2015年02期目次

  • Influence of aluminium hydroxide adjuvant on immune effect of inactivated enterovirus 71 vaccine containing various concentrations of antigen

    LI Hua;JIANG Guang-ju;YANG Ting;HE Xiao-juan;YUE Lei;XIE Tian-hong;YANG Shui-zhi;ZHU Fan-li;LONG Run-xiang;YANG Rong;LUO Fang-yu;XIE Zhong-ping;Institute of Medical Biology,Peking Union Medical College,Chinese Academy of Medical Science,Yunnan Provincial Engineering and Technological Research Center for Development of Vaccines against Major Infectious Diseases,Yunnan Provincial Key Laboratory for Development of Vaccines against Major Infectious Disease;

    Objective To analyze the influence of aluminium hydroxide adjuvant on immune effect of inactivated enterovirus 71(EV71) vaccine containing various concentrations of antigen. Methods ICR mice were randomly divided into nine groups,8 for each. The mice in six test groups were immunized with adjuvant-free and adjuvant-containing(1. 0 mg / ml aluminium hydroxide)inactivated EV71 vaccine at routine dosage(160 EU / 0. 1 ml EV71),1 / 2 dosage(80 EU / 0. 1 ml EV71)and 1 / 5 dosage(32 EU / 0. 1 ml EV71)respectively,while those in three control groups were immunized with aluminium hydroxide adjuvant and physiological saline and unimmunized(blank)respectively. Mice were immunized i. m. twice on days 0 and 28,of which the serum samples were collected 28 d after primary and booster immunizations respectively,and determined for neutralizing antibody titer by microneutralization test in vitro,and for IL-2,IL-4,IL-6,IL-17 A,IL-10,IFNγ and TNF levels by flow cytometry. Results The serum antibody positive rate was significantly higher in mice 28 d after primary immunization with adjuvant-containing vaccine at 1 / 2 dosage than in those with adjuvant-free vaccine(P < 0. 05),while showed no significant difference in other groups(P > 0. 05). Both the serum antibody positive rates of mice 28 d after booster immunization at routine dosage were 100%,while the GMTs were1 ∶ 152. 2 and 1 ∶ 139. 6 respectively. The GMTs of antibodies induced by adjuvant-containing vaccines at 1 / 2 dosage showed significant difference with those by adjuvant-free vaccines(P < 0. 05). However,no significant differences were observed in positive rates and GMTs of antibodies in other groups(P > 0. 05). The vaccines at routine,1 / 2 and 1 / 5dosages induced Il-6,IL-10,IFNγ and TNF,which showed significant difference with those in adjuvant and physiological saline control groups(P < 0. 05). However,no IL-2,IL-4 or IL-17 A was detected. Conclusion Inactivated EV71 vaccine induced specific anti-EV71 antibody as well as IL-6,IL-10,IFNγ and TNF,of which the effect was enhanced by adjuvant.

    2015 02 v.28 [Abstract][OnlineView][Download 208K]

  • Preparation and immune effect of chitosan-alginate microcapsules containing Vibrio harveyi antigen OmpK in orange-spotted groupers

    REN Yan;ZHANG Xiao-jiang;TAO Jia-fa;LI Ning-qiu;SHI Cun-bin;WU Shu-qin;Peal River Fisheries Research Institute,Chinese Academy of Fishery Sciences,Key Lab of Fishery Drug Development,Ministry of Agriculture, Key Lab of Aquatic Animal Immune Technology,Guangdong Province;

    Objective To prepare chitosan-alginate microcapsules containing the outer membrane protein(Omp K) of Vibrio harveyi and determine its immune effect in orange- spotted groupers by oral route. Methods The Omp K of V. harveyi was entrapped with chitosan-alginate microcapsules by emulsification-reemusification method,and the prepared microcapsules were determined for size,entrapment rate and antigen loading. Orange-spotted groupers were immunized twice with the prepared microcapsules by oral route at an interval of 2 weeks,while Omp K and blank controls were set up. The antibody levels in sera and intestine mucosa supernatant were determined by ELISA. The orange-spotted groupers were challenged i. p. with 1. 0 × 107 CFU / ml of V. harveyi 30 d after immunization,and the relative percent survivals(RPSs) of various groups were calculated. Results The chitosan-alginate microcapsules were in spherical shape at diameters of 5. 0 ~ 20. 0 μm,with a mean of 10. 6 μm. The mean encapusulation efficiency and loading percent of microspheres were 71. 26% and 1. 67% respectively. The maximum antibody titer in sera of orange-spotted groupers immunized with the prepared microcapsules was 29,which was significantly higher than that in Omp K group(P < 0. 01),while the absorbance(A450-A630)of hindgut mucus was higher than those in Omp K and blank control groups(P < 0. 01),and the RPS(62. 5%) was higher than that in Omp K control group(P < 0. 01). Conclusion The prepared chitosanalginate microcapsules showed good immune effect in orange-spotted groupers,which was feasible to be used as a delivery carrier of oral vaccine for fish.

    2015 02 v.28 [Abstract][OnlineView][Download 263K]

  • Complete genome sequencing of Zhong Ⅲ2 strain of live attenuated poliovirus vaccine

    DONG Li-juan;XIE Zhong-ping;LI Hua;YANG Ting;LONG Run-xiang;YUE Lei;YANG Rong;LUO Fang-yu;Yunnan Center for Disease Control and Prevention;

    Objective To sequence the complete genome of Zhong Ⅲ2strain of live atteanuted poliovirus vaccine,and analyze for homologies to those of P3 / Leon / 37 and Sabin Ⅲ strains. Methods The four large and eight small fragments which were overlapping and covered full-length genome of Zhong Ⅲ2strain were amplified by RT-PCR. The PCR products of intermediate fragments were sequenced directly,while the fragments at the end of genome were subjected to TA cloning and sequencing. The c DNA fragments obtained by two methods were spliced respectively,and the obtained fulllength c DNA sequence were analyzed for homologies to those of P3 / Leon / 37 and Sabin Ⅲ strains. Results The fulllength c DNAs of Zhong Ⅲ2strain,at a length of 7 432 bp,were obtained by both the methods,of which the base sequences were completely in agreement. Both the homologies of complete genome sequence of Zhong Ⅲ2strain to those of P3 / Leon / 37 and Sabin Ⅲ strains were 99%. There were 19 variations of base sequences in Zhong Ⅲ2and P3 / Leon /37 strains,3 in non-encoding region and 16 in encoding region. The six gene mutations in encoding region resulted in the variation of amino acid sequence. However,there were 28 variations of base sequences in Zhong Ⅲ2and Sabin Ⅲstrains,13 in non-encoding region and 15 in encoding region,and the three gene mutations in encoding region resulted in the variation of amino acid sequence. Conclusion The full-length of Zhong Ⅲ2strain of live attenuated poliovirus vaccine was 7 432 bp,while the homology to SabinⅢ strain was 99%.

    2015 02 v.28 [Abstract][OnlineView][Download 2183K]

  • Genetic stability and nucleotide difference of varicella-zoster virus Oka strain subcultured in human diploid cell SV-1 strain

    MENG Fan-hong;YU Wei;ZHANG Wei;YANG Li-wei;HE Yu-jun;GAO Qiang;HUANG Jin-feng;YIN Wei-dong;Sinovac(Dalian)Vaccine Technology Co., Ltd.;

    Objective To investigate the genetic stability of varicella-zoster virus(VZV)Oka strain during subculture in human diploid cell SV-1 strain, and analyze the complete gene sequences and differential sites of Oka strain of various passages. Methods Oka strain was subcultured in SV-1 cells to passage 48, and determined for virus titer. The complete genes of passages 33, 35, 38, 39 and 48 were sequenced, of which the data were spliced and analyzed by using DNASTAR.Lasergene. v 7. 1 software. Results The virus titers of Oka strain of passages 33 ~ 48 were 4. 000 ~ 4. 625 lg CCID50/ ml,with an arithmetic mean of 4. 292 lg CCID50/ ml. The full-length of genome of Oka strain was 125 114 bp, while the GC content was 46%. The homology of nucleotide sequences of Oka strain of passages 33, 35, 38, 39 and 48 was 99. 99%,while that of the five passages to standard vaccine strain V-Oka was 99. 96%. One differential site was observed in the complete gene sequence of Oka strain subcultured to passage 38 in MRC-5 and SV-1 cells. High homologies were observed in the gene sequences of Oka strain of various passages. However, as compared with those of vaccine strains Vari-vax and Varilrix, wild strain Dumas and parent strain p-Oka, the homology of gene sequence of Oka strain to that of vac-cine strain V-Oka strain was high. Conclusion VZV Oka strain showed high adaptability and genetic stability in SV-1 cells.

    2015 02 v.28 [Abstract][OnlineView][Download 280K]

  • Expression of E protein of West Nile virus by baculovirus expression vector system/insect cell system and identification of expressed product

    LI Ling;WANG Hua-lei;CAO Zeng-guo;JIN Hong-li;ZHENG Xue-xing;FENG Na;ZHAO Guo-xing;LI Qian;LI Nan;ZHAO Yong-kun;WANG Tie-cheng;GAO Yu-wei;YANG Song-tao;XIA Xian-zhu;College of Veterinary Medicine,Jilin University;

    Objective To express the envelope(E) protein of West Nile virus(WNV) by using baculovirus expression vector system(BEVS)/ insect cell(IC)system and identify the expressed product. Methods WNV E gene was cloned into baculovirus donor plasmid p Fast Bac1 by molecular cloning technique,and the constructed recombinant plasmid p Fast BacE was transformed to competent E. coli DH10 Bac. The obtained shuttle plasmid Bac-E was transfected to sf9 cells. Recombinant baculovirus Ac MNPV-E containing WNV E gene was harvested and determined for expression of E protein by indirect immunofluorescence assay(IFA) and Western blot. Results Both PCR and restriction analysis proved that recombinant donor plasmid p Fast Bac-E was constructed correctly. The sf9 cells were enlarged,round,granulated then fell off the culture plate and floated about 96 h after transfection. The harvested Ac MNPV-E of passage 1 was identified as positive by PCR. Green fluorescence was observed around sf9 cells infected with recombinant baculovirus Ac MNPV-E.The expressed protein showed specific binding to rabbit anti-WNV E polyclonal antibody and mouse anti-His monoclonal antibody,showing a protein band with relative molecular mass of about 53 000. Conclusion WNV E protein was successfully expressed by BEVS / IC system,which laid a foundation of development of a method for rapid diagnosis of WNV as well as novel vaccines.

    2015 02 v.28 [Abstract][OnlineView][Download 648K]

  • Effect of sodium chloride content on crystalline form of freeze-dried human coagulation factor Ⅷ

    JIANG Gui-xiang;ZHANG Wei;ZHU Guang-zu;LV Ying-nian;Guangdong Shuanglin Bio-pharmaceutical Co. , Ltd. , the Engineering Center of Blood Products in Guangdong;

    Objective To investigate the relationship between sodium chloride content and crystalline form of freeze-dried human coagulation factor Ⅷ(FⅧ)and provide a basis for the quality control of FⅧ. Methods Highly purified FⅧ was prepared by anion-exchange chromatography, then added with sodium chloride to low(40 mmol / L), moderate(80 mmol / L)and high(160 mmol / L) concentrations respectively and lyophilized in vacuum, using the freeze-dried F Ⅷ containing160 mmol / L sodium chloride and 100 mmol / L glycine as control. The appearance of freeze-dried FⅧ was examined visually, while the crystalline form was observed by scanning electron microscopy(SEM), the thermodynamic property was evaluated by differential scanning calorimeter(DSC), and the activity, moisture content and reconstitution time according to the requirements in Chinese Pharmacopoeia(Volume Ⅲ, 2010 edition). Results Along with the increasing sodium chloride concentration, the appearance of FⅧ changed from loose hole-net structure to dense lamellar structure, the eutectic point was lowered, and clotting activity decreased, and time for reconstitution increased, and the moisture content in-creased. However, the appearance of FⅧ using glycine as a stabilizer showed a loose hole-net structure, of which the eutectic point and clotting activity increased, and the time for reconstitution and moisture content decreased. Conclusion The crystalline form of lyophilized FⅧ was distinctly influenced by sodium chloride content. The sodium chloride content should be adjusted while the stabilizer should be added during formulation of F Ⅷ solution to control the crystalline form of freeze-dried FⅧ.

    2015 02 v.28 [Abstract][OnlineView][Download 717K]

  • Prediction of primary structure of high molecular protein twitchin by bioinformatics

    BAO Yu-long;ZHENG Yuan-qiang;DING Feng;LV Lu;XUE Cheng-fang;HAN Xin-rong;SHI Yan-chun;Inner Mongolia Key Laboratory of Molecular Biology, Inner Mongolia Medical University;

    Objective To predict the exon-intron structure of high molecular protein twitchin and determine its primary structure and domain. Methods Bioinformatics was used to explore the presence or absence of twitchin gene in the genome of Lottia gigantean and predict the exon-intron structure of the gene. The primary structure and domains of twitchin protein were identified by the prediction of m RNA. Based on the sequence alignment with homologous proteins,the position in phylogenetic tree was determined. Results The coding sequence of twitchin protein was located at a220 000 bp region(sites 149 839-367 346 of 「sca_38」)in the genome of L. gigantean. The predicted m RNA of twitchin protein consisted of 13 000 bp, while the deduced relative molecular mass was about 480 000. The twitchin protein consisted of Ig, Fn, kinase domains and unique sequences, which shared high homology with that from mussel.Conclusion The twitchin existed in L. gigantean, and the primary structure of twitchin protein was predicted and determined.

    2015 02 v.28 [Abstract][OnlineView][Download 544K]

  • Mechanism of metabolic product CK of ginsenoside in induction of apoptosis of ovarian cancer CAOV3 cells

    GAO Yan;LIN Ying-jia;LI Yang;JIN Ying-hua;Education Ministry Key Laboratory of Molecular Enzyme Engineering, College of Life Science,Jilin University;

    Objective T o investigate the inhibitory effect of metabolic product CK of ginsenoside on proliferation of ovarian cancer CAOV3 cells as well as its mechanism in induction of cell apoptosis. Methods The inhibitory effect of CK at various concentrations(0, 1. 0, 2. 5, 5. 0, 10. 0, 20. 0 and 50. 0 μg / ml)on proliferation of ovarian cancer CAOV3 cells was determined by MTT assay, while the apoptosis of CAOV3 cells induced with CK at various concentrations(0,2. 5 and 5. 0 μg / ml) by flow cytometry. The morphological change of cells during apoptosis was observed by 4 ′, 6-diamidino-2-phenylindole(DAPI)staining, while the activation of Caspase-3,-8 and-9 by in vitro Caspase activity assay,and the cleavage of poly(ADP-ribose) polymerase(PARP) by Western blot. Results CK showed significantly dosedependent inhibitory effect on the proliferation of CAOV3 cells, with an IC50 of 3. 6 μg / ml. The counts of apoptotic cells and the cells with typical morphological features of apoptosis increased with the increasing CK concentration. Caspase-3,-8 and-9 were activated. The activity of Caspase-3 increased with the increasing CK concentration, while the band of cleaved PARP was getting dark. Conclusion CK showed toxic effect on ovarian cancer CAOV3 cells, which inhibited the cell proliferation in a dose-dependent mode. CK inhabited the proliferation of CAOV3 cells by promoting the cell apoptosis involving endogenous and exogenous pathway.

    2015 02 v.28 [Abstract][OnlineView][Download 837K]

  • Preparation of biotin-labeled polyclonal antibody against Brucella abortus

    LI Li;JU Wen;YIN De-hui;MENG Ri-zeng;LI Juan;SONG Xiu-ling;XU Kun;Deparement of Health Laboratory, School of Public Health, Jilin University;

    Objective To prepare and identify the polyclonal antibody against Brucella abortus 544 A. Methods B. abortus544 A strain was resuscitated, cultured and enriched, then inactivated. New Zealand white rabbits were immunized s. c.with the prepared inactivated vaccine on back in several sites for 5 times, of which the heart blood was collected. Sera were separated, from which polyclonal antibody was crudely extracted by salting out with ammonium sulphate and further purified by protein affinity chromatography, then determined for purity by SDS-PAGE, for titer and specificity by indirect ELISA, and for protein content by Bradford method, and labeled with biotin. Results The prepared rabbit polyclonal antibody against B. abortus 544 A showed a high purity and a good specificity, of which the titer was 1 ∶ 256 000, the protein content was 2. 83 mg / ml, and the labeling efficiency was 3. 09 of biotin to antibody. Conclusion Biotinla beled polyclonal antibody against B. abortus 544 A was prepared successfully, which laid a foundation of development of immunological detection kit for B. abortus.

    2015 02 v.28 [Abstract][OnlineView][Download 247K]

  • Safety of live attenuated measles and rubella combined vaccine in 206 children allergic to eggs

    HAN Bai-hui;SHI Nian-min;LIU Fang;WANG Rui;Beijing Chaoyang District Center for Disease Control and Prevention;

    Objective To observe the safety of live attenuated measles and rubella(MR) vaccine in children allergic to eggs. Methods A total of 206 children allergic to eggs at age of 8 ~ 17 months in Chaoyang District,Beijing City were immunized with live attenuated MR vaccine in February to March of 2013,using 242 normal children as control. A retrospective cohort analysis on the incidence of adverse reactions after immunization was performed by telephone survey.Results The total response rate in this survey was 87. 3%. A total of 391 children were investigated,including 184 allergic to eggs and 207 as control. The adverse reaction rates in children allergic to eggs and in control group were 10. 3%and 8. 2%,while the local reaction rates were 1. 6% and 1. 0%,the abnormal rates of vital signs were 6. 0% and 4. 8%,and systemic reaction rates were 3. 8% and 5. 8%,respectively,which showed no significant difference(P > 0. 05).Of the cases of adverse reactions,3 were defined as coincidental reactions,including 2 in children allergic to eggs and one in control group,all of which were diagnosed as exanthema subitum. Conclusion No increased adverse reaction rate after immunization with live attenuated MR vaccine was observed in children allergic to eggs as compared with that in normal children.

    2015 02 v.28 [Abstract][OnlineView][Download 135K]

  • Surveillance and analysis of adverse events following immunization in Huangshan City, Anhui Province, China during 2010 2013

    ZHANG Xiang-yang;WANG Ping;WANG Wei-wei;The Center for Disease Control and Prevention of Huangshan City,Anhui Province;

    Objective To analyze the occurrence features of adverse events following Immunization(AEFI)in Huangshan City, Auhui Province, China during 2010 ~ 2013 and evaluate the safety of vaccination. Methods The data on AEFI in Huangshan City during 2010 ~ 2013 were analyzed by descriptive epidemiological method. Results A total of 116 cases of AEFI were reported in the city during 2010 ~ 2013, including 99(85. 34%)cases of general events, 11(9. 48%)cases of abnormal events, 5(4. 31%)cases of coincidental event and one case(0. 86%) of vaccination accident. The AEFI report coverage rate in counties was 100. 00%, while the report and investigated rates within 48 h were 93. 10% and97. 14% respectively. The sex ratio of the cases was 1. 27 ∶ 1. A portion of 43. 10% of the cases occurred in the infants at ages of not more than one year. The reported incidence of AEFI was 7. 39 / 100 000, while those of general and abnormal events were 6. 30 / 100 000 and 0. 70 / 100 000 respectively. The general events were mainly fever as well as redness,swelling and indurations of injection sites, while the abnormal events were mainly anaphylactic rash and BCG-associated lymphadenitis. Conclusion The quality of AEFI surveillance is still low and uneven between various counties. AEFIs are usually occurred in young children and those after inoculation with the vaccines belonging to the National Immunization Programme(NIP). The report rates of various vaccines are within the expected range, indicating high safety.

    2015 02 v.28 [Abstract][OnlineView][Download 158K]

  • Screening of national reference strains for evaluation of protective effect of drugs against tuberculosis

    DENG Hai-qing;CHEN Bao-wen;YANG Lei;ZHANG Ting;LU Jin-biao;WU Xiao-cui;WAN Kang-lin;HUANG Chang-jiang;WANG Guo-zhi;Institute of Environmental Safety and Health Risks Evaluation, Wenzhou Medical University;

    Objective To screen the national reference Mycobacterium tuberculosis(MTB) strains for evaluation of protective effect of drugs against tuberculosis(TB). Methods A total of 169 M. tuberculosis clinical isolates were tested for sensitivities to streptomycin(STR), isoniazid(INH), rifampin(RFP) and ethambutol(EMB) by using mycobacterial growth indicator tuber(MGIT)960 system. The resistance-associated mutant sites of isolates resistant to INH and / or RFP were sequenced. All the 169 isolates were genotyped by Spoligotyping, and those belonging to Beijing MTB family were further genotyped by multiple loci VNTR analysis(MLVA). Results Of the 169 clinical isolates, 118 were resistant to STR, while 143 to INH, 129 to RFP, 95 to EMB, 120 to multiple drugs(at least NIH and RFP), 5 to INH alone, and 7 to RFP alone. However, 11 isolates were sensitive to all the four drugs. The mutations of kat G gene were observed in 76. 9%,while those of inh A gene in 27. 3%, and those of aph C gene in 25. 2% of the 143 isolates resistant to INH, while muta-tions of at least two genes in 31 isolates. However, the mutations of rpo B gene at sites 531, 526 and 516 were observed in67. 4%, 17. 8% and 16. 3% of 129 isolates resistant to RFP, respectively, while the mutation in at least two sites in 10 isolates. Of the 169 isolates, 111 were of Beijing genotype, and 58 were of non-Beijing genotypes, including CAS(2 strains), EAI(7 strains), H(11 strains), LAM(6 strains), MANU(6 strains), T(7 strains), U(1 strain)and X(3strains) lineages, as well as 15 new genotypes not included in the Spol DB4. 0 database. All the 111 isolates of Beijing genotype were divided into four groups by MLVA, among which group Ⅱ accounted for 68. 5% and was the most representative one. Twenty-seven multiple drug-resistant(MDR) and 5 sensitive isolates, as well as 2 isolates resistant to INH alone and 3 isolates resistant to RFP alone were screened. Conclusion Sensitive and MDR isolates as well as those resistant to a single drug were successfully screened.

    2015 02 v.28 [Abstract][OnlineView][Download 405K]

  • Optimization of procedure for lyophilization of oral recombinant Helicobacter pylori vaccine

    ZHANG Jing;WENG Liang;ZOU Xue;LI Jian-xu;LIU Dan;PENG Chao;GE Zi-qiang;XIA Ming-peng;LIU Ya-long;YAN Jin-jin;GUO Zhi-yan;Wuhu Kangwei Biotechnology Co., Ltd.;

    Objective To optimize the procedure for lyophilization of of oral recombinant Helicobacter pylori vaccine so as to decrease the breakage rate of containers during lyophilization. Methods Twelve batches of oral recombinant H. pylori vaccine were subjected to lyophilization test by changing the technological parameters at steps of pre-freezing, sublimation drying and dehydration drying, and the procedure for lyophilization was optimized using the breakage rate of con-tainers based on the qualifications of appearance, moisture content, p H value and sterility. Results The procedure for lyophilization of oral recombinant H. pylori vaccine was optimized as follows:The vaccine was frozen to-45 ℃ within 2 h and maintained for 4. 5 h at step of pre-freezing, then subjected to periodic warming for 34 h at step of sublimation drying,and was maintained at 28 ℃ for 12 h at step of dehydration drying. The appearance, moisture content, p H value and sterility of the oral recombinant H. pylori vaccine were within the acceptable range, while the breakage rate of containers was zero. Conclusion The optimized procedure for lyophilization of oral recombinant H. pylori vaccine was stable and reliable, which laid a foundation of large-scale production in future.

    2015 02 v.28 [Abstract][OnlineView][Download 143K]

  • Development and verification of double antibody sandwich ELISA for quantitative determination of conjugates in quadrivalent meningococcal polysaccharide vaccine

    ZHU Tao;DENG Jie;DENG Xin;SHAO Zhong-qi;MAO Hui-hua;YU Xue-feng;Tianjin University of Science and Technology;

    Objective To develop and verify a double antibody sandwich ELISA for quantitative determination of the con-jugates in groups A, C, W135 and Y meningococcal polysaccharide vaccine. Methods A double antibody sandwich ELISA was developed using monoclonal antibody against CRM197, an avirulent mutant of diphtheria toxoid, as coating antibody, and HRP-labeled monoclonal antibody against groups A, C, W135 and Y meningococcal polysaccharide as cap-ture antibody, then determined for optimal linear range and limit of detection(LOD), and verified for specificity, accuracy and reproducibility. Results The linear range and LOD of the developed double antibody sandwich ELISA were 3. 75 ~120 ng / ml and 3. 75 ng / ml for group A meningococcal polysaccharide(GAMP)-CRM197 conjugate, 1. 875 ~ 30 ng / ml and 0. 938 ng / ml for group C meningococcal polysaccharide(GCMP)-CRM197, 7. 5 ~ 240 ng / ml and 7. 5 ng / ml for group W135 meningococcal polysaccharide(GWMP)-CRM197, and 1. 875 ~ 60 ng / ml and 0. 938 ng / ml for group Y meningococcal polysaccharide(GYMP)-CRM197, respectively. The developed method was suitable for determination of polysaccharide antigen contents in the corresponding conjugates, which showed no cross reactions with the polysaccharide antigens of other groups. The recovery rates of polysaccharide antigens of four groups were 80% ~ 120%, whil e the coefficients of variation(CVs)of the corresponding polysaccharide antigens were less than 15%. Conclusion The developed double antibody sandwich ELISA showed high specificity, accuracy and reproducibility, which might be used for the quantitative determination of polysaccharide antigens in quadrivalent meningococcal polysaccharide vaccine.

    2015 02 v.28 [Abstract][OnlineView][Download 349K]

  • Live attenuated varicella vaccines(VZV 84-7 strain)prepared by various processes

    LI Li;HOU Liang-yu;MA Guang;DING Xiu-hong;LI Yong-zhong;HA Xiu-jie;ZENG Hao;LI Yi;ZOU Wei-ran;LIN Chong-jian;SHAN Yu-jie;LIU Ye;HUANG Lin;Chengdu Institute of Biological Products Co.,Ltd.;

    Objective To prepare live attenuated varicella vaccines(VZV 84-7 strain)by cell factory instead of traditional roller system. Methods Six batches of 2BS cells were cultured in cell factory and 3 L roller separately,and counted by fully automatic Countstar cell counter,based on which the cell quality was evaluated by the cell count in unit area,survival rate and mean cell diameter after digestion. Meanwhile,the heat stability of vaccines were evaluated by determination of virus titers in bulk,final bulk and final product after storage at 37 ℃ for 7 d by plaque assay. Results The mean count in unit area and mean survival rate of six batches of cells prepared by cell factory were slightly higher,while the change range of mean diameter was less than those by culture in 3 L roller. The mean titers of bulk and final product of six batches of vaccine prepared by cell factory were 5. 58 and 4. 78 lg PFU / 0. 5 ml respectively,while the mean titer of final product after storage at 37 ℃ for 7 d was 4. 13 lg PFU / 0. 5 ml,which were higher than those culture in 3 L roller. Conclusion The titer,yield and stability of live attenuated varicella vaccines(VZV 84-7 strain)prepared by cell factory increased,while the intensity of labor and risk of contamination during production decreased,indicating that cell factory was suitable for large-scale production of live attenuated varicella vaccine(VZV 84-7 strain).

    2015 02 v.28 [Abstract][OnlineView][Download 606K]

  • Development of pilot purification procedure and analysis of activity of recombinant human lactoferrin from milk of transgenic cattle

    ZHAO Jian-min;WANG Jian-wu;MENG Ru-jie;FU Ming-bo;WANG Hui-min;TANG Bo;CHEN Min;LI Ning;College of Food Science and Nutritional Engineering, China Agricultural University;

    Objective To develop a pilot purification procedure for recombinant human lactoferrin(rh LF)from the milk of transgenic cattle and analyze the activity of purified rh LF. Methods The rh LF in the milk of transgenic cattle was filtrated through ceramic membrane to remove the bacteria and spores, then subjected to cation exchange chromatography to separate casein micelles, then added with non-ionic detergent and metal complexing agent to remove the endotoxin,concentrated by ultrafiltration, desalted and lyophilized. The obtained purified rh LF was analyzed for purity by SDS-PAGE, SEC-HPLC and RP-HPLC, for concentration by ELISA, for endotoxin content by limulus test, or secondary structure by circular dichroism technique, and determined for iron-binding capacity and bacteriostatic activity. Results The purified rh LF reached a purity of not less than 96%, of which the endotoxin content was less than 0. 5 EU / mg, while the overall recovery rate was 75%, the secondary structure was similar to that of natural h LF, and the iron-binding capacity and bacteriostatic activity were normal. Conclusion The developed pilot purification procedure was simple to handle and highly effective, which was suitable for the large-scale production of rh LF.

    2015 02 v.28 [Abstract][OnlineView][Download 830K]

  • Comparison of culture of hepatitis A virus in vessels of various materials

    SHAO Cong-wen;YANG Xiao-lei;LIANG Hai-xiao;CAO Jie;ZHANG Ming;YANG Jian-bo;FENG Chang-zeng;ZHENG hui-wen;CHEN Meng-jiao;YU Jian-kun;YANG Jing-si;Institute of Medical Biology,Chinese Academy of Medical Sciences and Peking Union Medical College,Yunnan Research Center of Vaccine Engineering Technology on Severe Infectious Diseases,Yunnan Key Laboratory of Vaccine Research and Development on Severe Infectious Diseases;

    Objective To comp are the difference in culture of hepatitis A virus(HAV) in neutral glass spinner and polystyrene cell factory. Methods Human embryonic lung diploid cells(KMB17)were simultaneously cultured in neutral glass spinner and polystyrene cell factory respectively and inoculated with HAV H2 strain,then observed for morphological change under inverted microscope. Samples were taken from the cells on days 0,7,14,18,22,26 and 30 after inoculation and determined for virus titer by ELISA,based on which the kinetics of virus proliferation was analyzed. Results There was no difference in growth states and morphologies of cells cultured in neutral glass spinner and polystyrene cell factory,while the proliferation kinetics were similar. No significant difference was observed between the virus yields per unit volume of the harvests from vessels of two materials. However,the area of KMB17 cells cultured in polystyrene cell factory was 7. 28 times of that in neutral glass spinner. Conclusion Both human diploid cells and HAV grew well in neutral glass spinner and polystyrene cell factory,indicating that cell factory might be used as a novel cell culture technique for large-scale production of vaccine instead of spinner.

    2015 02 v.28 [Abstract][OnlineView][Download 568K]

  • Separation of various plasma components by purification with one step column chromatography

    LA Wen-jun;WANG Yan;ZHOU Jing;LIN Xiao-fa;ZHANG Li-jun;School of Applied Chemistry and Biotechnology,Shenzhen Ploytechnic;

    Objective To separate various active components from source plasma by expanded bed adsorption,a column chromatography for one-step adsorption and stepwise desorption,so as to improve the comprehensive utilization of plasma.Methods Source plasma was loaded onto chromatographic column at a constant flow rate using agarose(tungsten carbide)-DEAE as an adsorption medium to adsorb the components to be separated. The active components were separated stepwise by elution with buffers of various compositions at various flow rates and elution volumes,based on which the key factors in scale-up of procedure were preliminarily investigated. Results Albumin,immunoglobulins,fibrinogen and α-1proteinase inhibitor were separated in turn from source plasma by EBA. The column height and elution rate were key influencing factors of separation effect during scale-up of the procedure. Partial viruses were removed by the technique,which decreased the virus titer by about one order of magnitude. Conclusion With a high utilization level of plasma and a short operation cycle,the one-step column chromatography technique was easy to handle,low-cost and effective in removal of viruses,which might be used for industrial production instead of traditional cold ethanol fractionation.

    2015 02 v.28 [Abstract][OnlineView][Download 334K]

  • Influence of host genetic factors on immune response after vaccination

    LI Ying;YAO Yu-feng;SHI Li;Institute of Medical Biology,Chinese Academy of Medical Sciences & Peking Union Medical College,Yunnan Key Laboratory of Vaccine Research & Development on Severe Infectious Disease;

    With no other health intervention means could be rivaling,vaccine has got a profound influence on the reduction of morbidity and mortality in preventing infectious diseases worldwide. It is well known that the immune response to vaccines is associated with many factors,including age,gender,race,body mass index,quality of vaccine antigens,as well as dosage and method for injection. However,for a typical vaccine antigen being considered,the host genetic factors play an important role in the immune response to vaccine. Studies show that the single or multiple variation of a variety of genes directly or indirectly participating in immune response,such as the virus receptor genes on host cell membrane,innate Toll-like receptors(TLRs),cytokine and its receptor genes and human leukocyte antigen(HLA)gene,may be related to the vaccine-induced immune response level. This review describes the correlation of host genetic factors and immune response after vaccination.

    2015 02 v.28 [Abstract][OnlineView][Download 160K]

  • Application and immunogenicity of live attenuated hepatitis A vaccine

    WU Bing-bing;ZUO Zhi-ping;ZHANG Yu-xi;Jia Lei;Baoding Municipal Center for Disease Control and Prevention;

    Hepatitis A(Hep A)vaccine is an important measure to prevent and control Hep A. Live attenuated Hep A vaccine is widely used in China because of its low price and good immunogenicity. This paper reviews the application and immunogenicity of live attenuated Hep A vaccine.

    2015 02 v.28 [Abstract][OnlineView][Download 142K]

  • Progress in research on molecular modification of cytokine drugs

    LU Yuan;LI Li-yun;CHEN Wei;School of Pharmaceutics,Jiangnan University;

    Cytokines are proteins or small peptides produced by many cells,with strong biological activity,low immunogenicity and high therapeutic efficacy. However,it is found that cytokine drugs are easily degraded by human body,with a short half-life and a high clearance rate. The development and application of cytokine drugs are limited by the instability because of the special molecular structure of the protein(or peptide). Therefore,the molecular modification is needed to improve the physicochemical and pharmacokinetic properties of these drugs. This paper reviews the molecular modification of cytokine drugs in three aspects,i. e. chemical modification,the formation of cyclic peptides and genetic reconstruction.

    2015 02 v.28 [Abstract][OnlineView][Download 130K]


@2012 Editorial Department of "Chinese Journal of Biologicals"