pH value in the same modes.No significant differences were observed in the ultraviolet and fluorescent spectrums of rHSA of three batches.Conclusion The conformational change of rHSA at various pH values was basically consistent with that of pHSA,and the conformations of three batches of rHSA showed high consistency.

    2010 07 v.23 [Abstract][OnlineView][Download 334K]

  • Construction of Eukaryotic Expression Vector for Mouse MicroRNA miR-21 and Its Expression in Mouse Mesangial Cells

    ZHANG Zheng,PENG Hui-min,XU Xiao-ming,HE Xiao-yan(Department of Cell Biology and Genetics,Basic Medical College,Chongqing Medical University,Chongqing 400016,China)

    Objective To construct the eukaryotic expression vector for mouse microRNA miR-21 and express in mouse mesangial cells.Methods The miR-21 gene was chemically synthesized and cloned into expression vector pGenesil-1.The constructed recombinant plasmid pGenesil-miR-21 was transfected to mouse mesangial cells in mediation of Lipofectamine 2000,and the stably transfected clones were screened with G418,from which total RNA was extracted for determination of expression of miR-21 by real-time fluorescent quantitative RT-PCR technique.Results Both restriction analysis and sequencing proved that recombinant plasmid pGenesil-miR-21 was constructed correctly.The miR-21 gene was stably and highly expressed in the screened positive clones of mouse mesangial cells transfected with the constructed recombinant plasmid.Conclusion The eukaryotic expression vector for miR-21 was successfully constructed and highly expressed in mouse mesangial cells,which laid a foundation of further investigation of biological function of miR-21.

    2010 07 v.23 [Abstract][OnlineView][Download 190K]

  • Construction and Expression of Trp Operon Gene Mutant of E.coli

    LIN Wei-ping,LIU Xiao-ying,WU Jing-liang,GAO Zhi-qin,SUN Tong-yi(Department of Basic Medicine,Weifang Medical University,Weifang 261042,Shangdong Province,China)

    Objective To construct the Trp operon gene mutant of E.coli and increase the yields of anthranilic acid synthase and tryptophan synthase.Methods The mutant of Trp Operon gene(Trp OperonM)was amplified by DpnⅠ-dependent PCR,based on which recombinant plasmid pET-22b(+)-Trp OperonM was constructed,then identified by restriction analysis and sequencing and transformed to E.coli BL21(DE3)for expression under induction of IPTG.The crude extract of the recombinant E.coli was determined for the activities of anthranilic acid synthase and tryptophan synthase by colorimetry.Results The PCR product showed a Trp OperonM band at a length of about 7 000 bp on electrophoretic profile.Both restriction analysis and sequencing proved that recombinant plasmid pET-22b(+)-Trp OperonM was constructed correctly.As compared with those of empty E.coli BL21(DE3),the anthranilic acid synthase and tryptophan synthase activities of recombinant E.coli BL21(DE3)/pET-22b(+)-Trp OperonM increased by 4.5 and 5.2 folds respectively.Conclusion The Trp operon gene mutant of E.coli was successfully constructed,with increased activities of anthranilic acid synthase and tryptophan synthase,which laid a foundation of construction of recombinant E.coli highly producing tryptophan.

    2010 07 v.23 [Abstract][OnlineView][Download 201K]

  • Effect of β-Hydroxybutyrate on Transcription and Translation Levels of Carnitine Palmityl Transferase-Ⅰ in Bovine Hepatocytes Cultured In Vitro

    XU Chuang△,ZHANG Ri-he,XIA Cheng,ZHANG Hong-you,WANG Zhe(△College of Animal Science and Technology,Heilongjiang August First Land Reclamation University,Daqing 163319,Heilongjiang Province,China)

    Objective To investigate the effect of various concentrations of β-hydroxybutyrate(BHBA)on the transcription and translation levels of carnitine palmityl transferase-Ⅰ(CPT-Ⅰ)in the bovine hepatocytes cultured in vitro.Methods The transcription and translation levels of CPT-Ⅰ gene in bovine hepatocytes cultured in vitro after treatment with various concentrations(0、 0.3、0.6、1.2、2.4 and 4.8 mmol /L)of BHBA were determined by fluorescent quantitative PCR and ELISA respectively.Results Both transcription and translation levels of CPT-Ⅰ gene decreased with the increasing BHBA concentration,while decreased remarkably when the BHBA concentration was more than 1.2 mmol /L.Conclusion The BHBA at a high concentration inhibited the expression of CPT-Ⅰ in bovine hepatocytes cultured in vitro significantly,and might decrease the fatty acid oxidation.

    2010 07 v.23 [Abstract][OnlineView][Download 159K]

  • Activin A Promotes Survival of Fetal Mouse Cortex Neurons Cultured In Vitro

    MENG Xiao-dan△,SUN Yang,YU Fang,LIU Hai-yan,LIU Zhong-hui,GE Jing-yan(△Department of Immunology,Norman Bethune College of Medicine,Jilin University,Changchun 130021,China)

    Objective To investigate the promoting effect of activin A on the survival of fetal mouse cortex neurons cultured in vitro.Methods Seventeen-day-old fetal mouse brain nerve cells were cultured preliminarily,in which the expression of ActRⅡA was observed by immunocytochemical staining.The cells were divided into negative control,positive control and activin A groups,and cultured with 5% fetal bovine serum-DMEM /F12 medium,4 ng /ml nerve growth factor(NGF)and 5 ng /ml activin A respectively,in which the survivals of neurons were determined by trypan blue staining on days 1,3,5,7 and 9 after culture.Results ActRⅡA was expressed in the fetal mouse brain nerve cells cultured in vitro.In negative control group,the surviving neurons decreased in a time-dependent mode,and no surviving neurons were detected on day 9 after culture.However,in positive control and activin A groups,partial neurons still survive on day 9 after culture.Conclusion Activin A may maintain the long-term survival of fetal mouse cortex neurons.

    2010 07 v.23 [Abstract][OnlineView][Download 380K]

  • Separation,Purification and Zymological Property of Fibrinolytic Enzyme UFE-Ⅱ from Urechis unicinctus

    CHU Jin-xin△,CAI Wen-di,HAN Bao-qin,LIU Wan-shun,DU Chang-qing,TAN Yong-lin(△Department of Preclinical Medicine,Weifang Medical College,Weifang 261053,Shandong Province,China)

    Objective To separate and purify fibrinolytic enzyme UFE-Ⅱwith a single component from Urechis unicinctus and analyze its zymological property.Methods UFE-Ⅱ was purified from the coelomic fluid of Urechis unicinctus by centrifugation,ultrafiltration,ion exchange chromatography and gel filtration,determined for purity and relative molecular mass by Native-PAGE,SDS-PAGE and Q-Tof-MS,for enzyme activity with Folin-phenol reagent using casein as a substrate,then analyzed for zymological property.Results The purified UFE-Ⅱ showed activity in hydrolysis of fibrin.The specific activity in hydrolysis of casein,purification fold and recovery rate of the purified UFE-Ⅱ were 375.6 U /mg,10.3 and 14.0% respectively.UFE-Ⅱ was a single component with a purity of more than 99% and a relative molecular mass of 24 329.The optimal reaction temperature and pH value of UFE-Ⅱ were 45℃ and 7.0 respectively.Ca2+,Mn2+ and Fe2+ were strong activators,while Fe3+,Cu2+ and Pb2+ showed certain inhibitory effect on the enzyme activity of UFE-Ⅱ.SBTI and PMSF completely inhibited the activity of UFE-Ⅱ,indicating that UFE-Ⅱ was a serine protease.Chymotrypsin inhibitor showed partial inhibitory effect,while leupeptin,aprotinin and benzamidine showed weak inhibitory effect on the enzyme activity of UFE-Ⅱ.Conclusion UFE-Ⅱ was successfully separated and purified from Urechis unicinctus and analyzed for zymological property,which was worthy of further development.

    2010 07 v.23 [Abstract][OnlineView][Download 305K]

  • Prokaryotic Expression and Preliminary Purification of Bos taurus Lysozyme

    FU Shi-xin,QI Chang-xue,LUO Chun-hai,ZHANG Li,WANG Yao(College of Animal Sciences and Technology,Heilongjiang August First Land Reclamation University,Daqing 163319,Heilongjiang Province,China)

    Objective To express Bos taurus lysozyme(LYZ1)in prokaryotic cells and preliminarily purify the expressed product.Methods The CDS sequence of LYZ1 gene was designed and synthesized,then cloned into expression vector pET-32a.The constructed recombinant plasmid pET-32a-LYZ1 was transformed to E.coli BL21(DE3),and the positive clones were screened for expression under induction of IPTG.The expressed product was preliminarily purified and identified by SDS-PAGE and Western blot.Results PCR,restriction analysis and sequencing proved that recombinant plasmid pET-32a-LYZ1 was constructed correctly.SDSPAGE proved that the expressed protein,with a relative molecular mass of about 32 000,mainly existed in a form of inclusion body,contained more than 70% of total somatic protein and reached a purity of 95% after purification.Western blot showed specific binding of the expressed fusion protein to mouse anti-His Tag McAb.Conclusion LYZ1 was successfully expressed in prokaryotic cells and preliminarily purified,which laid a foundation of further study.

    2010 07 v.23 [Abstract][OnlineView][Download 254K]

  • Immunogenicity of Recombinant Enterotoxigenic E.coli Vaccine in SD Rats

    WANG Rong-rong,HAO Ya-ning,MA Yong-ping,LI Jiang,LI Shi-bin(Medical Molecule and Tumor Research Center,Department of Biochemistry and Molecular Biology,College of Basic Medicine,Chongqing Medical University,Chongqing 400016,China)

    Objective To observe the immunogenicity of recombinant ente rotoxigenic E.col(iETEC)vaccineinSDrats.Methods SD rats were immunized with the recombinant bifidobacterium strain containing heat-labile enterotoxin B subunit(LTB)of ETEC antigen for 4 times by oral route,using those immunized with PBS as control.The IgA in supernatant of feces and IgG in sera of rats were determined by ELISA,the expression of sIgA in jejunum by immunohistochemical assay,and the activity of enterotoxin by intestine hypodesmus test.Results Specific IgA was induced in the supernatant of feces of rats,and IgG in sera,while specific sIgA in enteric tract.Immunohistochemical assay proved that the antibody secretion level increased significantly with the increasing doses of vaccine.The rats immunized with the vaccine were protected against the challenge with LT antigen.However,obvious enteric hydrops were observed in the rats in control group.Conclusion The constructed recombinant ETEC vaccine showed good immune effect,which protected SD rats from ETEC infection.

    2010 07 v.23 [Abstract][OnlineView][Download 268K]

  • Hepatitis B Vaccine Coverage and Hepatitis B Morbidity Trend in 1992～2007 in Guangzhou City,China

    LI Zhi-qun,WANG Ming,XU Jian-xiong,CAI Yan-shan,LIU Jian-hua,CHEN Jian,FENG Xiao-e(The Center of Disease Control and Prevention in Guangzhou,Guangzhou 510080,China)

    Objective To analyze the hepatitis B(HB)vaccine coverage and HB morbidity trend 1992 ~ 2007 in Guangzhou City,China and provide a basis for the strategy of prevention and treatment of HB.Methods The coverage of HB vaccine as well as morbidity of HB in the children born in 1992 ~ 2007 in Guangzhou were investigated.Results The coverages of HB vaccine in 1992 ~ 1994 and 2002 ~ 2007 were 63.14% and 95.72% respectively.A total of 13 699 patients with HB,at ages of 1 ~ 16 years,were reported in 1992 ~ 2007,indicating a morbidity ranged from 26.21 /100 000 in 2007 and 122.74 /100 000 in 1992,which decreased by 78.65%.The morbidities decreased year by year,of which those in the patients at ages of 1 ~ 5,6 ~ 10 and 11 ~ 16 years decreased by 54.82%,75.04% and 83.04% respectively.Compared with those in 1992 ~ 1994 and 1995 ~ 2001,the HBsAg positive rate in 2002 ~ 2007 decreased by 80.80% and 79.13%,while anti-HBs positive rate increased by 8.70% and 13.15%,and anti-HBc positive rate decreased by 23.31% and 4.29%,respectively.Conclusion The popularization of HB vaccine in newborns in Guangzhou City showed satisfactory effect.

    2010 07 v.23 [Abstract][OnlineView][Download 179K]

  • Screening and Identification of Human Anti-bungarotoxin ScFv

    WANG Yu-xiao,ZHOU Li-jun,ZHANG Hai-rong,QU Jia,QIAO Yuan-yuan,ZHAO Xiao-hang,GAO Rong-kai(Cetral Lab,Navy General Hospital of PLA,Beijing 100048,China)

    Objective To screen human anti-bungarotoxin ScFv from large natural phage antibody library then identify.Methods Specific antibody was screened from large natural phage antibody library by a course of "adsorption-elution-amplification" using bungarotoxin as target antigen,then determined for binding activity by ELISA,for neutralizing activity by competitive inhibition ELISA with horse antiserum against bungarotoxin,and typed by DNA fingerprint analysis and sequencing.Results Five positive clones with binding activities to bungarotoxin were obtained after four rounds of panning,of which the highest inhibiting rate to neutralizing activity reached 8.5%.Three different DNA fingerprints were observed.Sequencing result proved that all the heavy chain variable regions of the three ScFv genes belonged to VH Ⅰ,while the light chain variable regions were VκⅡ,VκⅢ and VλⅡ.Conclusion The anti-bungarotoxin ScFv with neutralizing activity was obtained from large natural phage antibody library.

    2010 07 v.23 [Abstract][OnlineView][Download 191K]

  • Preventive and Therapeutic Effects of Polyclonal Antibody on Porcine Reproductive and Respiratory Syndrome

    LIAN Hai△,LIU Zhi-jun,GAO Feng,JIN Hong-tao,WANG Ze,ZHANG Zhong-yang,XIA Zhi-ping(△Key Laboratory of Jilin Province for Zoonosis Prevention and Control,Institute of Military Veterinary Medicine,Academy of Military Medical Sciences,Changchun 130062,China)

    Objective To observe the preventive and therapeutic effects of polyclonal antibody against porcine reproductive and respiratory syndrome virus(PRRSV) on PRRS.Methods Changbai piglets were preliminarily immunized with inactivated PRRSV vaccine and boosted with virulent PRRSV NVDC-JXA1 strain passage C05 to prepare the polyclonal antibody against PRRSV.The prepared polyclonal antibody was used for immunization of piglets,at various dosages,and its prevention and therapeutic effects were evaluated with the survival rate,serum ELISA antibody titer as well as the PRRSV gene in serum,lung and brain tissues.Results The survival rate of piglet immunized with the prepared polyclonal antibody at a dosage of 0.5 ml /kg body weight after challenge with 3 × 105.5 TCID50 of NVDC-JXA1 strain was 5 /7,indicating a certain preventive effect of antibody.The survival rates of piglets challenged with 3 × 105.5 TCID50 of NVDC-JXA1 strain were 5 /7 and 7 /7 after treatment with the prepared polyclonal antibody at dosages of 0.8 and 1.0 ml /kg body weight for 3 times respectively.However,4 /7 and 2 /7 of piglets treated with the antibody at a dosage of 1.0 ml /kg body weight were positive for PRRSV gene in their sera and lungs respectively.Conclusion The polyclonal antibody with an ELISA S /P value of more than 3 at a dosage of more than 0.5 ml /kg body weight showed a certain preventive and therapeutic effects on PRRSV infection in piglets.However,the asymptomatic piglets after immunization or treatment with the prepared antibody might still carry PRRSV.

    2010 07 v.23 [Abstract][OnlineView][Download 165K]

  • Inhibitory Effect of Fusion Protein IFNα2a-α-MSH on Growth of Melanoma in Mice

    WANG Xiao-xia△,ZHANG Ying-qi,YAN Zhen,XUE Xiao-chang,WANG Zeng-lu,ZHANG Yan-guo,YU Chunyan(△Department of Dermatology,Tangdu Hospital,Fourth Military Medical University,Xi'an 710038,China)

    Objective To observe the inhibitory effect of fusion protein IFNα2a-α-MSH(α-melanocyte-stimulating hormone) on the growth of transplanted melanoma in mice.Methods Melanoma cell strain B16 was inoculated s.c.to C57BL /6 mice.After the diameters of tumors were more than 4 mm,the mice were divided into four groups.The mice in group 1~3 were injected i.m.with physiological saline(negative control),IFNα2a(9 × 106 U /kg body weight)and IFNα2a-α-MSH(9 × 106 U /kg body weight)respectively,once a day for 11 d,while those in group 4 were injected i.p.with cyclophosphamid(20 mg /kg body weight,positive control) every other day for 11 d.The sizes of tumors were measured every other day.The mice were killed 24 h after the last injection,and their tumors were weighed,based on which the inhibitory rate to growth of tumors was calculated.Results IFNα2a-α-MSH inhibited the growth of melanoma in mice significantly,and its inhibitory effect was significantly stronger than that of IFNα2a.Conclusion IFNα2a-α-MSH showed strong inhibitory effect on the growth of melanoma in mice,which provided an experimental basis for the clinical therapy of melanoma with IFNα2a-α-MSH.

    2010 07 v.23 [Abstract][OnlineView][Download 201K]

  • Purification of Botulinum Type B Neurotoxin and Effect of Trypsin on Botulinum Neurotoxin

    ZHANG Xue-ping,LI Xiao-juan,HE Xing(Lanzhou Institute of Biological Products,Lanzhou 730046,China)

    Objective To purify botulinum type B neurotoxin,compare the characteristics of inactive and trypsin-activated botulinum type B neurotoxin and analyze the integrities of botulinum type B neurotoxin complex under acidic and basic conditions.Methods Neurotoxins were purified from inactive,trypsin-activated and heat-activated botulinum type B complexes by anion-exchange column chromatography,and served as groups A,B and C respectively.The trypsin-activated botulinum type B neurotoxin was dissolved under acidic condition and served as group D.The botulinum type B neurotoxins in various groups were determined for purities(specific toxicities)and analyzed by SDS-PAGE.Results The purities of neurotoxins after chromatography were higher than those of complexes before chromatography.However,the purities of activated neurotoxins were 2 ~ 3 times higher than those of inactive ones.Both reduced and non-reduced SDS-PAGE profiles of neurotoxins in group A showed three bands,i.e.neurotoxin,heavy chain and light chain,while those in group B showed only two bands,i.e.heavy and light chains.The neurotoxin in group C showed two bands,i.e.heavy and light chains on non-reduced SDS-PAGE profile,while showed only one band of light chain on reduced SDS-PAGE profile.Botulinum type B neurotoxin complex consisted of neurotoxin and non-toxic components under acidic condition.However,under basic condition,the neurotoxin and non-toxic components were separated.Conclusion Botulinum type B neurotoxin was successfully purified,of which the single chain was cleaved into double chain and the specific activity increased after treatment with trypsin.

    2010 07 v.23 [Abstract][OnlineView][Download 365K]

  • Inhibitory Effect of Docetaxel on Proliferation of Human Laryngeal Carcinoma Hep-2 Cells

    SUN Li-li,ZHENG Ying,GAO Hong,WANG Hong,WANG Xiao-chun,WANG Hong-dong,BAI Lan,JIN Hui(Department of Head and Neck Surgery,Tumor Hospital of Jilin Province,Changchun 130001,China)

    Objective To investigatetheinhibitoryeffectofdocetaxel(TXT)ontheproliferationofhuman laryngealcarcinoma Hep-2 cells.Methods Hep-2 cells were treated with TXT at various concentrations for 0,0.5,1,2 and 4 h separately.The proliferative activity of Hep-2 cells was determined by MTT method and compared with those of cells treated with adriamycin(ADM),5fluorouracil(5-Fu)and vincristine(VCR).The effect of TXT on apoptosis of Hep-2 cells was determined by flow cytometry.Results Compared with ADM,5-Fu and VCR,TXT showed high inhibiting activity to Hep-2 cells in dosage-and time-dependent modes at certain ranges.Meanwhile,TXT induced the apoptosis of Hep-2 cells effectively.Conclusion TXT inhibited the proliferation of Hep-2 cells significantly,which might be used as a potential drug for clinical therapy of laryngeal carcinoma.

    2010 07 v.23 [Abstract][OnlineView][Download 150K]

  • Preparation of Diagnostic Kit for Influenza A H1N1 Virus by Real-time Fluorescent PCR

    LI Li△,YANG Xiu-yun,ZOU Shu,ZHANG Xue-mei,LI Xiao-bo,LI Yu-xiang,WU Dong-lin,LI Jing,YANG Wen-chong,DU Li-na,LI Yong(△Changchun Brother Biotech Co.Ltd,Changchun 130062,China)

    Objective To prepare and verify a diagnostic kit for influenza A H1N1 virus by real-time fluorescent PCR.Methods Viral RNA was extracted from the nasopharyngeal swabs of suspected patients with influenza A(H1N1 strain)by magnetic beads-based kit and reversely transcribed to cDNA.The primers and probes specific to the M gene encoding matrix protein were designed according to the gene sequence of influenza A H1N1 virus(2009)published lately in NCBI for detection of influenza A virus,while those specific to HA and NA genes for influenza A H1N1 virus.Meanwhile,the primers and probes specific to human RNase P gene were designed for internal control.All the probes were Taqman probes,of which the 5'-terminus were labeled with FAM and 3'terminus with BHQ1.The reaction condition for real-time fluorescent PCR was optimized,based on which a diagnostic kit for influenza A H1N1 virus by real-time fluorescent PCR was assembled and verified for specificity,sensitivity,precision and stability.The detection results by the prepared kit were compared with those by commercial kit.Sixty-three swabs of suspected patients with influenza A were detected by the prepared kit.Results Influenza A H1N1 virus was accurately detected by the designed PCR primers and probes,and no cross reactions with influenza virus of other types or subtypes were observed.The sensitivity of the prepared kit was 0.004 hemagglutinin units /ml.The intra-and inter-coefficients of variation of detection results by the prepared kit were less than 2.5% and less than 5% respectively.The kit showed high stability at-20℃.The detection results of 20 swabs of suspected patients with influenza A(H1N1)strain by the prepared kit were consistent with those by commercial kit.A total of 63 swabs of suspected patients with influenza A were detected by the prepared kit,of which 36 were positive for pandemic influenza A H1N1 virus and 5 for common influenza A virus.Conclusion The prepared diagnostic kit for influenza A H1N1 virus by real-time fluorescent PCR shows high sensitivity,specificity,precision and stability,which may be used for rapid detection of the current influenza A H1N1 virus.

    2010 07 v.23 [Abstract][OnlineView][Download 217K]

  • Significance of Expressions of BAK and BAG-1 Proteins in Colorectal Carcinoma

    ZHANG Zhao-xia(Department of Pathology,Fenyang College,Shanxi Medical University,Fenyang 032200,Shanxi Province,China)

    Objective To investigate the significance of expressions of BAK and BAG-1 proteins in colorectal carcinoma.Methods The expressions of BAK and BAG-1 proteins in 80 colorectal carcinoma specimens,40 colorectal adenomas specimens and 25 adjacent normal colorectal tissue specimens were determined by immunohistochemical SP method,based on which the relationship between the expressions and clinical pathological characters of colorectal carcinoma was analyzed.Results Both BAK and BAG-1 proteins were over-expressed in colorectal carcinoma,with positive rates of 72.5%(58 /80)and 80%(64 /80)respectively.The expression level of BAG-1 was negatively related,while that of BAK was positively related to the differentiation degree of tumor.The positive rates of BAG-1 in colorectal carcinoma with and without metastasis in lymph node were 96.7%(29 /30)and 70%(35 /50) respectively,which showed significant difference(P < 0.01).Conclusion The overexpressions of BAK and BAG-1 were closely related to the differentiation degree,while that of BAG-1 to the metastasis in lymph node of colorectal carcinoma.The mechanism of colorectal carcinogenesis might be involved in the abnormality of several genes in the regulatory pathway of BAK and BAG-1.

    2010 07 v.23 [Abstract][OnlineView][Download 259K]

  • Determination of Disruption Rate of Recombinant Hansenula polymorpha Cells Expressing HBsAg

    WANG Xi△,YANG Xu-qin,LIU Ying-wei,XU Ning,MA Rui,ZHANG De-you,CHENG Ye,LI Cai-mei,LI Jin(△Beijing Tiantan Biological Products Co.Ltd,Beijing 100024,China)

    Objective To develop a method for rapid determination of disruption rate of recombinant Hansenula polymorpha(HP)cells expressing HBsAg.Methods The disruption rate of recombinant HP cells was determined by microscopic counting and capillary centrifugation after staining,and the protein contents in supernatants of cells before and after disruption were determined by Lowry method to evaluate the disruption rate indirectly,using the recombinant Saccharomyces cerevisiae(SC)cells as control.Results The disruption rate of cells was directly calculated by microscopic counting,and the result was reliable.However,difference was observed between the data determined by different persons.The disruption rate of cells determined by capillary centrifugation was similar to that by microscopic counting,however,the determination result by the former was more stable than that by the latter.The suspension of disrupted HP cells were well stained with Coomassie brilliant blue.The protein release rate determined by Lowry method was corresponding to the disruption rate of cells.Conclusion Microscopic counting,capillary centrifugation and Lowry method were suitable for evaluation of cell disruption rate,however,capillary centrifugation was rapid,simple and practical in actual production process.

    2010 07 v.23 [Abstract][OnlineView][Download 417K]

  • Optimization of Procedure for Activation of Group Y Meningococcal Polysaccharide

    LIU Mei-ying,CHEN Jing,LIU Yu,LI Ye-shan,LIN Hai-tao,ZHANG Yan-bin,WANG Ping,WANG Xue-wei,JIAO Xiao-ling,WANG Yi-ping(National Vaccine & Serum Institute,Beijing 100024,China)

    Objective To optimize the procedure for activation of group Y meningococcal polysaccharide and lay a foundation of preparation of groups A,C,W135 and Y meningococcal combined vaccine.Methods An orthogonal test with three factors at three levels was designed using polysaccharide derivation rate and free polysaccharide content as indicators.Polysaccharide was activated at various pH values for various minutes,with various amounts of activating agent added,then conjugated to TT.The polysaccharide-TT conjugate was purified by Sepharose 4FF chromatography,then subjected to biochemical assay,determined for immunogenicity and observed for stability,based on which the procedure for activation was optimized and verified.Results Using polysaccharide derivation rate as an indicator,pH value and amount of activating agent added showed significant effect on activation of group Y meningococcal polysaccharide(P < 0.05).However,using free polysaccharide content as an indicator,the effects of three factors on activation of the polysaccharide could not be evaluated(P > 0.05).Based on the determination results of various indexes and the stability of polysaccharide-TT conjugate,the procedure for activation of polysaccharide was optimized as follows:each microgram of polysaccharide was added with 1 mg of activating agent,and reacted at pH12 for 10 min.Three batches of group Y meningococcal polysaccharide-TT conjugate were prepared by the optimized procedure,of which all the quality indexes met the routine quality control standard for polysaccharide conjugate vaccine.Conclusion The group Y meningococcal polysaccharide-protein conjugate vaccine with low free polysaccharide content and high immunogenicity was prepared by the optimized polysaccharide activation procedure which showed high reproducibility.

    2010 07 v.23 [Abstract][OnlineView][Download 198K]

  • Development of A PCR Assay for Diagnosis of Toxoplasma gondii Infection Based on Repetitive 529 bp Fragment

    FANG Qiang△,XIA Hui,CHEN Xing-zhi,XU Jia-sen,WANG Xue-mei,QI Wen-juan,SUN Xin,SHEN Ji-long(△Department of Microbiology and Parasitology,Bengbu Medical College,Bengbu 233030,Anhui Province,China)

    Objective To develop a PCR assay for diagnosis of Toxoplasma gondii infection based on repetitive 529 bp fragment.Methods Primers were designed and synthesized according to the repetitive 529 bp fragment of genome of Toxoplasma gondii,based on which the fragment was amplified by PCR and inserted into vector pMD18-T.The constructed recombinant plasmid pMD18 /Tox529 bp was identified by sequencing,then diluted,calibrated and used as standard.The reaction system and condition for PCR were optimized,and the sensitivity and specificity of the developed PCR assay were verified.Results Sequencing proved that recombinant plasmid pMD18 /Tox529 bp was constructed correctly.The minimum detection limit of the developed PCR assay was 10 copies of target gene.Specific DNA bands were amplified by the developed PCR assay from the genome of Toxoplasma gondii.However,no specific DNA bands were amplified from the genomes of human,mouse,Plasmodium vivax,Plasmodium faciparum or Mycobacterium tuberculosis.Conclusion A highly sensitive and specific PCR assay for diagnosis of Toxoplasma gondii was developed,which might be used for the screening,clinical diagnosis and epidemiological investigation of Toxoplasma gondii infections in humans and animals.

    2010 07 v.23 [Abstract][OnlineView][Download 182K]

  • Development and Validation of Alternative Method for Determination of Haemagglutinin Content in Influenza Vaccine

    SHAO Ming,LI Juan,SONG Ying-li,CUI Xiao-yu,YUAN Li-yong,FANG Han-hua,LI Chang-gui,LI Feng-xiang,WANG Jun-zhi(National Institute for the Control of Pharmaceutical and Biological Products,Beijing 100050,China)

    Objective To establish an alternative method for determination of haemagglutinin(HA)content in influenza vaccine so as to solve the problem of quantitative determination of HA in vaccine bulk at early stage of outbreak of pandemic influenza.Methods Influenza vaccine bulk was treated with PNGase F under an optimized condition,and separated by reduced SDS-PAGE,in which the percentage of HA protein was determined by densitometric analysis.The HA content was calculated based on the percentage of HA and total protein content in test sample.The HA contents in seven batches of vaccine bulks were determined by the alternative method and traditional SRID method respectively,and the results were compared.Results The optimal total protein content of bulk for treatment with PNGase F was 400 μg /ml,and the optimal ratio of PNGase F to bulk was 1:50(v /v).After treatment,various protein bands in test samples were differentiated clearly by reduced SDS-PAGE,of which two HA subgroups were identified by sequencing,with the identical relative molecular masses to those expected.The coincidence rates of determination results of HA contents in seven batches of vaccine bulks by the alternative method and traditional SRID method were 87.90% ~ 122.20%.Conclusion An alternative method for determination of HA content in influenza vaccine is preliminarily developed,which may be used for the quantitative determination of HA in vaccine bulk when the WHO reference materials are not available in outbreak of pandemic influenza.

    2010 07 v.23 [Abstract][OnlineView][Download 335K]

  • Isolation,Culture and Identification of Rat Mesenchymal Stem Cells In Vitro

    YAN Wen-xing△,WANG Hong-yan,LIU Li-ping,DENG Li,ZHANG Hong-mei,CHEN Yu-bing(△Department of Radiotherapy,Second Hospital,Jilin University,Changchun 130021,China)

    Objective To develop the methods for isolation,culture and identification of rat mesenchymal stem cells(MSCs) in vitro and lay a foundation of a serial study on MSCs.Methods MSCs were isolated and cultured by direct wall adhesion culture of the whole bone marrow,then subcultured and observed for morphology under invert microscope.The proliferation of MSCs was determined by MTT method,based on which a growth curved was plotted.The MSCs of passage 3 were determined for cell cycle and phynotype by flow cytometry.The directional differentiation of MSCs to osteoblasts and fat-like cells were induced,and the differentiation ability was identified.Results After the whole bone marrow cells were cultured for 5 d,obvious proliferation of adherent cells were observed under microscope,and the cells were even in shape,most of which were spindle-shaped.The cells could be subcultured about 7 d after culture,and formed spindle-shaped fibroblast-like cells,i.e.MSCs after subculture for 2 ~ 3 passages.The growth curve of cells was in S shape.Flow cytometry showed that 76.01%,7.13% and 16.86% of MSCs were in G0 /G1,G2 /M and S phases respectively.No CD34 was expressed on the surface of MSCs.However,by using specified inducers,MSCs were differentiated to fibroblast-like and fat-like cells respectively.Conclusion The methods for isolation,culture and identification of rat MSCs in vitro was successfully developed,which might be used for evaluation of MSCs cultured in vitro.

    2010 07 v.23 [Abstract][OnlineView][Download 319K]

  • Determination of Residual Chloroform Content in Inactivated Hepatitis A Vaccine by Gas Chromatography

    LI Zhi-gang,LI Jing,GAO Qiang,SONG Li-li(Sinovac Biotech Co.Ltd.,Beijing 100085,China)

    Objective To determine the residual chloroform content in inactivated hepatitis A(HA)vaccine by headspace gas chromatography.Methods The residual chloroform content in inactivated hepatitis A vaccine was determined by gas chromatography with capillary column DB-WAX(30 m × 0.53 mm × 0.50 μm)and ECD detector,using nitrogen as a carrier gas by headspace sample loading.Standard curves were plotted based on the working solution of chloroform diluted with various matrixes.The method was verified for adaptivity,specificity,linearity,precision as well as quantitative and detection limits.Results The standard curves showed effect of matrix on the determination result.The separation degree between chloroform and other foreign matters in HA vaccine was 2.58.The determination result showed good linear relationship to chloroform concentration at a range of 20 ~ 100 ng /ml(R2 = 0.999 3).The method showed high precision,with a RSD of less than 5%.The detection and quantitative limits of the method were 1.2 and 4.8 ng /ml respectively.The recovery rates of test samples at high,moderate and low concentration,with addition of standard,were(95.6 ± 1.8)%,(95.5 ± 3.1)% and(101.9 ± 2.1)% respectively.Conclusion Headspace gas chromatography was simple and sensitive,and the determination result was accurate and reliable,indicating that the method was suitable for determination of residual chloroform content in inactivated HA vaccine.

    2010 07 v.23 [Abstract][OnlineView][Download 172K]

  • Advance in Research on Cytokine Vaccine

    LI Li,HUANG Shi-he,YANG Xiao-ming(Wuhan Institute of Biological Products,Wuhan 430060,China)

    Cytokines are highly active and multi-functional polypeptides which may regulate the functions of cells,produced by immune cells such as mononuclear macrophages,T lymphocytes,B lymphocytes and NK cells,and other relevant cells such as vascular endothelial cells,and fibroblasts.At pathological status,cytokines play important roles in regulating and modulating autoimmune diseases,chronic inflammations,tumors and HIV infection.Cytokines may be used for the prevention and treatment of some diseases.However,they may also cause and promote the onsets of some diseases.Regulatory therapy with cytokines includes supplement /addition and blocking /antagonism.The blocking /antagonism therapy with cytokines at early stage is performed by passive immunization with antibodies against cytokines,which shows significant disadvantages and shortcomings.Active immunization with cytokine vaccine may stimulate the production of autoantibodies to block the activity of cytokines,which is one of the most important development in regulatory therapy with cytokines in recent years.The advance in research on cytokine vaccine is reviewed in this paper.

    2010 07 v.23 [Abstract][OnlineView][Download 182K]

  • Progress in Research on Rotavirus Vaccine

    ZHANG Yan,WEI Hai-tao,CHEN Yuan-ding(Institute of Medical Biology,Chinese Academy of Medical Sciences and Peking Union Medical College,Kunming 650118,China)

    Rotavirus(RV)is the major pathogen of infantile diarrhea in the world.Great progresses have been made in research on RV vaccine,however,there are still many problems in the current vaccines,so it is important to further develop novel vaccines.Though the three kinds of current RV vaccines provide good protection against severe diarrhea,they fail to prevent RV infection.The progresses in researches on RV and its vaccine are reviewed in this paper.

    2010 07 v.23 [Abstract][OnlineView][Download 141K]

  • Progress in Research on Infantile Sensorineural Hearing Loss Caused by Congenital Cytomegalovirus Infection

    DING Ting-ting,WANG Ming-li(Department of Microbiology,Anhui Medical University,Hefei 230032,China)

    Congenital cytomegalovirus(CMV)infection is an important cause for infantile sensorineural hearing loss,meanwhile,the latter is also the most common sequel of the former.The symptoms of hearing loss may occur in both neonatal period and in a certain period after birth(at age of one year in general).The hearing of patients may be improved by antiviral therapy within the range of safety.The progress in research on infantile sensorineural hearing loss caused by congenital CMV infection is reviewed in this paper.

    2010 07 v.23 [Abstract][OnlineView][Download 113K]


@2012 Editorial Department of "Chinese Journal of Biologicals"