2014年06期目次

  • Early cellular immune response induced by domestic and imported recombinant hepatitis B vaccines prepared with yeast cells in mice

    HE Peng;QIU Shao-hui;FANG Xin;ZHONG Xi;LIANG Zheng-lun;SHEN Qi;HU Zhong-yu;National Institutes for Food and Drug Control;

    Objective To compare the difference of domestic and imported recombinant hepatitis B(HB)vaccines prepared with yeast cells in induction of cellular immune response in mice. Methods Domestic HB vaccines 1 and 2(Saccharomyces cerevisiae),imported vaccine 1(Saccharomyces cerevisiae),domestic vaccines 3 and 4(Hansenula polymorpha)and imported vaccine 2(Hansenula polymorpha),three batches for each,were inoculated s. c. into BALB / c mice,3 μg / 100 μl for each. The mice were killed 7 d after immunization,of whom the spleens were collected aseptically and prepared into splenocyte suspension from which mononuclear cells(MNCs)were isolated. HBsAg antigen specific IFNγ,IL-2,IL-4 and IL-5 levels produced by MNCs after stimulation in vitro with HBsAg as well as the IFNγlevel after stimulation in vitro with MHC Ⅰ polypeptide S28-39were determined by enzyme linked immunospot assay(ELISPOT). Results The IFNγ,IL-2 and IL-4 levels secreted by MNCs after stimulation in vitro with HBsAg in the mice inoculated with domestic vaccine 1 were significantly higher than those with domestic vaccine 2 and imported vaccine 1,while the IL-2 levels induced with domestic vaccines 1 and 2 were significantly higher than that with imported vaccine 1(each P < 0. 05). After stimulation of MNCs with S28-39,the IFNγ levels induced with the three vaccines prepared with S. cerevisiae showed no significant difference(P > 0. 05). However,after stimulation with HBsAg or S28-39,both the IFNγ and IL-4 levels induced with imported vaccine 2 were significantly higher than those with domestic vaccine 3,while the IL-2 and IL-5 levels induced by three vaccines prepared with H. polymorpha showed no significant difference(P > 0. 05). Conclusion Domestic recombinant HB vaccines prepared with S. cerevisiae induced early cellular immune response effectively.

    2014 06 v.27 [Abstract][OnlineView][Download 673K]

  • Expression of gC2 of herpes simplex virus type Ⅱ in CHO cells and purification and immune activity of expressed product

    ZHOU Jie;CAI Ran;XU Yue-yue;PAN Ying;LI Su-qin;SHANG Yan-hong;XU Gui-li;YUAN Jing-yu;MA Ying;ZHANG Su-fen;LI Yue-xi;Department of Biochemistry & Molecular Biology,School of Preclinical Medicine,Nanjing Medical University;

    Objective To express the extracellular domain fragment of gC2 of herpes simplex virus(HSV gC2)type Ⅱ in CHO cells,purify the expressed product and determine its immune activity. Methods The amino acid sequence of HSV type Ⅱ gC2 in GenBank was analyzed by ANTHEWIN software,from which the epitope-rich extracelluar fragments with good hydropathy were screened,optimized by OptimumGene software using prokaryotic cells- and eukaryotic cellspreferred codons,synthesized chemically,amplified by PCR and cloned into eukaryotic expression vector pMCE5. The constructed recombinant plasmid pMCE5-gC2 was transfected into CHO cells. The expressed protein gC2 was purified by affinity chromatography,and identified by SDS-PAGE and Western blot. Mice were immunized i. p. with the purified recombinant gC2 protein at weeks 0,2 and 4,using PBS as control,and determined for serum antibody level by indirect ELISA,for frequencies of splenic lymphocytes secreting IFNγ(Th1) and IL-4(Th2). Results Recombinant plasmid pMCE5-gC2 was constructed correctly as proved by PCR,restriction analysis(EcoRⅠ / NotⅠ) and sequencing. The purified gC2 protein,with a relative molecular mass of about 90 000,reached a purity of about 85% and a concentration of 40 μg / ml,which showed specific binding to mouse monoclonal antibody against His. High serum antibody level was induced in mice after immunization for 3 times,which showed significant difference with that before immunization(P <0. 001). The frequencies of mouse splenic lymphocytes secreting IFNγ and IL-4 after stimulation with specific antigen were(13. 46 ± 2. 83)/ 106 cells and(19. 20 ± 2. 48)/ 106 cells respectively,both of which were significantly higher than those in control group(P < 0. 001). Conclusion Recombinant gC2 protein was expressed in CHO cells,which induced good humoral and cellular immune responses in mice after purification. It laid a foundation of further preparation of HSV subunit vaccine.

    2014 06 v.27 [Abstract][OnlineView][Download 524K]

  • Genetic stability of FY23-KB strain as virus seed for production of inactivated enterovirus 71 vaccine(human diploid cells)

    ZHANG Ying;ZHAO Hong-ling;WANG Li-chun;LIU Long-ding;DONG Cheng-hong;LI Qi-han;Institute of Medical Biology,Chinese Academy of Medicine Science & Peking Union Medical College,Yunnan Key Laboratory of Vaccine Research and Development on Severe Infectious Diseases;

    Objective To investigate the genetic stability of FY23-KB strain as virus seed for production of inactivated enterovirus 71(EV71)vaccine(human diploid cells)after subculture in KMB-17 cells. Methods The sequences of VP1regions and virulence of passage 2 of vaccine strain FY23-KB in Vero cells(FY23-V-2) were compared with those of passages 12(FY23-KB-12), 14(FY23-KB-14)and 16(FY23-KB-16)in KMB-17 cells, and those of FY23-KB-14 with those of passage 19(FY23-KB-19), 20(FY23-KB-20)and 21(FY23-KB-21), based on which the genetic stability of vaccine strain was analyzed. Results Three base mutations were observed in VP1 regions of FY23-KB-12, 14 and 16 as compared with FY23-V-2, resulting in the variation of two amino acids, which were Ser→Pro at site 226 and Asn→Asp at site 282 respectively. However, 6, 8 and 11 base mutations were observed in the VP1 regions of FY23-KB-19, 20 and 21as compared with FY23-KB-14, most of which were non-sense mutations. The variations of amino acids were only observed in two sites(P134 and P230)of FY23-KB-21. All the titers of virus of various passages were 6. 5 ~ 7. 0 lgCCID50/ ml.Conclusion No significant changes were observed in VP1 sequence of FY23-KB strain after subculture in KMB-17 cells,while the virulence was homogeneous, indicating high genetic stability. It demonstrated the safety of inactivated EV71vaccine at molecular level.

    2014 06 v.27 [Abstract][OnlineView][Download 419K]

  • Construction and transcriptional activity of recombinant plasmid containing human interferon λ1 gene promoter luciferase reporter gene

    PANG Li-li;SHAO Chang-sheng;DUAN Zhao-jun;National Institute for Viral Disease Control and Prevention,Chinese Center for Disease Control and Prevention;

    Objective To construct the recombinant plasmid containing human interferon(IFN)λ1 promoter recombined luciferase reporter gene,and investigate the effect of plasmids IRF3-5D and HA-IRF7 on its transcriptional activity.Methods The promoter of human IFNλ1 gene was amplified by PCR using the extracted genomic DNA of BEAS-2B cells as a template,and inserted into the luciferase reporter vector pGL3-enhancer. The constructed recombinant plasmid IFNL1-luc was transfected to 293T cells,using plasmid IFN-β-luc and pGL3-enhancer as controls. The cells were infected with Sendai virus 24 h after transfection. The cells were collected before and 4,12,18 and 24 h after infection,and determined for luciferase activity. The cells were co-transfected with plasmids IFNL1-luc and IRF3-5D or HA-IRF7,and collected 36 h later for determination of luciferase activity. Results Both restriction analysis and sequencing proved that recombinant plasmid IFNL1-luc was constructed correctly. The folds of luciferase activation of cells transfected with plasmids IFNL1-luc and IFNβ-luc increased significantly and reached peak values of 1 000 and 2 300 18 h after infection with Sendai virus. However,the folds of luciferase activation of cells transfected with pGL3-enhancer were maintained at low levels at various time points after infection with Sendai virus. The activation folds of plasmids HAIRF7 or IRF3-5D to IFNL1-luc were significantly higher than that of pGL3-enhancer(P < 0. 05). Conclusion The recombinant plasmid IFNL1-luc containing human interferon(IFN)λ1 promoter recombined luciferase reporter gene was constructed successfully. Sendai virus as well as plasmids IRF3-5D and HA-IRF7 activated the transcription of IFNL1-luc,which laid a foundation of further study on the signaling pathway regulating IFNλ1 expression.

    2014 06 v.27 [Abstract][OnlineView][Download 682K]

  • Prediction of specific binding domain of rubella virus E1 protein and its receptor

    XING Jia-qiang;LIU Xiao-fan;DOU Qiang;LIN Jie;WEI Zhi-dong;Lanzhou Institute of Biological Products Co.,Ltd.;

    Objective To build the three-dimensional structure of rubella protein E1 and its receptor myelin oligodendrocyte glycoprotein(MOG) by homologous modeling,and predict the candidate residues of E1 binding to MOG by molecular docking technique. Methods The three-dimensional structure of rubella protein E1 and its receptor MOG were predicted by MODELLER program,while the active packet by CASTp server. The hydrophobicity of MOG was analyzed by ProtScale method. The motif of protein was searched by Hidden Markov Model HMM-based SMART program. The cavity medium structures of E1 and MOG were designed by PyMOL-APBS software,while the electrostatic potentials were analyzed.The binding sites of were searched by molecular docking program PLATINUM. Results E1 protein consisted of three domains,of which D1 and D3 were at the bottom of structure and formed a big packet,while the C-terminus and one side of D2 formed another big packet. However,other packets were mainly centralized on the fusion surface of upper half part of D2. The structural model of MOG was a β-sheet barrel consisting of eight antiparallel sheets,while all the amino acid residues in main chain conformation of predicted structure were located in the permissible domains. The amino acids at sites 1 ~ 17 of N-terminus formed a motif(low complexity region),while those at sites 46 ~ 127 formed an immunoglobulin V-type(IGv). The amino acid residues in low complexity region showed strong hydrophobicity. The five amino acid residues at N-terminus of MOG and a β-turn consisting of ARG101 ~ GLU107 formed a spike-like structure which projected towards an active pocket located in a fusion surface framed by FL1 and FL2,and bound to ALA86,TYR90,TYR101,PHE102,ASN103,GLY105,SER107,TYR109,ALA122,PHE123,HIS125 and SER126 through hydrophobic interaction,hydrogen bond and aromatic stack. Conclusion The specific binding domain of E1 and MOG was predicted by computer modeling technique,which laid a foundation of further study on the interaction.

    2014 06 v.27 [Abstract][OnlineView][Download 1004K]

  • Interleukin-6 promotes pathogenic damage in suckling mice infected with enterovirus 71

    WANG Li-chun;LIAO Yun;WANG Jing-jing;ZHANG Ying;LIU Long-ding;LI Qi-han;Institute of Medical Biology,Chinese Academy of Medical Sciences and Peking Union Medical College,Yunnan Key Laboratory of Vaccine Research & Development on Severe Infectious Diseases;

    Objective To investigate the promoting effect of interleukin-6(IL-6)on the pathogenic damage in suckling miceinfectedwithenterovirus71(EV71). Methods Suckling ICR mice were injected i. c. with IL-6 or anti-IL-6 antibody during infection with EV71,using the uninfected suckling mice injected with IL-6 and with PBS as control. The survival and physical sign of mice were observed daily for 11 d,based on which the survival rate was calculated. The viral loadings in brains of mice in various groups were determined by real-time PCR,while the pathogenic change by HE staining. Results Increased IL-6 content in brain promoted the pathogenesis of EV71 and the death of suckling mice.However,the injection i. c. with anti-IL-6 antibody showed a certain protective effect on the suckling mice infected with EV71. Conclusion IL-6 promoted the pathogenesis of EV71 to suckling mice,of which the increase in central nerve system might involve the immunopathological course of EV71 infection.

    2014 06 v.27 [Abstract][OnlineView][Download 679K]

  • Effect of esterified epigallocatechin gallate on apoptosis of breast cancer cells

    CHANG Li-jing;WANG Jing;WANG Peng-cheng;WANG Xuan-jun;SHENG Jun;College of Long Run Pu-erh Tea,Yunnan Agricultural University;

    Objective To investigate the effect of esterified epigallocatechin gallate(EGCG)on apoptosis of Myc-dependent MCF-7 and Myc-independent MDA-MB-231 strains of human breast cancer cells. Methods EGCG was esterified by lipase catalysis,purified by silica gel column chromatography for 2 times,identified by HPLC-MS,and analyzed for stability. The effect of esterified EGCG on apoptosis of MCF-7 and MDA-MB-231 cells were evaluated by flow cytometry.Results EGCG-C16 was obtained after modification and purification. All the relative molecular masses of esterified products at various retention time points in HPLC were 696. The hydrogen peroxide level produced by EGCG in common medium was significantly higher than that by EGCG in acidic medium and that by EGCG-C16 in common medium(P <0. 000 1),indicating that the stability of EGCG-C16 was higher than that of EGCG. EGCG-C16 showed significantly promoting effect on the apoptosis of MCF-7 cells,while showed no the effect on MDA-MB-231 cells. Conclusion Esterified EGCG promoted the apoptosis of Myc-dependent breast cancer cells specifically,which laid a foundation of further study on the anti-tumor effect of EGCG.

    2014 06 v.27 [Abstract][OnlineView][Download 1132K]

  • Preparation and immune effect of meningococcal immunogenic conjugates

    ZHANG Ying;ZHANG Shui-hua;LI Zhi-long;WANG Yan;School of Applied Chemistry and Biotechnology,Shenzhen Polytechnic;

    Objective To conjugate Neisseria meningitidis(Nm)groupsA,B,W135 and Y by using various carriers,and compare the immune effects of conjugates. Methods The polysaccharides of Nm groups A,W135 and Y were conjugated to CRM197. The seeds of Nm group B were subcultured,from which outer membrane protein was extracted,purified and conjugated to polysaccharide group C. The protein-polysaccharide conjugates were mixed with liposome by freezedried hydratation and immunized to guinea pigs,using alum as control,and determined for bactericidal titer. Results The conjugates showed various immune effects against Nm of the corresponding serogroups. Compared with that of direct mixture,the covalent conjugate of polysaccharide group C and outer membrane protein group B induced high bactericidal titer in guinea pigs. The immunopotentiation of conjugates using liposome as adjuvant was superior to that using alum as control,while the immune effects of conjugates groups A and Y were enhanced significantly(P < 0. 05). Conclusion The immunogenic conjugates of Nm prepared with various antigens by covalent conjugation showed higher bactericidal titer than those by direct mixing. In addition,liposome as an adjuvant was beneficial to the enhancement of immune effect of Nm vaccine.

    2014 06 v.27 [Abstract][OnlineView][Download 624K]

  • Optimization and safety of nano-emulsion adjuvant of influenza H3N2 vaccine

    ZHAO Lan-hua;LIU Hong-qing;CHUAN Hong-yun;DUAN Nan;YANG Shu;YANG Lei;DAI Zong-xiang;LI Ying-bo;Institute of Medical Biology,Chinese Academy of Medical Sciences & Peking Union Medical College;

    Objective To optimize the formula of nano-emulsion as an adjuvant of influenza H3N2 vaccine and investigate the influencing factors of adjuvant effect using water-loading and mode of vaccine loading as indexes.Methods ICR mice were divided into seven groups. The mice in A1,A2,A3 and A4 groups were injected i. m. with the mixture of nano-emulsion adjuvant and influenza H3N2 split vaccine containing 1. 8 μg hemagglutinin(HA),in a volume of 0. 2 ml,on days 0 and 21. The vaccines in A1,A2 and A3 groups were added after emulsion,while that in A4group before emulsion. The water-loadings in A1,A2,A3 and A4 groups were 66. 7%,80. 0%,85. 7% and 80. 0%respectively. However,the mice in blank control group were injected with physiological saline,while those in vaccine control group with 1. 8 μg HA,and those in aluminum adjuvant control group with 0. 2 mg aluminum hydroxide + 1. 8 μg HA. Blood samples were collected every week starting from week 1 after the first injection and determined for haemagglutination inhibition(HI)antibody titer,based on which the immune response levels in various groups were compared. The maximal tolerance dose(MTD)of nano-emulsion was analyzed by the change of bodyweights of mice within one week after the first injection and maximal dose test. The sizes and dispersion coefficients of nano-emulsion particles with various water-loadings were analyzed by Melvin granulometer. The stability was evaluated by pseudo ternary phase diagram method. Results The HI titer of mice in vaccine control group at week 5 after the first injection showed significant difference with those in A1,A2 and A3 groups(P < 0. 05),while showed no significant difference with that in A4 group(P > 0. 05). The HI titer in A3 group reached the maximum at week 5 after the first injection,which was about 4 times of that in vaccine control group,and were significantly higher than those in aluminum adjuvant control,A1 and A2 groups(each P < 0. 05). Loading or embedding of vaccine with nano-emulsion showed no significant influence on the growth of mice,with a MTD of more than 25 ml / kg. The mean sizes of nano-emulsion sizes were about10 ~ 100 nm,which showed no significant difference in A1,A2 and A3 groups,indicating a good uniformity. The formula of nano-emulsion,with a large range of water-loading,was suitable for loading of sufficient antigen,which showed high stability. Conclusion The nano-emulsion with a water-loading of 85. 7%,on which vaccine was loaded by adsorption,show an ideal adjuvant effect and high safety.

    2014 06 v.27 [Abstract][OnlineView][Download 798K]

  • Prokaryotic expression of cholera toxin subunit A gene and preparation of monoclonal antibody

    CHEN Jian;WEI Yu-xing;XIE Xiao-yan;ZHU Zhao-qin;WAN Yan-min;XU Jian-qing;Wenzhou Medical University;

    Objective To express cholera toxin subunit A gene in prokaryotic cells,purify the expressed product,establish a hybridoma cell line and prepare the monoclonal antibody(mAb)against cholera toxin subunit A(CTA).Methods Full-length CTA gene was cloned into vector pET30a(+),and the constructed recombinant plasmid pET30aCTA was transformed to E. coli BL21(DE3)and induced with IPTG for 6 h. The expressed product was analyzed by12% SDS-PAGE and purified by Ni2+affinity chromatography,with which C57BL / 6 mice were immunized to prepare mAb by hybridoma cell technique. The prepared mAb was determined for specificity by Western blot,and for blocking effect on CT toxicity by in vitro blocking test. Results PCR and restriction analysis proved that recombinant plasmid pET30a-CTA was constructed correctly. The expressed CTA protein,with a relative molecular mass of 25 000 ~ 35 000,reached a purity of more than 90% after purification. A hybridoma cell line(G6C3-D10) secreting mAb against CTA was obtained,and the secreted mAb bound to CTA specifically,which showed an obvious band with a relative molecular mass of 25 000 ~ 35 000. The mAb reached a titer of 54 in culture supernatant of hybridoma cells and,after being mixed with CT toxin,inhibited the increase of cyclic adenosine monophosphate(cAMP)induced by CT significantly(P <0. 01). Conclusion CTA gene was expressed in prokaryotic cells and purified,and the hybridoma cell line secreting mAb with ability of blocking CT toxicity was established,which provided a reference for further study on mechanism of toxicity and adjuvanticity of CTA.

    2014 06 v.27 [Abstract][OnlineView][Download 660K]

  • Construction of eukaryotic expression vector for human SIAH2 gene and its effect on proliferation of breast cancer MDA-MB-231 cells

    TAO Wei;LI Jin-cheng;ZHANG Jia-yi;BAI Jian-yin;SUN Xiao;Department of Breast Surgery,The First Affiliated Hospital of Liaoning Medical University;

    Objective To construct the eukaryotic expression vector for human SIAH2 gene and investigate its effect on the proliferation of breast cancer MDA-MB-231 cells. Methods Full-length SIAH2 gene was amplified from MDA-MB-231 cells by PCR and cloned into expression vector pcDNA3. 1-hisB. The constructed recombinant plasmid pcDNA-3. 1-hisB-SIAH2 was transfected to MDA-MB-231 cells. The expression of SIAH2 protein in cells was determined by Western blot,while the location by IFA,the distribution of cell cycle by flow cytometry,and the proliferation activity of cells by MTT assay. Results Restriction analysis(EcoRⅠ / XhoⅠ)and sequencing proved that recombinant plasmid pcDNA3. 1-hisB-SIAH2 was constructed correctly. The expression level of SIAH2 protein in the cells transfected with the recombinant plasmid increased significantly(P < 0. 05). The expressed protein was located in nuclei,of which the percentage at S phase increased significantly. The proliferation activity of MDA-MB-231 cells 48,72 and 96 h after transfection increased significantly. Conclusion The eukaryotic expression vector for SIAH2 gene was constructed successfully,and SIAH2 protein was expressed in MDA-MB-231 cells. SIAH2 protein promoted the proliferation of MDAMB-231 cells significantly,indicating its important role in the pathogenesis and progress of breast cancer.

    2014 06 v.27 [Abstract][OnlineView][Download 665K]

  • Cloning and prokaryotic expression of P40 gene of Neospora caninum surface antigen

    YANG Bin-tong;ZHAO Quan;HE Peng-fei;CHANG Le;GONG Peng-tao;LI Jian-hua;YANG Ju;LI He;ZHANG Guo-cai;ZHANG Xi-chen;College of Animal Science and Technology,Jilin Agricultural University;

    Objective To clone the P40 gene of Neospora caninum surface antigen and express in prokaryotic cells.Methods P40 gene of the N. caninum surface antigen was amplified by PCR and cloned into prokaryotic expression vector pET-28a. The constructed recombinant plasmid pET-28a-P40 was transformed to E. coli Rosseta(DE3)for expression under induction of IPTG. The expressed product was identified by SDS-PAGE and Western blot. Results The prokaryotic expression vector pET-28a-P40 was constructed correctly as proved by PCR and restriction analysis. The expressed recombinant P40 protein,with a relative molecular mass of about 40 000,recognized the positive mouse serum against N. caninum effectively. Conclusion P40 gene of the N. caninum surface antigen was expressed successfully in E. coli Rosseta(DE3),which laid a foundation of preparation of N. caninum vaccine and development of methods for serologic diagnosis of neosporosis.

    2014 06 v.27 [Abstract][OnlineView][Download 634K]

  • Construction of RNAi expression vector targeting unknown differentially expressed UG1 gene of high-yield strain of Monierella isabellina

    CHANG Yuan-yuan;YAO Di;YU Chang-qing;College of Food Science,Heilongjiang Bayi Agricultural University;

    Objective To construct a RNAi expression vector targeting unknown differentially expressed UG1 gene of high-yield strain of Monierella isabellina. Methods DsReD RNAi expression vector pREDi was constructed using red fluorescent protein gene DsReD as a report gene,and transformed to M. isabellina stably expressing DsReD gene by PEG /CaCl2-mediated protoplast transformation,based on which the RNAi expression vector pUG1i targeting differentially expressed UG1 gene of high-yield M. isabellina strain was constructed,and the M. isabellina transfected with pREDi and pUGli were analyzed by Northern blot. Results Restriction analysis and sequencing proved that plasmids pREDi and pUGli were constructed correctly. The M. isabellina strain transformed with pREDi was colorless. However,the expressions of UG1 and DsReD in recipient M. isabellina transfected with plasmid pUGli were inhibited at gene level.Conclusion RNAi expression vector targeting unknown differentially expressed UG1 gene of high-yield strain of M. isabellina was constructed successfully,which laid a foundation of co-silencing DsReD and target gene by RNAi and further study on the effect of UG1 gene on synthesis of ARA and analysis of gene function.

    2014 06 v.27 [Abstract][OnlineView][Download 837K]

  • Effect of combined genotype of estrogen receptor,follicle stimulating hormone beta subunit,prolactin receptor and retinol-binding protein 4 on litter size of pigs

    WANG Li-xin;SU Yu-hong;ZHANG Qiang;FAN Zheng-zheng;Husbandry and Veterinary Academy of Liaoning Medical University;

    Objective To investigate the effect of combined genotype of estrogen receptor(ESR),follicle stimulating hormone beta subunit(FSH-β),prolactin receptor(PRLR) and retinol-binding protein 4(RBP4) on litter size of pigs.Methods ESR,FSH-β,PRLR and RBP4 genes were amplified from the Large Yorkshire,Landrace and Duroc pigs in nine pig breeding farms in Shenyang Region,Liaoning Province,China by PCR,and tested for polymorphism by PCRRFLP,based on which the effect of single and combined genotypes of the four genes on litter size were analyzed. Results Genotypes AA,AB and BB were observed in the four genes,and each of the genes were in a genetic equilibrium. The analysis of single genotype showed significant difference in the litter sizes of Landrace and Large Yorkshire pigs of geno-type FSH-β(P < 0. 05),and proved BB as a superior genotype. However,no significant differences were observed in the litters of the other genotypes(P > 0. 05). The superior genotypes of ESR,PRLR and RBP4 genes were AA,BB and AB respectively. Combined analysis of four genes proved AABBBBAB as the dominant combination. Conclusion The genetic efficacy of combined genotype was significantly higher than those of single genotype,which was helpful to the markerassisted selection of litter size of pigs.

    2014 06 v.27 [Abstract][OnlineView][Download 744K]

  • Degradation of cholesterol with Lactobacillus plantarum M1-UVs29 in vitro

    GUO Feng;FAN Lei;LIAO Yu-xin;QIAN Li-li;YU Chang-qing;College of Food Science,Heilongjiang Bayi Agricultural University;

    Objective To investigate the ability of Lactobacillus plantarum M1-UVs29 in degradation of cholesterol under various conditions. Methods The dry powder of M1-UVs29 was activated and inoculated to liquid MRS,MRS-bile salt,MRS-cholesterol and MRS-bile salt-cholesterol media respectively,of which the growth curves were plotted. The abilities of M1-UVs29 in degradation of cholesterol under various conditions(amounts of cholesterol and bile salt added and bacterial seeds inoculated)were evaluated using the degradation rate of cholesterol as an index. Results Bile salt showed inhibitory effect on the growth of M1-UVs29,while cholesterol remitted the inhibitory effect. The degradation rate of cholesterol increased stably with the cholesterol content in medium. When the bile salt content was 0. 2%,and the cholesterol content was 400 μg / ml,the cholesterol degradation rate reached 34. 76%. However,the cholesterol degradation rate increased irregularly with the increasing bile salt content. When the cholesterol content was 100 μg / ml,and the bile salt content was 0. 4%,the cholesterol degradation rate reached 32. 85%. When the bile salt content was more than 0. 5%,the cholesterol degradation rate decreased remarkably. When the cholesterol content in medium was 100 μg / ml,bile salt content was 0. 2%,and the amount of bacterial seeds was 2 × 108 cfu / ml,the cholesterol degradation rate reached the maximum of 37. 30%. Conclusion L. plantarum M1-UVs29 showed stable ability in degradation of cholesterol,which provided a reference for its further application.

    2014 06 v.27 [Abstract][OnlineView][Download 650K]

  • Expression of recombinant human VEGF121KDR/rGEL fusion toxin in E. coli and purification and activity of expressed product

    SUN Hong-yan;LIU Ai-jun;SHEN Yun-fei;LI Yue;LIU Min-xia;WU Fu-jun;SHEN Yan-jie;ZHOU Man-xiang;Shanxi Kangbao Biological Product Co.,Ltd.;

    Objective To express recombinant human vascular endothelial growth factor(VEGF)121KDR/ rGEL fusion toxin,specific to VEGF receptor 2(VEGFR2)KDR,in E. coli,purify the expressed product and determine its biological activity. Methods The gene sequence of VEGF121KDR/ rGEL fusion toxin were cloned into prokaryotic expression vector pET-32a(+),and the constructed recombinant plasmid pET-32a(+)-VEGF121KDR/ rGEL was transformed to E. coli Origami(DE3)for expression under induction of IPTG. The expressed protein was purified by SP-Sepharose FF and NiSepharose FF chromatography,then digested with thrombin,further purified by Q-Sepharose FF chromatography,and determined for cytotoxicity to PAE / FLT1 and PAE / KDR cells on which VEGFR1 / FLT1 and VEGFR2 / KDR were highly expressed. Results Restriction analysis and sequencing proved that recombinant plasmid pET-32a(+)-VEGF121KDR/rGEL was constructed correctly. The fusion protein of thioredoxin(Trx)as a tag and rhVEGF121KDR/ rGEL,with a relative molecular mass of about 62 000,was expressed in a soluble form,and contained about 20% of total somatic protein.However,the relative molecular mass of purified rhVEGF121KDR/ rGEL fusion toxin,after removal of tag,was about43 000,while the purity was more than 98%. The median inhibitory concentrations(IC50)of VEGFR1-positive PAE / FLT cells was 278 nmol / L,which was more than 800 times of that of VEGFR2-positive PAE / KDR cells(0. 32 nmol / L).Conclusion The rhVEGF121KDR/ rGEL fusion toxin was successfully expressed in E. coli,which showed strong killing effect on vascular endothelial cells overexpressing VEGFR2. It laid a foundation of further study on the anti-tumor effect of the fusion toxin and development of novel drugs targeting tumors.

    2014 06 v.27 [Abstract][OnlineView][Download 717K]

  • Role of TGF-β1/Smad7 in enhancement of cholesteryl ester hydrolase expression by resveratrol

    ZHANG Feng-xiang;HE Cheng-ye;LI Chen-guang;SI Zhong-yi;Department of Cardiac Surgery,The First Hospital Affiliated to Liaoning Medical College;

    Objective To investigate the role of TGF-β1 / Smad7 in enhancement of cholesteryl ester hydrolase(CEH)expression by resveratrol. Methods Mouse mononuclear macrophage RAW264. 7 strain was transformed to foam cells by induction with oxidized low density lipoprotein(oxLDL),and grouped for 2 times,i.e. normal control,resveratrol,LY2157299 and resveratrol + LY2157299 groups in the first time,and normal control,resveratrol,Ad-Smad-7 and resveratrol + Ad-Smad-7 groups in the second time. The cells in various groups were treated for 48 h,then determined for cholesteryl outflow rate by cholesteryl outflow test,for bubblization by oil red O staining,for transcription levels of pSmad-7 and CEH mRNAs by RT-PCR,and for expression levels of p-Smad-7 and CEH proteins by Western blot. Results As compared with those in normal control group,the cholesteryl outflow rate in resveratrol group increased significantly(P < 0. 05),while those in resveratrol + LY2157299,LY2157299,resveratrol + Ad-Smad-7 and Ad-Smad-7 groups decreased significantly(P < 0. 05); the bubblization levels of macrophages in resveratrol group decreased significantly(P <0. 05),while those in resveratrol + LY2157299,LY2157299,resveratrol + Ad-Smad-7 and Ad-Smad-7 groups increased significantly(P < 0. 05); the mRNA transcription and protein expression levels of CEH in resveratrol group increased significantly(P < 0. 05),while those of p-Smad-7 decreased significantly(P < 0. 05). However,both the mRNA transcription and protein expression of CEH in resveratrol + LY2157299,LY2157299,resveratrol + Ad-Smad-7 and Ad-Smad-7groups were inhibited significantly(P < 0. 05),while those of p-Smad-7 were enhanced significantly(P < 0. 05). Conclusion Resveratrol might enhance the expression of CEH by TGF-β1 / Smad-7 signaling pathway.

    2014 06 v.27 [Abstract][OnlineView][Download 1076K]

  • Application of trend analysis in evaluation of blood screening diagnostic kit for HIV

    SONG Ai-jing;HUANG Wei-jin;XU Si-hong;NIE Jian-hui;LIU Xiu-hua;ZHAO Chen-yan;LIU Qiang;WANG You-chun;National Institutes for Food and Drug Control;

    Objective To investigate the stability of trend analysis used for the evaluation of blood screening diagnostic kit for HIV. Methods The coincidence rates of negative results of reference S4 for cutoff value of sensitivity,HIV-1sample with moderate reactivity(P18),HIV-2 sample with low reactivity(P19)and negative reference were determined by consecutive batches of blood screening diagnostic kits from manufacturers A,B and C,based on which Levey-Jennings trend analysis curve was plotted,and the mean and standard deviation(SD)of each sample were calculated. The change trend of test indexes of diagnostic kits from various manufacturers were observed serving mean ± 2 SD as a warning limit and mean ± 3 SD as an action limit. Results The quality of kit A was stable,with any batch of which the determination results of samples S4,P18 and P19 were not beyond the action limit. The coincidence rates of negative results of negative reference with kits B and C were shown in the trend analysis. However,the determination results of sample P18 with kit B of two batches were beyond the action limits,while those of samples S4,P18 and P19 by kit C of eight consecutive batches were on the same sides of mean. In accelerated stability test at 37 ℃,the change trends of determination results of samples P18,P19 and S4 determined by 32 batches of kit C were in agreement. The results of accelerated stability test at 37 ℃ and stability test at 4 ℃ of sample S4 showed significant difference(P < 0. 01). The results of accelerated stability tests of samples S4,P18 and P19 from various manufacturers showed significant difference with those from Na-tional Institutes for Food and Drug Control(NIFDC)(each P < 0. 01). Conclusion The qualities of domestic blood screening diagnostic kits for HIV were stable,of which the change could by found by trend analysis. Trend analysis may be used for the evaluation on stability of in vitro diagnostic kits for HIV.

    2014 06 v.27 [Abstract][OnlineView][Download 1471K]

  • Investigation on tetanus antibody levels in healthy plasma donors

    SUN Wen;LU Xiao-wu;XIANG Qing-jun;JIANG Sha;WANG Xiao-bing;Wuhan Institute of Biological Products Co. Ltd.;

    Objective To investigate the tetanus antibody levels in healthy plasma donors in Songzi City,Hubei Province,China. Methods A total of 539 healthy plasma donors in Songzi City,at ages of 18 ~ 55 years,were randomly selected and divided into five age groups(not more than 35,36 ~ 40,41 ~ 45,46 ~ 50 and 51 ~ 55 years),of whom plasma samples were collected and determined for serum tetanus antibody level by indirect ELISA. Results The tetanus antibodies of 479 of the 539 donors reached protective levels,with a positive rate of 88. 9% and an average total content(ATC)of(2. 350 ± 3. 246)IU / ml. The tetanus antibody positive rates in various groups were nearly 90%,which showed no significant difference(P > 0. 05). The positive rate in the donors at ages of not more than 35 years was the highest(89. 8%),while that in those at ages of 51 ~ 55 years was the lowest(87. 8%). However,the ATC was the highest in the donors at ages of 46 ~ 50 years[(2. 622 ± 3. 518)IU / ml],while was the lowest in those at ages of51 ~ 55 years[(1. 870 ± 2. 947)IU / ml]. The distributions of antibody levels in the donors at ages of 41 ~ 45 and 51 ~55 years showed significant difference(P < 0. 05),while those in other age groups showed no significant difference(P >0. 05). Conclusion Most of healthy adults at ages of 18 ~ 55 years in Songzi City showed the immunity against tetanus.

    2014 06 v.27 [Abstract][OnlineView][Download 496K]

  • Safety of domestic influenza split virion vaccine for adult use

    AO Rui;FANG Gang;MA Qian-li;WANG Jin;YANG Zhong-nan;ZHANG Min;CHEN Hai-ping;Sichuan Center for Disease Prevention and Control;

    Objective To observe the safety of domestic influenza split virion vaccine for adult use. Methods The adverse events following immunization(AEFIs) in populations at ages of not less than 3 years in Sichuan Province in2013 after inoculation with influenza split virion vaccine manufactured by Changchun Institute of Biological Products Co.,Ltd.(CIBP)and by other manufacturers as control were investigated retrospectively. Results Thirty-six cases of AEFIs were reported in Sichuan Province in 2013 after inoculation with the vaccine manufactured by CIBP,indicating an incidence of 11. 78 / 105. The incidences of general and abnormal reactions were 8. 5 / 105and 2. 62 / 105respectively.However,the composition ratios of adverse reactions in elder age groups after inoculation with the vaccine manufactured by CIBP were higher than those with control vaccines. The incidences of general reactions including fever,duration and redness and swelling as well as abnormal reactions except urticaria after inoculation with vaccine manufactured by CIBP were lower than those with control vaccines. The abnormal reactions were not clustered in the subjects inoculated with vaccines of certain batches. Conclusion The influenza split virion vaccine for adult use manufactured by CIBP showed high safety.

    2014 06 v.27 [Abstract][OnlineView][Download 531K]

  • Laboratory evaluation on test method for neutralizing antibody against coxsacievirus A16

    MAO Qun-ying;SHAO Jie;LUO Zhen;GAO Fan;AN Wen-qi;HAO Chun-sheng;GAO Qiang;YANG Er-xia;HAN Jin-le;LI Zhen-ping;WANG Yi-ping;LIANG Zheng-lun;XU Miao;National Institutes for Food and Drug Control;

    Objective To evaluate the intra- and inter-reproducibility of test method for neutralizing antibody against coxsacievirus A16(CA16)in various laboratories. Methods The test method and experimental standard for neutralizing antibody against CA16 were developed according to the WHO requirements for test method for neutralizing antibody against poliovirus. Twenty-two serum samples were collected and tested for neutralizing antibody against CA16 in strain G10 of genotype A and strains W190,33,203,P11 and CS02 of genotype B separately to evaluate the intra- and interreproducibility of the method in laboratories A ~ G. The influences of these strains on test result of neutralizing antibody against CA16 were compared in laboratories A ~ E. Results The intra-coefficients of variation(CVs) of test results of17 of the 22 samples were 0 ~ 11. 4%,indicating good intra-reproducibility of the method. However,except three samples with low titers and one with high titter,the difference of GMTs of other serum samples was within 3. 0 folds among seven laboratories,which was acceptable. The test results of strains W190,33,203,P11 and CS02 showed similar tendencies to that of strain G10. The test results of strains W190 and 33 in the same laboratory showed no significant difference with that of strain G10. However,the test results of strains 203,P11 and CS02 were significantly lower than that of G10,indicating a certain difference in biological activities of strains of genotypes A and B. Conclusion The test method for neutralizing antibody against CA16 of strain G10 were used effectively in the cooperative laboratories,which was beneficial to the standardization of CA16 neutralizing antibody. Howerer, the applicabilidy of the method needs to be further assessed and confirmed.

    2014 06 v.27 [Abstract][OnlineView][Download 947K]

  • Optimization of chemically defined serum- and protein-free medium for MDCK cells by Plackett-Burman experiment design combined with response surface analysis

    HUANG Ding;LI Wen-ming;ZHAO Liang;FAN Li;YE Zhao-yang;LIU Xu-ping;TAN Wen-song;LUO Jian;CHEN Ze;The State Key Laboratory of Bioreactor Engineering,East China University of Science and Technology;

    Objective To optimize chemically defined serum- and protein-free medium for MDCK cells by Plackett-Burman experiment design combined with response surface analysis. Methods For component screening and quantitative optimization,23 groups of nutrients were investigated by the Plackett-Burman design and response surface analysis based on an in-house protein containing serum-free medium serving the specific growth rate,time duration and maximum viable cell density as response values. Chemical defined serum- and protein-free medium was formulated with the components at optimal concentrations,with which MDCK cells were cultured in cell culture plate and stirring bottle,and the response values were evaluated. Results Four factors promoting the growth of MDCK cells significantly,cysteine,threonine,leucine and glucose,were screened by Plackett-Burman test. Threonine and lecucine were helpful to increasing the specific cell growth rate,while cysteine and glucose to increasing maximum viable cell density. The chemical defined serum-free and protein-free medium supported the suspending growth of MDCK cells effectively. The specific growth rate and maximum viable cell density of MDCK cells in single-cell suspension culture reached 0. 058 / h and 32. 65 × 105cells / ml,which are 2. 15 and 3. 09 times higher than those in basal medium,respectively. Conclusion The chemically defined serum- and protein-free medium for MDCK cells was optimized by Plackett-Burman experiment design combined with response surface analysis,which laid a foundation of industrial production of influenza vaccine by using MDCK cells.

    2014 06 v.27 [Abstract][OnlineView][Download 1802K]

  • Optimization and verification of determination method for residual deoxysodium cholate content in pneumococcal polysaccharide

    SUN Sheng-jun;HUI Zeng-di;ZHAO Zhe;LU Wei;CAI Fang;GAO Qiang;Beijing Sinovac Biotech Co.,Ltd.;

    Objective To optimize the determination method for residual deoxysodium cholate content in pneumococcal polysaccharide. Methods The determination method for residual deoxysodium cholate content in bulk of pneumococcal polysaccharide was verified for suitability by the method in Chinese Pharmacopoeia(Volume Ⅲ,2010 edition). The absorbance of reference after incubation in 70 ℃ water bath for 20,40,60 and 120 min and in 80 ℃ water bath for 30 min were determined,based on which the reaction temperature and time were optimized. The optimized method was verified for linearity,reproducibility and accuracy. Results The residual deoxysodium cholate content in two batches of bulks of pneumococcal polysaccharide after incubation in 70 ℃ water bath for 20 min were determined,and the result showed recoveries of 8. 6% ~ 240. 8%,indicating that the suitability was unqualified. The condition for reaction was optimized as incubation in 80 ℃ water bath for 30 min. The deoxysodium cholate contents at a range of 10 ~ 80 μg / ml showed good linearity with the absorbances,with a R2 value of more than 0. 99. The CVs of determination results of three batches of references in reproducibility test were less than 8%. The recovery rates of deoxysodium cholate at low,moderate and high concentrations,added to 23 kinds of bulks of pneumococcal polysaccharide,were 80% ~ 120%.Conclusion The optimized method was simple,which showed good linearity,accuracy and reproducibility,and might be used for determination of residual deoxysodium cholate content in pneumococcal polysaccharide.

    2014 06 v.27 [Abstract][OnlineView][Download 578K]

  • Study on effect of recombinant endostatin on endothelial cells by atomic force microscope

    HE Wei;WANG Qun;LU Hui-li;ZHANG Jing;HAN Hui-li;Changchun Institute of Biological Products Co.,Ltd.;

    Objective To study the effect of recombinant endostatin(rhES)on endothelial cells by atomic force microscope(AFM). Methods The proliferative activities of human umbilical vein endothelial ECV304 cells treated with rhES at various concentrations(0. 05 ~ 2. 4 μg / ml)were determined by MTT assay. ECV304 cells were treated with 0. 8 and2 μg / ml rhES respectively,then observed for overall morphology by AFM,and for local morphology on surface by SPI3800 New DFM. Results The rhES inhibited the proliferation of ECV304 cells significantly(P < 0. 001),and decreased the thickness of adherent ECV304 cells,both in dose-dependent modes. The surface of cells after treatment with rhES changed from smooth to rough,on which some small processes were formed. However,the surface structure of ECV304 cells after treatment with rhES showed an irregular change. Conclusion The samples for atomic force microscopy were easy to prepare,while the resolving power was high,indicating that the method was suitable for in situ observation of adherent cells.

    2014 06 v.27 [Abstract][OnlineView][Download 942K]

  • Method for and advance in research on neutralizing epitope of EV71

    GAO Wen-chao;GUO Tai;WANG Yu-lin;Department of Enterovirus Vaccine,National Institutes for Food and Drug;

    As a member of Enterovirus family in Picornaradae,enterovirus type 71(EV71)is a major pathogen of hand,foot and mouth disease(HFMD)in Asian-Pacific region. EV71 causes severe neuropathy,resulting in high mortality in the children at ages of less than 6 years. There are no effective vaccines to prevent EV71 infection at present,so it is urgent to developing vaccines and drugs against the virus. Virus neutralizing epitope is the material basis of specific immune response,of which the study on structure and function is vital for development of vaccines,especially the preparation of subunit vaccines and screening of drug target. This paper reviews the method for research on EV71 epitope and the advance in research.

    2014 06 v.27 [Abstract][OnlineView][Download 528K]

  • Application of PD-1 antibody in therapy of tumors

    CAO Jia-bin;WEI Jing-shuang;NCPC New Drug Research and Development Co. Ltd.,State Key Laboratory of Antibody Drug Development;

    Tumors are closely related to immune system and they can escape immune surveillance by stimulating immune inhibitory receptors,which can be blocked by the inhibitory receptors on the T cell. This provides a potential method for anti-tumor immune therapy. PD-1 is an inhibitory receptor with sustained expression in T cell dysfunction,of which the primary function is to limit the T cell effector as the stop signal. Blocking PD-1 signal is effective in helping exhausted T cells,which plays an important role in promoting the anti-tumor immune response. This review summarizes PD-1 and its ligands,PD-1 / PD-Ls signaling pathway as well as the application of PD-1 antibody in the therapy of tumors.

    2014 06 v.27 [Abstract][OnlineView][Download 765K]

  • Progress in research on immunotherapy with cytokine-induced killer cells

    WU Nian;FENG Hu-yi;Department of General Surgery,Chongqing Fifth People's Hospital;

    Adoptive immunotherapy holds great promises in the scenario of potential new approaches for the treatment of solid tumors refractory to conventional therapies. Cytokine-induced killer(CIK)cells are a heterogeneous subset of ex-vivo expanded T lymphocytes which present a mixed T-NK phenotype and are endowed with a MHC-unrestricted antitumor activity. Previous reports have suggested that treatment with CIK cells may benefit the patients with various types of tumors. This paper provides an overview of progress in research on the biological characters,action mechanism and clinically curative effect of CIK cells.

    2014 06 v.27 [Abstract][OnlineView][Download 535K]

  • Advance in study on general principles of nomenclature of biologicals in China

    REN Yue-ming;ZHANG Shi-bin;Chinese Pharmacopoeia Commission;

    The management of drug name is of an important significance,of which the standardization is the key to strengthening the surveillance of drugs and protecting the public health. The current General Principles of Nomenclature of Biologicals is complicated,which needs to be further standardized. This paper reviews the principles,advances and problems in nomenclature of the rapeutic biotherapeutics in China.

    2014 06 v.27 [Abstract][OnlineView][Download 525K]


@2012 Editorial Department of "Chinese Journal of Biologicals"