2015年03期目次

  • Immune effects of inactivated rotavirus vaccine combined with inactivated poliovirus vaccine

    NIU Xiang-lian;ZHANG Guang-ming;WU Jin-yuan;MI Kai;XIE Li;SUN Xiao-qin;LI Hong-jun;SUN Mao-sheng;Institute of Medical Biology, Chinese Academy of Medical Science;

    Objective To evaluate the immunogenicity and interaction of co-vaccination with inactivated rotavirus vaccine(IRV)and inactivated poliovirus vaccine(IPV)in rats. Methods Wistar rats were randomly divided into co-vaccination,alternative vaccination as well as IPV and IRV groups. The rats in co-vaccination group were vaccinated with IPV at first and, 4 weeks later, vaccinated twice with IPV and IRV in right and left posterior limbs respectively, at an interval of 4weeks. The rats in IPV and IRV groups were vaccinated with three doses of IPV and two doses of IRV respectively, each at an interval of 4 weeks. However, the rats in alternative vaccination group were vaccinated with IPV at first and, 4 weeks later, alternatively vaccinated with IPV and IRV for 6 weeks, each at an interval of 2 weeks. Venous blood samples were collected 4 weeks after each vaccination, from which sera were separated and determined for neutralizing antibody levels against rotavirus(RV)and poliovirus by microneutralization test, and for specific Ig G level against RV by ELISA. Results Both geometric mean titers(GMTs)of neutralizing antibody and specific Ig G against RV of rats after vaccination with two doses of IRV increased significantly, while the antibody positive conversion rates were both 100%. However, after three doses of IPV, the GMTs of neutralizing antibodies against poliovirus of types Ⅰ, Ⅱ and Ⅲ increased significantly, while all the antibody positive conversion rates were 100%. No significant differences were observed in the levels of neutralizing antibodies against RV and poliovirus as well as specific Ig G against RV in various groups(P > 0. 05). Conclusion Both IRV and IPV showed good immune effects in rats, which showed no interference when administered in combination.

    2015 03 v.28 [Abstract][OnlineView][Download 281K]

  • Consistency of various batches of inactivated enterovirus 71 vaccine

    ZHANG Ying;LIU Long-ding;DONG Cheng-hong;WANG Li-chun;LIAO Yun;GUO Lei;WANG Jing-jing;LIANG Yan;FENG Min;CUI Wei;LI Qi-han;Institute of Medical Biology, Chinese Academy of Medicine Sciences & Peking Union Medical College,Yunnan Key Laboratory of Vaccine Research and Development on Severe Infectious Diseases;

    Objective To investigate the consistency of immunogenicity and safety of full course of vaccination with inac-tivated enterovirus 71(EV71) vaccine(human diploid cells) of various batches. Methods Three batches of inactivated EV71 vaccine were determined for antigen content, free formaldehyde content, aluminum content, endotoxin content,residual antibiotic content, abnormal toxicity and potency. A random and double-blind trial was carried out on the three batches of vaccine in 900 children at ages of 6 ~ 72 months. The children were immunized on days 0 and 28, of whom serum samples were collected on days 0(before immunization) and 56, and determined for neutralizing antibody titer against EV71 by micro-CPE method. Immediate reactions were recorded within 30 min, while the recruiting local and systemic symptoms within 0 ~ 7 d, and the non-recruiting adverse reactions within 0 ~ 28 d, after each dose. The severely adverse events were reported during the whole observation. Results All the control test results of three batches of final products of inactivated EV71 vaccine were within the range of qualification. The antibody positive conversion rates of children after immunization with two doses of inactivated EV71 vaccine of Lots 20120101, 20120102 and 20120203 were98. 00%, 95. 67% and 98. 67%, while those of susceptible children(with neutralizing antibody titers of less than 1 ∶ 8)were 96. 45%, 92. 44% and 97. 60%, respectively. However, the 4-fold increasing rates of antibody titers of unsusceptible children(with neutralizing antibody titers of not less than 1 ∶ 8)after immunization with the three batches were 67. 94%,65. 63% and 71. 43%, while the GMTs of antibody were 283. 79, 231. 48 and 285. 78, respectively, which showed no sig-nificant difference(each P > 0. 05). The adverse reactions and severely adverse events induced by the vaccine of the three batches showed no significant difference(P > 0. 05). Conclusion The production procedure of inactivated EV71 vaccine was stable as proved by comparison of quality control indexes and induced antibody levels of three batches of vaccine.

    2015 03 v.28 [Abstract][OnlineView][Download 302K]

  • Immunological evaluation on live Brucella vaccine for intradermal injection

    CHEN Cheng;WEI Dong;LI Ke-mei;FU Li-li;HUANG Chang-jiang;WANG Guo-zhi;National Institutes for Food and Drug Control;

    Objective To evaluate the humoral and cellular immune responses as well as protective immunity of live Brucella vaccine for intradermal injection in mice. Methods Thirty mice were randomly divided into three groups, ten for each, and immunized i. d. with live Brucella vaccine at low(5 × 107 bacteria)and high(2 × 108 bacteria)and physiological saline, each in a volume of 0. 1 ml, respectively. Serum samples were collected 4 weeks after immunization, while the splenic lymphocytes were prepared. The Ig G titer against Br-PPD in sera was determined by ELISA, while the splenic lymphocytes secreting IFNγ and IL-4 were counted by ELISPOT. The IFNγ level secreted by mouse splenic lymphocytes after stimulation in vitro was determined by ELISA. The T cell subsets were tested by flow cytometry. The immunized mice were challenged with attenuated Brucella melitensis M5 strain, and the immune protection of vaccine was evaluated by the bacterial count in spleen. Results The GMTs of Ig G against Br-PPD in sera of mice immunized with vaccine at low and high dosages were 642 and 557 respectively. The counts of splenic lymphocytes secreting IFNγ and IL-4 as well as the secreted IFNγ contents after stimulation with Br-PPD of mice immunized with the vaccine at low and high dosages showed no significant difference with(each P < 0. 05), while the proportions of CD4+ IFN-γ+ and CD4+ IL-4+ lymphocytes were significantly higher, than those in control group(each P < 0. 05). Both the bacterial counts in spleens of mice immunized with vaccine at low and high dosages were 0, while that in control group was(5. 13 ± 0. 16) log10 CFU. Conclusion Live Brucella vaccine for intradermal injection showed strong humoral and cellular responses and protective immunity in mice.

    2015 03 v.28 [Abstract][OnlineView][Download 293K]

  • Expression of truncated human papilomavirus 58 L1 protein in insect cells

    LIU Jing-hui;LIU Yu-lin;CHU Di;CHE Wei-wei;Changchun Institute of Biological Products Co. Ltd.;

    Objective To construct a recombinant baculovirus with truncated human papillomavirus 58 L1 gene by using Bac-to-Bac expression system and express in Sf9 cells. Methods HPV 58 L1 gene was collected from NCBI and aligned by Clustal X2 software to obtain the relatively conserverd target gene sequences. The gene was synthesized, based on which truncated HPV58 L1-1 gene was amplified by PCR and cloned into vector p Fast Bac-1. The constructed recombinant plasmid p FB-HPV 58 L1-1 was transformed to E. coli DH10 Bac. The obtained recombinant strain Bacmid-HPV 58 L1-1was transfected to Sf9 cells to obtain the passage 1(P1) of recombinant baculovirus BV-HPV 58 L1-1 which was determined for titer by rapid titration after propogation. The recombinant baculovirus was infected to Sf9 cells, and the ex-pressed HPV58 L1-1 protein was identified by Western blot and transmission electron microscopy. Results PCR and sequencing proved that Bacmid-HPV58L1-1 was constructed correctly. The recombinant baculovirus of P2 reached a titer of1. 0 × 108 pfu / ml. The expressed HPV 58 L1-1 showed a specific band with relative molecular mass of about 55 000 on Western blot profile, and assembled spontaneously into virus-like particles(VLPs)under electron microscope. Conclusion HPV 58 L1-1 was successfully expressed in insect cells and assembled spontaneously into VLPs, which laid a foundation of study on prophylactic HPV vaccine.

    2015 03 v.28 [Abstract][OnlineView][Download 638K]

  • Construction of E. coli displaying streptococcus Gap C1 on its surface by RED homologous recombination

    YANG Xi-jing;ZHANG Jian-xin;CHEN Xiao-ting;TANG Wei;YU Si-miao;LIU Wei;YAO Di;WU Zhi-jun;YU Li-quan;ZHU Zhan-bo;CUI Yu-dong;College of Animal Science and Technology, Heilongjiang Bayi Agricultural University;

    Objective To construct an E. coli strain displaying the streptococcus Gap C1-150 aa(Gap C1)on its surface by RED homologous recombination and lay a foundation of further construction of surface display recombinant strains of E. coli.Methods Primers were designed according to the known gap C gene sequence(GI:30348860)in Gen Bank, and the target genes were amplified by PCR using plasmids p KD3 and p QE30 / lpp-omp A-gap C as templates, then fused, transformed to E. coli HB101 containing plasmid p KD46, and recombined into the genome by using RED system encoded by p KD46.Recombinant E. coli HB101 LOG, in which plasmid p KD46 was removed, was obtained by screening with ampicillin(Amp)and chloramphenicol(Cm), identified by PCR, Western blot, flow cytometry, fluorescent microscopy, and determined for bioactivity. Results Recombinant E. coli HB101 LOG was constructed correctly as proved by PCR, on the surface of which foreign protein Gap C1-150 aawas expressed. HB101 LOG showed obvious green fluorescence under laser confocal mi croscope, while HB101 showed no green fluorescence. Both the growth curves of HB101 LOG and HB101 were of the characteristics of E. coli. Conclusion A recombinant E. coli strain displaying streptococcus Gap C1-150 aawas successfully constructed by using RED system.

    2015 03 v.28 [Abstract][OnlineView][Download 522K]

  • Expression of human papillomavirus L1 proteins in E. coli

    YANG Xu;XIA Ye;LI Ya-dong;JIN Xiao-mei;LONG Qiong;SUN Wen-jia;HUANG Wei-wei;LIU Cun-bao;YAO Yu-feng;MA Yan-bing;Institute of Medical Biology, Chinese Academy of Medical Sciences & Peking Union Medical College, Yunnan Key Laboratory of Vaccine Research & Development on Severe Infectious Diseases, Yunnan Engineering Research Center of Vaccine Research & Development on Severe Infectious Diseases;

    Objective To investigate the influence of various factors in E. coli on expression of high-risk human papillomavirus(HPV)L1 proteins and lay a foundation of development of low-cost and abroad vaccines against multi-genotypes of HPV. Methods Recombinant plasmids for non-fusion and fusion expressions of HPV L1 protein as well as that for fusion expression of HPV L1 and glutathione-S-transferase(GST) were constructed with non-fusion gene, different modes of fusion gene, HPV L1 sequence of different genotypes and the L1 sequence of identical genotype and different characters,and transformed to various host bacteria(E. coli DH5α and E. coli BL21), and induced with IPTG at various temperatures(37 and 20 ℃). The fusion protein were analyzed for expression and solubility by 12% SDS-PAGE, and identified by Western blot. Results SDS-PAGE showed that GST-HPV16 L1 fusion protein was highly expressed in E. coli DH5α,while no non-fusion expression of target protein or expression of Trx fusion protein was observed. Host strain showed no significant influence on the expression levels of GST fusion proteins. The expressed GST fusion proteins existed in a form of inclusion body at the culture temperature of 37 ℃. However, at 20 ℃, partial expressed proteins existed in a soluble form. The expression levels of various HPV16 L1 gene sequences were in agreement. Like HPV16 L1, both HPV18 L1 and HPV58 L1 genes after optimization of yeast codon were highly expressed in forms of GST fusion proteins. Conclusion Major L1 proteins from multi-genotypes of HPV were highly expressed in soluble and GST-fusion forms in E. coli, which laid a foundation of disassembling of virus-like particles(VLPs)in vitro and provided a reference for the high expression of other heterologous genes in E. coli.

    2015 03 v.28 [Abstract][OnlineView][Download 624K]

  • Screening of prokaryotic enhancer-like sequences and identification of its domains

    CUI Cheng-cheng;WANG Ying-ming;BI Yan-hong;LI Pan;YANG Si-da;HUANG Fen;ZENG Wei-kun;JING Shen-rong;Medical Faculty, Kunming University of Science and Technology;

    Objective To screen prokaryotic enhancer-like sequences from the microorgans enriched in collected sewage and soil samples to construct expression vectors, and identify its domains by construction of depletion mutant. Methods The major capsid protein gene L1(named as L11)of truncated human papillomavirus(HPV)was linked to chloramphenicol acetyltransferase(CAT) gene, and used as a reporter gene for screening of sequences with activity of enhancer from the mi croorgans enriched in collected sewage and soil samples, based on which the expression vector containing enhancer-like sequences was constructed, and the expressed HPV L11 protein was determined for enhancing activity. The depletion mutant of ER1 was constructed, by which the effect of various mutants on expression of L11 protein was evaluated to de termine the domains. Results An enhancer-like sequence was screened from the samples, which increased the resistance of bacterial strain to chloramphenicol by 5 folds; and the expression level of HPV L11 protein by 1. 78 folds. The activity-enhancing region was located in 117 ~ 317 bp. Conclusion An enhancer-like sequences was successfully screened from the collected sewage and soil samples, and the expression vectors carrying the sequences increased the ex-pression level of target protein.

    2015 03 v.28 [Abstract][OnlineView][Download 592K]

  • Preparation and purification of chimeric virus-like particles of HCV neutralizing epitopes and HBV S antigen

    SHU Fang;WANG Xiao-yan;LEI Ying-feng;LIN Fang;WANG Xi;LI Bin;LIU Chong;ZHANG Hui-zhong;WEI San-hua;Department of Clinical Laboratory Medicine, Tangdu Hospital, Fourth Military Medical University;

    Objective To prepare, purify and identify the chimeric virus-like particles(VLPs) of HCV neutralizing epitopes and HBV S antigen. Methods Four recombinant plasmids for chimeric expression of HCV neutralizing epitopes and HBV S antigen, p CI-HBSE1, p CI-HBSE2, p CI-HBSE3 and p CI-HBSE4, were transfected to HEK293 T cells in mediation of liposome respectively, and the culture supernatants were collected 48 h later. The obtained self-assembled HBV VLPs chimerized with HCV neutralizing epitopes, named as VLPs-SE1, VLPs-SE2, VLPs-SE3 and VLPs-SE4 respectively,were purified by sucrose density gradient centrifugation, concentrated by dialysis, observed by electron microscopy, deter-mined for HBs Ag content by electrochemical luminescence assay, and tested for reactions with the sera of patients infected with HCV by ELISA. Results Four kinds of chimeric VLPs were observed by electron microscopy, each at an size of about 22 nm. The HBs Ag content in VLPs-SE2 with the highest concentration reached 5. 51 × 103 ng / ml, which met the needs for further study. The serum neutralizing antibody levels of patients infected with HCV determined using pooled VLPs as coating antigen were slightly higher than those using single VLPs. Conclusion The chimeric VLPs of HCV neutralizing epitopes and HBV S antigen was prepared successfully, which laid a foundation of further evaluation of the neu-tralizing antibody and its protective effect induced by VLPs in vivo.

    2015 03 v.28 [Abstract][OnlineView][Download 504K]

  • Fusion expression and purification of bovine Clostridium perfringens α-ε fusion toxin gene

    YIN Hui;ZHAO Da;CHEN Wei-hong;GAO Jia-bin;QIAO Bo;CHEN Nan-nan;HU Xu;LIANG Hong-ru;JIANG Dong-jun;WANG He;ZHU Zhan-bo;College of Animal Science and Veterinary Medicine, Heilongjiang Bayi Agricultural University;JCollege of Animal Science and Veterinary Medicine, Heilongjiang Bayi Agricultural University;

    Objective To express and purify the fusion gene α-ε of bovine Clostridium perfringens. Methods Partial αtoxin gene fragment was amplified from the genomic DNA of C57-8 strain of Clostridium perfringens type A by PCR and inserted into plasmid p ET28a-ε. The constructed recombinant plasmid p ET28a-α-ε was transformed to competent E. coli BL21(DE3)plys S and induced with IPTG. The expressed recombinant protein was purified by Ni-Agarose His tag purification kit and identified by Western blot. Results Both PCR and restriction analysis proved that recombinant plasmid p ET28a-α-ε was constructed correctly, of which 99% of the gene sequence was consistent with that expected. The expressed recombinant protein, with a relative molecular mass of about 78 000, mainly in a form of inclusion body, contained22. 7% of total somatic protein, and reached a purity of 91. 2%, which showed specific binding to polyclonal antisera against mouse C. perfringens, indicating a good reactogenicity. Conclusion Recombinant plasmid p ET28a-α-ε was successfully constructed, and recombinant α-ε fusion protein was expressed in E. coli BL21(DE3) plys S. The expressed fusion protein showed good reactogenicity, which might be used as a candidate antigen for recombinant subunit vaccine against cattle enterotoxemia.

    2015 03 v.28 [Abstract][OnlineView][Download 445K]

  • Prokaryotic expression and purification of multicopy Gn RH analogue

    YANG Yang;FU Jia-fang;XU Xiao-bin;WANG Shi-li;Shandong Medicinal Biotechnology Center,Key Laboratory for Biotech-Drugs, Ministry of Public Health;

    Objective To express gonadotropin releasing hormone(Gn RH) analogue in prokaryotic cells and purify the expressed product. Methods The 8Gn RH analogue was synthesized, based on which the recombinant plasmid containing16 Gn RH analogue tandem repeats was constructed by isocaudamer technology. The 8Gn RH and 16 Gn RH analogues were inserted into prokaryotic expression vector p GEX- 2t, and the constructed recombinant plasmids were transformed to E. coli BL21. The recombinant 8Gn RH analogue or 16 Gn RH analogue expressed after induction with IPTG under the optimized condition was purified by Glutathione Sepharose 4B column chromatography, and identified by SDS-PAGE and Western blot. Results Both restriction analysis and sequencing proved that recombinant plasmids p GEX-2t-8Gn RH analogue and p GEX-2t-16 Gn RH analogue were constructed correctly. The expression level of target protein in supernatant reached a peak value after induction with IPTG at a final concentration of 0. 2 mmol / L at 37 ℃ for 2 h. The expressed recombinant protein, with a relative molecular mass of about 37 000, showed specific binding to rabbit monoclonal antibody against Gn RH. The relative molecular mass and concentration of purified recombinant protein were about 1 400 and 260 μg / ml respectively. Conclusion Recombinant plasmid with multicopy Gn RH analogue gene was successfully constructed and highly expressed in E. coli, which laid a foundation of production of Gn RH analogue by multicopy method.

    2015 03 v.28 [Abstract][OnlineView][Download 495K]

  • Construction and identification of recombinant adenovirus with sh RNA of phosphoatase and tensin homolog gene

    FU Shi-xin;DENG Qing-hua;HAN Ying-ying;YANG Wen-tao;LI Xiao-bing;WANG Zhe;Department of Clinical Veterinary Medicine,College of Animal Science and Veterinary Medicine,Heilongjiang Bayi Agricultural University;

    Objective To construct a recombinant adenovirus with sh RNA of phosphoatase and tensin homolog(PTEN)gene,and observe its effect on the expression level of PTEN m RNA in hepatocytes of newborn calf. Methods The psh RNAs of four PETN genes were transfected to primary hepatocytes of newborn calf in mediation of Lipofectamine2000,of which those with the most satisfactory transfection efficacy were screened and inserted into shuttle vector p Shuttle-EGFP-CMV. The constructed recombinant shuttle plasmid p Shuttle-GFP-PTEN-sh RNA and adenovirus backbone vector p Adxsi were digested with restriction endonuclease and connected. The constructed recombinant adenovirus plasmid p Adxsi-GFP-PTEN-sh RNA was linearized with Pac Ⅰ and transfected to human kidney K293 cells for packaging. The obtained recombinant adenovirus Adxsi-GFP-PTEN-sh RNA was subjected to three cycles of propagation and determined for titer. The expression level of PTEN m RNA in calf hepatocytes was determined by fluorescent quantitative PCR.Results The expression levels of PTEN m RNAs in psh RNA1 and psh RNA3 groups decreased by 44% and 68%respectively(P < 0. 05)indicating p GPU6 / GFP / Neo-PTEN-sh RNA3 as the sptimal plasmid for transfection. Recombinant adenovirus plasmid p Adxsi-GF-PTEN-sh RNA was constructed correctly as proved by restriction analysis. The titer of recombinant adenovirus Adxsi-GFP-PTEN-sh RNA after packaging and three cycles of propagation were 1. 2 × 1010 pfu / ml.The expression levels of PTEN m RNA in hepatocytes of newborn calf were 42% and 51% of those in blank and negative control groups respectively(P < 0. 05). Conclusion Recombinant adenovirus Adxsi-GFP-PTEN-sh RNA was prepared successfully,which decreased the expression level of PTEN in hepatocytes of newborn calf and laid a foundation of further study on prevention and treatment of fatty liver in cows.

    2015 03 v.28 [Abstract][OnlineView][Download 419K]

  • Enhancement of thermo-stability of nitrile hydratase by homologous-fragment swapping

    FANG Yue-qin;CUI Wen-jing;CUI You-tian;CHEN Yan;ZHOU Zhe-min;School of Environment and Civil Engineering,Jiangnan Univeristy;

    Objective To improve the thermo-stability and catalytic activity of thermo-sensitive nitrile hydratase(NHase,EC 4. 2. 1. 84)of Psedomonas putida NRRL-18668 by molecular modification using computer-aided semi-rational design.Methods Homologous target fragments were screened by site-targeted amino acid recombination(STAR) software and molecular dynamic modeling using the NHases from Pseudonocardia thermophila JCM3095 and Comamonas testosterone 5-MGAM-4D as templates respectively. Seven chimeric NHases were generated by homologous fragment swapping on the corresponding fragment of NHase from Psedomonas putida NRRL-18668, and determined for thermo-stability and activity.The secondary structures of wild-type NHase and chimeric NHase 3AB were analyzed by circular dicroism(CD)spectrometry. Results Compared with that of wild-type NHase, the thermo-stabilities of seven chimeric NHases(1A, 2B,2C, 2BC, 3AB, 3AC and 3ABC) after treatment at 50 ℃ for 10 min increased by 1. 5 ~ 3. 5 folds. The thermo-stability and activity of NHase 3AB increased by 3. 5 and 1. 4 folds respectively. The α-helixe contents in secondary structures of wild-type NHase and chimeric NHase 3AB were(34. 56 ± 3. 21)% and(36. 88 ± 1. 41)%, while the β-sheet contents were(19. 78 ± 2. 14)% and(18. 69 ± 1. 74)%, respectively. Conclusion The thermo-sensitive NHase was rationally engineered to sets of thermo-stable chimeric enzyme using homologous fragment swapping by substituting themo-sensitive domains with homologous thermo-stable domain, of which the specific activity increased without alteration in secondary structure.

    2015 03 v.28 [Abstract][OnlineView][Download 542K]

  • Relationship between MIP-1a and persistent immune protection of vaccinia virus Tiantan strain

    NIU Yan;ZHANG Liang-yan;LI Heng-bin;XING Li;SHI Xin-fu;YU Shu;Department of Microbiology and Immunology, Inner Mongolia Medical College;

    Objective To analyze the relationship between MIP-1a and persistent immune protection of vaccinia virus Tiantan strain(VTT). Methods Ten C57 / BL6 mice and ten B6.129P2-Ccl3 model mice with MIP-1a gene knock out(KO)were immunized i. m. with VTT at a dosage of 2 × 107 PFU / ml respectively, and excited with VTT at the same dosage 30 and 180 d later. The mice were killed 3 d after excitation, of which the splenic lymphocytes were collected and determined for peripheral CD44+CD62L-CD4+effector memory T(Tem) cells by flow cytometry. The effect of deletion of MIP-1a on persistent immune memory was analyzed by staining of tetramer loading vaccinia virus-specific CD8+T cell epitopes(VACV-B8R20-27 Kb). Results The phenotypes of obtained CD4+ memory T cells were mainly CD4+CD44+CD62L-, i. e. Tem, while those of CD8+memory T cells in a small quantity were mainly Tem(CD44 +CD62L-). The percentages of VTT-specific CD8+T cells in B6.129P2-Ccl3 mice 30 and 180 d after excitation were significantly lower than those in C57 / BL6 mice. However, the percentage of tetramer+CD8+T cells 30 d was significantly higher than that180 d after excitation. Conclusion MIP-1a was associated with the persistent immune memory induced by vaccinia virus, which provided a reference for prolonging the immune memory of vaccine.

    2015 03 v.28 [Abstract][OnlineView][Download 430K]

  • Migration and differentiation of human umbilical stem cord blood stem cells in rats with focal cerebral ischemia

    ZHAO Kai;JIA Yu-chen;Hohhot First Hospital;

    Objective To observe the migration and differentiation of human umbilical cord blood stem cells(HUCBSCs)in rats with focal cerebral ischemia, and investigate the possible action mechanism of HUCBSCs in recovery of neural function of the rats. Methods Rat model of middle cerebral artery occlusion(MCAO) reperfusion was established by suture-occluded method. Twenty model rats were randomly divided into control(n = 5)and test(n = 15)groups. The rats in test groups were transplanted i. v. with 1 ml(2 × 106 cells) of HUCBSCs labeled with DAPI / CM-Dil 24 h after establishment of model, while those in control group with 1 ml of physiological saline. Modified neurological severity score(m NSS)was assessed before transplantation and 3, 7 and 15 d after transplantation, while the brain tissues of rats were prepared into frozen section and observed for morphology by HE staining, for migration and distribution of HUCBSCs by fluorescence microscopy, and for expressions of Nestin and neuron specific enolase(NES)by IFA. Results The m NSS of rats in test group was significantly lower than that in control group 7 and 15 d after transplantation(P < 0. 05). The pathological lesion in brain tissues of rats in test group 15 d after transplantation was relieved significantly as compared with that in control group. Positive cells were observed in and around the lesion site in injured hemisphere in test group3 d after transplantation, which increased as time went on. However, the number of cells migrated to the lesion site increased 7 d and reached the peak value 15 d after transplantation. The numbers of positive cells at various time points showed significant difference(P < 0. 05). The percentages of cells positive for CM-Dil / NSE-FITC and CM-Dil / NestinFITC in brain tissues of rats 15 d after transplantation were 85. 2% and 81. 6% respectively. Conclusion HUCBSCs transplanted intravenously may survive in rats and be migrated to the cerebral ischemic lesion site. With the potential of differentiation to neurocytes, HUCBSCs may promote the recovery of neural function of rats with focal cerebral ischemia.

    2015 03 v.28 [Abstract][OnlineView][Download 513K]

  • Preparation and application of monoclonal antibodies against capsular polysaccharides of Neisseria meningitides serogroups A, C, W135 and Y

    ZHANG Ping-ping;DENG Jie;ZHANG Xiao-gang;SUN Zhe;ZHU Tao;Ab Max Biotechnology Co. Ltd.;

    Objective To prepare monoclonal antibodies(Mc Abs) against capsular polysaccharides of Neisseria meningitides serogroups A, C, W135 and Y(shortened as GAMP, GCMP, GWMP and GYMP respectively), and apply to immunoturbidimetric assay. Methods BALB / c mice were immunized with the conjugates of N. meningitides capsular polysaccharides to avirulent variant CRM197 of diphtheria toxin, i. e. GAMP-CRM197, GCMP-CRM197, GWMP-CRM197 and GYMP-CRM197 respectively, of which the spleen cells were fused with myeloma SP2 / 0 cells. Hybridoma cell strains were screened by indirect ELISA, tested for specificity and determined for the titer of purified antibodies. One clone of each group was selected for immunoturbidimetric assay. Results Twenty, eighteen, nine and thirteen strains secreting Mc Abs against GAMP, GCMP, GWMP and GYMP were obtained respectively, of which five against GAMP, five against GCMP, three against GWMP and five aginst GYMP showed high specificities, without cross reactions with CRM197 or polysaccharides of other three serogroups. The titers of purified antibodies against GAMP, GCMP, GWMP and GYMP were more than 1 ∶ 4 000 000, more than 1 ∶ 200 000, 1 ∶ 40 000 and 1 ∶ 40 000 respectively. Mc Abs 6B7 against GAMP and 2H4 against GCMP were suitable for immunoturbidimetric assay within a standard concentration range of 2 ~10 μg / ml, with R2 values of 0. 990 9 and 0. 991 9 respectively. However, Mc Abs 5F1 against GWMP and 7C6 against GYMP were suitable for the assay within standard concentration ranges of 4. 33 ~ 21. 7 and 1 ~ 8 μg / ml, with R2 values of 0. 851 8 and 0. 998 7, respectively. Conclusion Monoclonal antibodies against GAMP, GCMP, GWMP and GYWP were successfully prepared and applied to immunoturbidimetric assay.

    2015 03 v.28 [Abstract][OnlineView][Download 496K]

  • Influencing factors of high immune response in newborns induced by hepatitis B vaccine in Haidian District, Beijing

    FU Ji-ye;DING Qi;SHI Ru-jing;WANG Ju-guang;ZHANG Wei;Beijing Haidian District Center for Diseases Control and Prevention;

    Objective To investigate the immune effect of newborns who received a full course of vaccination with 10 μg hepatitis B vaccine(Hep B)prepared with yeast cells within 12 months after birth in Haidian District, Beijing City, China,and to analyze the influencing factors of high anti-HBs response. Methods The parents of infants who lived in Beijing for not less than 6 months and received a full course of vaccination with Hep B within 12 months after birth were investigated for the general status of infants, including Hep B vaccination, bodyweight, height, bodyweight on birth, premature birth,mode of delivery, feeding pattern, as well as their mother's statuses of hepatitis B surface antigen(HBs Ag) and hepatitis e antigen(HBe Ag)and their father's status of HBs Ag. Venous blood samples were collected from the infants, 2 ml for each,from which sera were separated and determined for anti-HBs and HBs Ag. A non-conditional multivariate Logistic regression model was used to analyze the influencing factors of high anti-HBs(not less than 1 000 IU / L)response rate and estimate the odds ratio(OR) and 95% confidence interval(CI) of various factors. Results A total of 691 infants at ages of 7 ~15 months were investigated, of whom 666 were included into the analysis. All the infants were negative for HBs Ag, of whom 99. 8%(665 / 666)were positive for anti-HBs(not less than 10 IU / L). Low(the anti-HBs level was not less than10 IU / L but less than 100 IU / L), moderate(the anti-HBs level was not less than 100 IU / L but less than 1 000 IU / L)and high responses were observed in 1. 2%, 34. 0% and 64. 8% of the 665 infants positive for anti-HBs. Logistic regression model indicated high anti-HBs response appeared more in male infants after a full course of vaccination with Hep B than in female infants(OR = 1. 396, 95% CI: 1. 010 ~ 1. 930). However, the high anti-HBs response rate in infants whose serum samples were collected at least 60 d after the third vaccination(Hep B3)were lower than those within 60 d(OR = 0. 326,95% CI: 0. 186 ~ 0. 573). Conclusion High anti-HBs positive rate was observed in the infants receiving a full course of vaccination with Hep B within 12 months after birth in Haidian District, Beijing City. In the anti-HBs positive infants, high response rate decreased with the increasing time interval between collection of serum samples and the third vaccination,which was higher in male infants than in female ones.

    2015 03 v.28 [Abstract][OnlineView][Download 516K]

  • Safety evaluation on influenza vaccine(split virion)in population at ages of more than 60 years during 20112012 in Beijing

    LIU Xiao-qin;MA Yan-li;YANG Zhong-nan;ZHANG Min;HUANG Xing-xiao;XU Bin;CHEN Hai-ping;China National Biotec Group Company Limited;

    Objective To evaluate the safety of influenza vaccine(split virion)manufactured by Beijing Tiantan Biological Products Co., Ltd, in population at ages of more than 60 years during 2011 ~ 2012 in Beijing City. Methods The data on adverse events following immunization(AEFIs) cases in people at ages of more than 60 years in Beijing City during2011 to 2012, after free vaccination with influenza vaccine(split virion), were collected by AEFI information system, and analyzed by descriptive methodology. Results Of the 536 812 recipients of vaccine manufactured by Beijing Tiantan Biological Products Co., Ltd, 10 cases of AEFIs were reported, including 6 cases of adverse reactions(3 cases of general reactions and 3 cases of abnormal reactions), indicating an incidence of adverse reactions of 1. 12 / 100 000. However, of the 701 820 recipients of vaccines by other manufacturers, 21 cases of AEFIs were reported, including 10 cases of adverse reactions(6 cases of general reactions and 4 cases of abnormal reactions), indicating an incidence of adverse reactions of1. 42 / 100 000. No significant differences were observed in reported incidences of total adverse reactions, general reactions and abnormal reactions(P > 0. 05). Conclusion The influenza vaccine(split virion) manufactured by Beijing Tiantan Biological Products Co., Ltd showed high safety, which might be used for the prevention of influenza in the relevant population.

    2015 03 v.28 [Abstract][OnlineView][Download 407K]

  • Determination of osmolality of live attenuated Japanese encephalitis vaccine and two kinds of meningococcal polysaccharide vaccine by freezing point osmometer

    JI Hong;DING Rui;LIU Jing;MA Xun;QI Jin;WANG Wei;Beijing Institute for Drug Control;

    Objective To determine the osmolality of live attenuated Japanese encephalitis vaccine as well as group A and group ACYW135 meningococcal polysaccharide vaccines and the relevant diluents. Methods According to method in Chinese Pharmacopoeia(Volume Ⅲ, 2010 edition), 20 batches of commercial live attenuated Japanese encephalitis vaccine, 55 batches of group A meningococcal polysaccharide vaccine and 7 batches of group ACYW135 meningococcal polysaccharide vaccine as well as the corresponding diluents were determined for osmolality by freezing point osmometer.Results The osmolality of three kinds of vaccines ranged from 260 ~ 700 m Osmol / kg, which showed significant difference. Significant differences were observed in the osmolality of live attenuated Japanese encephalitis vaccine, but not in the other two kinds of meningococcal polysaccharide vaccines, in inter- and intra-assays. However, the osmolality of diluents attached with three kinds of vaccines also showed significant differences. Conclusion It is necessary to include the determination of osmolality into the routine quality control of the three kinds of vaccines.

    2015 03 v.28 [Abstract][OnlineView][Download 448K]

  • Development of sucrose cushion centrifugation method for large-scale purification of poliovirus

    ZHAO Bing-xin;DAI Su-ping;PAN Xiao-xia;XIA Wen-yue;WEN Yu-ling;LIAO Guo-yang;CEHN Yuan-ding;Institute of Medical Biology, Chinese Academy of Medical Sciences & Peking Union Medical College;

    Objective To develop a sucrose cushion centrifugation method for large-scale purification of poliovirus(PV).Methods PV liquid was harvested after propagation in a large quantity, concentrated with PEG, extracted with trifluorotrichloroethane, then purified by sucrose cushion centrifugation and cesium chloride density gradient centrifugation.Guinea pigs were immunized with PV of types Ⅰ, Ⅱ and Ⅲ respectively, of which serum samples were collected and determined for neutralizing antibody titer by micro-neutralization test. Results The particles of PV purified by sucrose cushion centrifugation showed typical morphology, of which the background was clear. In addition to the intact and smooth virus particles, a large quantity of empty virus particles were observed, indicating a satifactory purification effect. However,though the PV purified by cesium chloride density gradient centrifugation also showed typical morphology, intact virus particles were in a large quantity. The genomic nucleic acids were of obvious characters, of which the background was clear. No obvious difference was observed between the genomic nucleic acids of PV purified by two methods. The infec-tious titers and neutralizing antibody titers induced by PV of various types purified by sucrose cusion centrifugation were higher than those by cesium chloride density gradient centrifugation. Conclusion The PV purified by sucrose cushion centrifugation showed high purity and immunogenicity, indicating that the method was suitable for large scale purification of PV or other viruses.

    2015 03 v.28 [Abstract][OnlineView][Download 527K]

  • Development of a novel method for determination of residual toxicity of diphtheria toxoid

    MA Xiao;TAN Ya-jun;DONG Guo-xia;TIAN Lin;HOU Qi-ming;National Institute for Food and Drug Control, Key Laboratory of the Ministry of Health for Research on Quality and Standardization of Biotech Products;

    Objective To develop a novel method for determination of residual toxicity of diphtheria toxoid, instead of the current skin test in rabbits. Methods The residual toxicity of diphtheria toxoid was determined by Vero cell assay. The method was verified for specificity, sensitivity(minimum detection limit) and reproducibility, by which the determination result was compared with that by skin test in rabbits. Results Vero cell assay was specific to diphtheria toxoid, of which the minimum detection limit was 10-8LD50. The method showed high reproducibility, of which the sensitivity was 10 000 times higher than that of skin test in rabbits. Conclusion Vero cell assay may be used for determination of residual toxicity of diphtheria toxoid and toxicity reversion test. The method showed high specificity, of which the sensitivity was higher than that of skin test in rabbits. It was suitable for the safety evaluation on DT-based combined vaccines(DT combos).

    2015 03 v.28 [Abstract][OnlineView][Download 426K]

  • Progress in research on recombinant varicella-zoster virus vaccine

    LI Zhi-jun;HU Xin-yue;YIN Yu-tian;JIANG Ling-ling;LI Bo;Department of Epidemiology and Biostatistics, School of Public Health,Jilin University;

    The safety, efficacy and usage of varicella-zoster virus(VZV)vaccines have been accepted widely. Because of its mild adverse reaction and abroad genome, VZV strain may be used as a safe vector for recombinant vaccine, in which a large exogenous gene may be inserted. Since a single or multiple antigen genes may be inserted into the nonessential regions for replication in VZV strain, the vaccine strain as a vector may not only prevent the incidence of varicella and zoster, but also prevent and treat various infectious diseases in children and older person. This paper reviews the progress in research on construction and immune effect of recombinant VZV vaccine.

    2015 03 v.28 [Abstract][OnlineView][Download 438K]

  • Progress in research on recombinant vaccine against allergic disease

    ZHANG Ying-ying;LI Hui-qiang;Department of Clinical Laboratory Diagnosis, Tianjin Medical University;

    Allergic disease is an important health problem worldwide. Under most circumstances, it is difficult for patients to avoid contacting with allergens. Therefore, the specific immunotherapy has become an important measurement.With the deep understanding of epitopes and the development of recombinant protein expression, a novel vaccine for specific immunotherapy, recombinant vaccine, has been recognized by researchers and widely used. Recombinant vaccine has many advantages over the traditional one, such as safety, efficiency and specificity, with which the goal of individualized therapy may be achieved. This paper reviews the specific immunotherapy(SIT), recombinant vaccine as well as preparation and clinical trial of this kind of vaccine.

    2015 03 v.28 [Abstract][OnlineView][Download 422K]

  • Clinical progression of treatment of chronic hepatitis B with antiviral drugs

    JU Ping;CAI Wei-wang;Department of Laboratory Medicine, The Second Hospital of Huangshi City;

    Chronic hepatitis B is an infectious diseases which caused by hepatitis B virus. The virus impairs liver func-tion and, if it is not controlled timely, causes cirrhosis and fulminant hepatitis which threats the health of patients serious-ly. Antiviral therapy is the main method for treatment of chronic hepatitis B at present, while interferon, polysaccharide compounds and nucleotide analogues are the main antiviral drugs. This paper reviews the status of chronic hepatitis B, an-tiviral drugs and treatment protocol of chronic hepatitis B.

    2015 03 v.28 [Abstract][OnlineView][Download 402K]
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@2012 Editorial Department of "Chinese Journal of Biologicals"