2019年12期目次

  • Molecular characteristics and virulence of partial strains of leptospiral vaccine

    XU Ying-hua;DU Zong-li;XIN Xiao-fang;YE Qiang;National Institutes for Food and Drug Control,Key Laboratory of the Ministry of Health for Research on Quality and Standardization of Biotech Products;

    Objective To analyze the molecular characteristics and virulence of partial strains of leptospiral vaccine.Methods The molecular characteristics of four attenuated leptospiral vaccine strains were analyzed by multi locus variable number tandem repeat analysis(MLVA) combined with pulsed field gel electrophoresis(PFGE) technologies,while the virulence by challenge test in guinea pig model. Results The four attenuated vaccine strains contained four different MLVA types. PFGE showed 8 ～ 13 pieces in the bacterial chromosome after treatment with Not Ⅰ. Histopathologic analysis demonstrated that the main organs of infected animals presented the typical Leptospira infection lesions after challenge with all the attenuated vaccine strains. Conclusion These findings further understood the unique molecular characteristics and virulence in guinea pigs of four attenuated vaccine strains,which provided the most powerful experimental data for improving the current quality control of vaccine strains.

    2019 12 v.32 [Abstract][OnlineView][Download 333K]

  • Preparation of rabies vaccine for human use in 40 L bioreactor

    LI Xu;ZHOU Hao;PAN Xiao-feng;LI Xiao-feng;YU Hao;WAN Bing;Liaoning Chengda Biological Co.LTD;

    Objective To prepare rabies vaccine for human use in a large scale by cell culture in 40 L bioreactor.Methods Rabies vaccine was prepared by perfused culture of Vero cells inoculated with rabies virus PV2061 strain in bioreactor CelliGen 510. The virus bulk was continuously harvested, then concentrated, inactivated and purified.Samples were taken in various steps of the production process for the relevant tests. Results The density of cultured cells reached 1. 2 × 10~7 cells/mL. The virus bulk could be continuously harvested for about 16 d after inoculation and reached a highest titer of 7. 4 LgLD50/mL. After purification by column chromatography,the removal rate of foreign protein reached more than 98%. The residual host cell DNA content in final product was not more than 50 pg/dose,while the titer was not less than 4. 5 IU/dose. The yield of vaccine in each bioreactor reached 130 000 doses.Conclusion CelliGen 510 bioreactor could be used for the large-scale production of rabies vaccine for human use,and the final product met the requirements in Chinese Pharmacopoeia(Volume Ⅲ,2015 edition).

    2019 12 v.32 [Abstract][OnlineView][Download 457K]

  • Cross-blockade antibody spectrum of predominant genotypes of norovirus

    HAN Zi-bo;ZHANG Xue-feng;LIU Zhao-ming;ZHANG Hao;TANG Fang;LI Qi-ming;The Sixth Laboratory,National Vaccine and Serum Institute;

    Objective To analyze the cross-reactivity and cross-blockade activities among five predominant genotypes(GⅠ. 1,GⅡ. 2,GⅡ. 3,GⅡ. 4,GⅡ. 17)of GⅠand GⅡ group norovirus(NoV),and to provide basic data for the development of route and the clinical trial design of NoV vaccines. Methods Mice were immunized with virus-like particles(VLPs)of NoV GⅠ. 1,GⅡ. 2,GⅡ. 3,GⅡ. 4,GⅡ. 17 VLPs,adsorbed onto aluminum adjuvant,at a dosage of 3 μg respectively,and boosted on day 28 after the first dose,using adjuvant as control. The mice were killed on day 56,of which the serum samples were collected and determined for specific IgG antibodies against various genotypes of NoV by indirect ELISA,while the blockade antibodies by an ELISA-based receptor-blockade assay. Results All the GMTs of cross-reactive IgG antibodies between GⅠ. 1 and GⅡgenotypes were less than or close to the cut-off value(titer =40). Either GⅡ. 2 and G Ⅱ. 3 VLPs induced cross-reactive IgG antibodies against other genotypes,at GMTs of 494 ～2 792. GⅡ. 4 VLPs induced GⅡ. 2 IgG at a GMT of 1 660 as well as low level GⅡ. 3(GMT = 226)and GⅡ. 17(GMT =147)IgGs. GⅡ. 17 VLPs induced GⅡ. 2 and GⅡ. 3 IgGs at GMTs of 6 401 and 830 respectively. However,all the GMTs of cross blockade antibodies between GⅠ. 1 and various GⅡgenotypes were less than the cut-off value(BT_(50)= 20).GⅡ. 2 VLPs induced cross blockade antibodies against GⅡ. 3 and GⅡ. 4,while GⅡ. 3 VLPs induced that against GⅡ. 2,and G Ⅱ. 4 and G Ⅱ. 17 VLPs induced those against G Ⅱ. 2 and G Ⅱ. 3,in sera. All the GMTs of above-mentioned blockade antibodies were 31 ～ 73,of which those against the same genotypes in various groups showed no significant difference. G Ⅱ. 2,G Ⅱ. 3 and G Ⅱ. 4 showed no cross-blockade antibody against G Ⅱ. 17. Conclusion No crossreactivity or cross-blockade antibodies were found between NoV GⅠ. 1 and GⅡ genotypes,while limited cross-reactivity and cross-blockade antibodies among partial genotypes in GⅡ group.

    2019 12 v.32 [Abstract][OnlineView][Download 293K]

  • Application of Design of Experiment in optimization of condition for prokaryotic expression

    WANG Hong;TONG Fang;ZHANG Qing;PANG Li-li;LI Zhi-peng;LIU Qing-you;National Institute for Viral Disease Control and Prevention Chinese Center for Disease Control and Prevention;

    Objective To optimize the condition for prokaryotic expression by the Design of Experiment(DOE).Methods The condition for induction of expression of recombinant plasmid pET32 a-Ferritin in E. coli BL21(DE3),including time,inducer concentration and original bacterial density,were optimized by One-way ANOVA and DOE respectively,under which the expression efficacies were compared. Results The optimal condition for One-way ANOVA was induction with 0. 5 mmol/L IPTG at a bacterial density(A600)of 0. 6 for 4 h. However,the optimal condition for DOE was induction with 1. 0 mmol/L IPTG at a bacterial density(A600)of 0. 8 for 10 h. The DOE scheme was more convenient and accurate,and the expression efficacy was higher. Conclusion DOE is a more accurate and convenient tool to obtain the optimal induction conditions of fusion protein. This study offers a new scheme for optimization of prokaryotic expression system,and provides a reference for the application of DOE schemes in the field of biological engineering.

    2019 12 v.32 [Abstract][OnlineView][Download 444K]

  • Screening of specific IK1 agonists based on multi-source data and cardiac action potential profile

    LI Pan;ZHAO Xiao-ze;QIAO Xi;HE Pei-feng;LU Xue-chun;YU Qi;LIU Qing-hua;Department of Pathophysiology,Shanxi Medical University;

    Objective To screen novel specific agonists of inward rectifier potassium channel(I_(K1))based on multi-source data and myocardial action potential(AP)profile. Methods Human colon cancer HCT116 cells were incubated with 1(Za_1 group)and 40 μmol/L(Za_40 group)specific I_(K1) agonist zacopride for 24 h respectively,and then subjected to transcriptome sequencing(RNA-seq) by Illumina Hi Seq system. The data on differentially expressed genes in Za_1,Za_40 and control(Ctl) groups were introduced into a drug repositioning platform based on multi-source data to screen the analogues of zacopride. The effects of drugs on resting potential(RP)and AP in left ventricular myocytes isolated from adult male SD rats were monitored by patch clamp technique. Results There were 226 differentially expressed genes(DEGs)between Ctl and Za_1 groups,while 302 DEGs between Ctl and Za_40 groups. The top 100 drugs from similarity ranking in each group screened by drug repositioning platform were intersected and subjected to text association analysis.Finally,seven drugs were identified as potential zacopride analogues without literature report,which were randomly numbered as Drugs 1 ～ 7. Validation test showed that Drugs 4,6 and 7 could hyperpolarize RP and shorten the action potential duration(APD),which were in line with the characteristics of specific I_(K1) agonist. However,Drugs 1 and 3 showed no significant effect on AP. Drug 5 significantly prolonged the APD. Drug 2 prolonged APD at low concentrations while short-ened APD at high concentration. Drug 6,alcuronium,hyperpolarize RP and shorten the APD in a dose-dependent manner at concentrations of 1 ～ 50 μmol/L,which was in line with the characteristics of specific I_(K1) agonist to AP. Conclusion The drug screening method based on multi-source data and ventricular AP profile is conducive to quickly and effectively identify the potential pharmacologic tools for ion channel,especially I_(K1) agonists,and provide more possibilities for establishing the new anti-arrhythmia strategy and method.

    2019 12 v.32 [Abstract][OnlineView][Download 639K]

  • Optimized expression of virus-like particles of human papillomavirus 52 L1 protein in Pichia pastoris and purification and immunogenicity of expressed product

    TONG Guang-jie;WANG Wen-wei;CAI Bei-bei;WANG Bei;HU Hai-tao;LOU Jue-ren;Shanghai Institute of Biological Products Co.,Ltd.;

    Objective To express human papillomavirus 52(HPV52)L1 protein in Pichia pastoris,purify the expressed product and evaluate its immunogenicity. Methods The codon of wild-type gene of HPV52 L1 was optimized by synonymous codon substitution,then the multi-copy expression vector was constructed and transformed to P. pastoris. The clones for high expression of virus-like particles(VLPs)of HPV52 L1 protein were obtained through screening for largescale fermentation culture in 15 L fermenter. The culture supernatant was purified by cation-exchange chromatography and size-exclusion chromatography. The obtained VLPs were observed for size and shape by dynamic light scattering and transmission electronic microscopy,and evaluated for immunogenicity by pseudovirus neutralization test in mice and rats.Results HPV52 L1 VLPs showed a homogeneous single component in solution,at a hydrated size of 91. 37 nm,which were homogeneous spherical empty particles at a size of about 50 nm under microscope,similar to those of natural virus particles of HPV52. The relative molecular mass of HPV52 L1 VLPs was about 56 000,while the purity was more than95% and the yield was 3. 6 mg/L. The VLPs showed specific binding to mouse polyclonal antibody against HPV L1,of which the ED50 was 0. 010 μg in mice. However,the VLPs induced a neutralizing antibody titer of 106 in rats.Conclusion HPV52 L1 VLPs were successfully expressed in P. pastoris,which showed good immunogenicity. It laid a foundation of development of relevant prophylactic vaccines.

    2019 12 v.32 [Abstract][OnlineView][Download 467K]

  • Colonization and preliminary evaluation of Helicobacter pylori in BALB/c mice

    LIU Yu;ZHONG You-xiu;CHEN Jing;TANG Chong-fa;WANG Xue-wei;WANG Ping;ZHANG Yan-bin;WEI Bo;LIU Mei-ying;National Vaccine and Serum Institute;

    Objective To establish a stable animal model with long-term gastric colonization by infection of BALB/c mice with Helicobacter pylori SS1 strain by oral route,and analyze the immune response. Methods The mice were administered with H. pylori SS1 strain by lavage for 3 times,and sampled periodically for gastric H. pylori culture test,PCR identification,histological section staining,rapid urease test,pathological examination of gastric tissue,serum antiH. pylori antibody test and cellular immunoassay. Results The numbers of H. pylori colonized stably in 6 week-old mice after infection for 32 weeks and in 3 week-old mice after infection for 12 weeks were about 10~4 CFU/g gastric tissue.Serum IgG against H. pylori reached the peak value 10 weeks after infection and maintained hereafter. No specific sIgA was detected in the feces. Flow cytometry proved that the percentages of CD4~+ IL-17~+,CD4~+ IFNγ+ and CD8~+ IFNγ+lymphocytes in intestinal intraepithelial lymphocytes(IELs)showed a declined trend in the 3 week-old mice 2 weeks after infection. The histopathological changes in the stomach was observed in the mice 6 weeks after infection. Conclusion Long-term stable colonization model of H. pylori in BALB/c mice was established,and histophathological change and significant serum antibody response were observed,while no specific sIgA was detected in feces. Th1 and Th17 responses showed declining trend in 3 week-old mice. These results laid a foundation of evaluation of candidate antigens of the therapeutic vaccine.

    2019 12 v.32 [Abstract][OnlineView][Download 495K]

  • Antioxidant activity and structure of flavonoid compounds extracted from raspberry root

    DIAO Jing-jing;CAO Rong-an;LI Chao-yang;LI Liang-yu;National Coarse Cereals Engineering Research Center,Heilongjiang Bayi Agricultural University;

    Objective To analyze the antioxidant activity and structure of flavonoid compounds extracted from raspberry root. Methods The condition for extraction of flavonoid compounds from raspberry root by ultrasonic/microwave coordinative extraction optimized by response surface test. The product obtained under the optimized condition was purified by gradient elution with deionized water as well as 20%,40%,60% and 80% alcohol. The abilities of various eluents in scavenging 1,1-diphenyl-2-picrylhydrazyl(DPPH) and 2,2'-azinobis-3-ethylbenzothia zoline-6-sulphonic acid(ABTS)radicals were determined,and the compounds with high antioxidant activity were analyzed for structure. Results The optimal power of microwave,ratio of liquid to solid,temperature and time for extraction were 431 W,20. 3 ∶ 1,72. 2 ℃and 30. 46 min respectively. Under the optimized condition,the yield of extract was(10. 05 ± 0. 2)%,while the purities of flavonoid compounds in the eluents of 40%,60% and 80% alcohol reached 80%. The IC50 of the extract in scavenging DPPH and ABTS radicals were 0. 34 and 0. 59 times of those of Vc at the same concentration respectively. The flavonoid compounds mainly consisted of five components,i.e. procyanidins B_1,procyanidins C_1,catechins,fisetinidol-(4β,8)-catechin and procyanidins B2. Conclusion The flavonoid compounds with high antioxidant activities were successfully extracted and identified,which provided a theoretical basis for application of raspberry root.

    2019 12 v.32 [Abstract][OnlineView][Download 353K]

  • Effects of microcapsule-based scaffolds for controlled-release of bone morphogenetic protein 2 and vascular endothelial growth factor on differentiation of bone marrow-mesenchymal stem cells to osteoblasts

    REN Qian-gui;ZHANG Kun;REN Wei;SUN Tao;LI Yong-feng;MA Li-bo;The Emergency Department,Second Affiliated Hospital of Inner Mongolia Medical University;

    Objective To investigate the effects of microcapsule-based scaffolds for controlled-release of recombinant human bone morpho-genetic protein-2(rhBMP-2)and vascular endothelial growth factor(VEGF)on differentiation of bone marrow mesen-chymal stem cells(bMSCs) to osteoblasts. Methods The microcapsule-based scaffolds with rhBMP-2 on the outer surface and VEGF encapsulated were prepared with polylactide-poly(ethylene glycol)-polylactide(PELA) by emulsion solvent volatilization method. The concentrations of rhBMP-2 and VEGF released by microcapsule-based scaffolds were determined by ELISA. The activities of bMSCs on days 3,7 and 14 after culture with microcapsule-based scaffolds were determined by MTT assay. The effects of microcapsule-based scaffolds on the expression levels of MAPK pathway-associated proteins and alkaline phosphatase(ALP)during differentiation of bMSCs to osteoblasts were evaluated by Western blot. Results On the second day after culture in PBS,about 60% of rhBMP-2 and about 32% of VEGF were released from the microcapsule-based scaffolds. The microcapsule-based scaffolds showed no significant effect on the activity of bMSCs with the increasing culture time(P > 0. 05). The expression levels of phosphorylated ERK1/2 and ALP were significantly higher on day 14 after culture than on days 3 and 7(P < 0. 05),and significantly higher on day 7 than on day 3(P < 0. 05). However,the expression levels of phosphorylated JNK and phosphorylated p38 on days 3,7 and 14 showed no significant difference(P > 0. 05). Conclusion The microcapsule-based scaffolds for controlledrelease of rh BMP-2 and VEGF induced differentiation of bMSCs to osteoblasts by a possible mechanism of activating MAPK pathway.

    2019 12 v.32 [Abstract][OnlineView][Download 287K]

  • Preparation of recombinant human ciliary neurotrophic factor,modification with polyethylene glycol and effect on body weight,glucose and lipid metabolism of ob/ob obese mice

    ZHENG Xiu-yu;LIU Sen;LI Su-zhen;ZHANG Xi-yan;CHAO Hua;WANG Jian;Lab No.4,National Vaccine & Serum Institute;

    Objective To prepare recombinant human ciliary neurotrophic factor(rhCNTF) mutant [rhCNTF(R~(63)15)],modify with polyethylene glycol(PEG) to obtain long-acting molecule P-rh CNTF(R~(63)15),and investigate the their effects on body weight as well as glucose and lipid metabolism of ob/ob obese mice.Methods The rhCNTF(R~(63)15) was expressed in recombinant E.coli BL21(DE3) and purified by ammonium sulfate precipitation as well as Butyl HP and QHP chromatography in a row.The free sulfhydryl group on rhCNTF(R~(63)(15) C17 was modified with PEG modifier Y-40 KD-MAL-mPEG and further purified by QH P chromatography.The obtained P-rhCNTF(R~(63)15) was identified by SDS-PAGE,and analyzed for purity,modification rate,secondary structure characteristics,modification site,in vitro activity and in vivo pharmacokinetics.The ob/ob obese mice were injected s.c.with P-rhCNTF(R~(63)15) at dosages of 0.05,0.1 and 0.2 mg/kg respectively every4 d,using rhCNTF(R~(63)15) and excipient as controls,each at a dosage of 0.1 mg/kg,once a day.The ob/ob mice were fed with high-fat forage,using C57 BL/6 J mice fed with common forage as control.The body weight,food intake as well as glucose and lipid metabolisms were measured.Results The purities of rhCNTF(R~(63)15) and P-rhCNTF(R~(63)15) were97.4% and 99.3%,while the mean specific activities were 9.5×10~5 and 6.9×10~5 IU/mg,and relatively specific activities were 100% and 72.6%,respectively.The modification rate of rhCNTF(R~(63)15) reached more than 90%.Most of the rhCNTF(R~(63)15) was single modified product,of which the secondary structure was not influenced by modification with PEG,and modification site was located in the C17 of DLCSR peptide fragment.The concentrations of P-rhCNTF(R~(63)15)in blood 72 h after subcutaneous and intravenous injections decreased to the levels before injection.Compared with that in excipient control group,the bodyweights of mice injected with P-rhCNTF(R~(63)15) at dosages of 0.05,0.1 and 0.2 mg/kg decreased by 10%,11% and 21% respectively at end of the treatment course(P <0.05),while that with rhCNTF(R~(63)15) decreased by 30%(P <0.01).The food intakes of mice 15 d after treatment with rhCNTF(R~(63)15) and P-rhCNTF(R~(63)15) at various dosages decreased significantly(P <0.05),then recovered to the normal level gradually.The random blood glucose levels of mice 8 d after treatment with P-rhCNTF(R~(63)15) at various dosages showed a certain dosedependent decrease,while that in rhCNTF(R~(63)15) group decreased 4 d after treatment.The areas under curve(AUC_(0～2 h)) and intraperitoneal fat weights of mice treated with P-rhCNTF(R~(63)15) at a dosage of 0.2 mg/k_g and with rhCNTF(R~(63)15)decreased significantly(P <0.05),while the subcutaneous fat levels of mice treated with rhCNTF(R~(63)15) and P-rhCNTF(R~(63)15) at various dosages decreased significantly(P <0.01).Conclusion Highly purified rhCNTF(R~(63)15) and PrhCNTF(R~(63)15) were prepared,both of which showed good curative effects on the over weight,hyperglycemia and high bod_y fat weight in ob/ob mice.However,P-rhCNTF(R~(63)15) was more long-acting than rhCNTF(R~(63)15).

    2019 12 v.32 [Abstract][OnlineView][Download 445K]

  • Expression of Mycobacterium tuberculosis Rv0674 protein and preparation of polyclonal antibody

    YANG Yan-hui;DONG Li-jun;LIANG Zhong-zhe;LIU Tong;ZHANG Wei;YANG Yu-rong;YANG Zhi-wei;Ningxia Medical University;

    Objective To express Mycobacterium tuberculosis(MTB) Rv0674 protein,prepare polyclonal antibodies(PcAbs)and determine their titers. Methods Rv0674 gene was amplified from MTB H37 Rv genome by PCR and cloned into expression vector pEASY-Blunt E1. The constructed recombinant plasmid was transformed to E. coli BL21(DE3)and induced with IPTG under the optimized condition. The expressed Rv0674 protein was purified by His trapTM HP affinity chromatography and injected i.p. into BALB/c mice. The prepared PcAbs were tested for titer and specificity by ELISA and Western blot respectively. Results Recombinant plasmid pEASY-E1-Rv0674 with correct Rv0674 gene sequence was constructed,and a strain for high expression of Rv0674 was screened. The condition for expression was optimized as induction with 1. 0 mmol/L IPTG at 28 ℃ for 8 h. A soluble Rv0674 protein with a relative molecular mass of about 26 500 was obtained,which induced PcAbs in BALB/c mice. The PcAbs showed good antigen-binding properties,of which the titers were more than 1 ∶ 51 200. Conclusion Highly purified Rv0674 protein was obtained,and PcAbs against Rv0674 was prepared at first,which laid a foundation of further study on function of Rv0674 protein and its application in the clinical diagnosis of tuberculosis(TB).

    2019 12 v.32 [Abstract][OnlineView][Download 404K]

  • Characteristics of antibody levels against varicella-zoster virus in healthy children in Tongzhou District,Beijing

    ZHAO Chun-yan;SHI Jing;ZHANG Ling;SUN Yuan-jie;ZHANG Jian-ming;XU Ying;DENG Yan-chun;WANG Bao-lan;ZOU Lin;WANG Feng-jun;ZHOU Jing-lin;College of Public Health,Capital Medical University;

    Objective To investigate the IgG antibody level against varicella-zoster virus(VZV)among children at ages of less than 15 years in Tongzhou District,Bejing City,China after the immune strategy of two doses of live attenuated varicella vaccine(VarV)was implemented for 5 years,and provide a reference for improving the immunization with VarV.Methods Healthy children at ages less than 15 years,who have lived in 10 communities in Tongzhou District for more than 6 months,were investigated by questionnaire and check of vaccination certificate,of whom the basic information and vaccination history were collected by Beijing Immunization Planning Information Management System,and the VZV IgG levels were determined by ELISA. Results A total of 380 children were investigated,of whom the total positive if VZVIgG was 38. 68%(147/380),while the GMC was 77. 12 m IU/m L. The vaccination rates of one and two doses of VarV were41. 05% and 20. 00% respectively. The antibody titers in infants at ages of less than 12 months were negatively related to the age,with a correlation coefficient of-0. 130(P < 0. 05). However,both the positive rate and GMC of VZV-IgG in the children at ages of not less than 12 months increased with the increasing age and dose. The positive rate and GMC of VZV-IgG were the highest in the children with vaccination history of two doses of VarV. In the 156 children with vaccination history of one do se of VarV,the antibody positive rate decreased with the increasing time duration after vaccination. However,in the 74 children with vaccination history of two doses of VarV,no significant differences were observed between the positive rate of and GMC of VZV-IgG at various time points after the last vaccination,and between those vaccinated at various time intervals(each P > 0. 05). Conclusion The VarV coverage rate and VZV-IgG positive rate were relatively low in the children at ages if less than 15 years in Tongzhou District after the immunization strategy of VarV was implemented for 5 years. Efforts should be made to enhance the popularization of VarV vaccination.

    2019 12 v.32 [Abstract][OnlineView][Download 180K]

  • Immunogenicity and safety of two-dose and booster immunization schedules of domestic varicella vaccine

    LUO Sheng;JIANG Da-lei;HUANG Ya-ling;HUANG Xiao-yu;QIN Hui-ying;WU Xing-juan;TAO Hang;YANG Jin;Center for Disease Prevention and Control of Wuming District,Nanning City;

    Objective To investigate the immunogenicity and safety of two-dose and booster immunization schedules of domestic live attenuated varicella vaccine(VarV). Methods From July 2015 to December 2017,300 healthy children at ages of 1 ～ 12 years,without vaccination history of VarV,were enrolled in two-dose immunization group,while 300 healthy children at ages of 4 ～ 13 years,who had been vaccinated with one dose of VarV,were enrolled in booster immunization group(immunization intervals were 1 ～ 3 and 4 ～ 6 years respectively),in six vaccination centers in Wuming District,Nanning City,Guangxi Zhuang Autonomous Region,China. The 1 st dose and the 2 nd dose of VarV were vaccinated on days 0 and 90 ～ 104 respectively. However,the children in the booster immunization group were boosted with one dose of VarV on day 0 after enrollment. Venous blood samples were collected from the children in two-dose immunization group before and 6 weeks after vaccination of the 1 st dose as well as 6 weeks after the 2 nd dose,and from those in booster immunization group before and 6 weeks after the booster immunization. Membrane antigen fluorescent antibody assay was used to detect the varicella zoster virus(VZV)antibody titer in sera. The immunization success rate,antibody geometric mean titer(GMT) and increasing fold of GMT in two groups were compared,while the systemic and local adverse reactions were observed within 42 d after vaccination. Results A total of 887 doses of VarV were vaccinated to 600 children,with a vaccination rate of 98. 56%. A total of 578 children were included into immunogenicity evaluation,while 887 doses into safety evaluation. The immunization success rate of the 2 nd dose in two-dose immunization group was 98. 93%,which was significantly higher than that of the 1 st dose(86. 83%)(P < 0. 001). However,in booster immunization group,the immunization success rate of children who were boosted 1 ～ 3 years(93. 96%)showed no significant difference with that 4 ～ 6 years(88. 51%)after the 1 st dose(P > 0. 05). Both the GMTs after the 1 st and the 2 nd doses in two-dose immunization group were significantly higher than those before vaccination(P < 0. 001),which increased by 9. 33 and 25. 12 folds respectively,while the GMT after the 2 nd dose was significantly higher than that after the 1 st dose(P < 0. 001). However,in booster immunization group,both the GMTs of children who were boosted at various time intervals after the 1 st dose were significantly higher than those before booster immunization(each P < 0. 001),which increased by 7. 93 and 7. 01 folds respectively,while the GMT of children boosted 1 ～ 3 years was significantly higher than that 4 ～ 6 years after the 1 st dose(P < 0. 05). The adverse reactions after vaccination with VarV were mainly fever,while no local adverse reactions were observed. The adverse reaction rates in booster immuni-zation group and two-dose immunization group were 2. 67% and 2. 89% respectively,which showed no significant diffe-rence(P > 0. 05). Conclusion Domestic VarV has great immunogenicity and safety in both two-dose and booster immunization schedules,which is suitable for popularization in the target children.

    2019 12 v.32 [Abstract][OnlineView][Download 182K]

  • Post-marketing safety evaluation on inactivated poliovirus vaccine(Vero cells)made from Sabin strain

    LIU Xiao-qin;CHEN Hai-ping;ZHANG Zhen-guo;HAN Sha-sha;SHI Xuan-wen;SUN Li;China National Biotec Group Company Limited;

    Objective To evaluate the post-marketing safety of inactivated poliovirus vaccine(Vero cells) made from Sabin strain(IPV-Sabin). Methods The data of adverse events following immunization(AEFI) with IPV-Sabin in population in Hebei Province from September 29,2017 to January 25,2018 were collected from Hebei Centers for Disease Control AEFI Information System,and the class and reported incidence of AEFI were analyzed retrospectively.Results A total of 378 cases of AEFI were reported after vaccination with IPV-Sabin among the 545 256 doses of IPVSabin vaccine,378 cases(incidence 69. 33/100 000 doses) of AEFI were reported,including 364 cease of general reaction(incidence 66. 76/100 000 doses),7 cases of abnormal reaction(incidence 1. 28/100 000 doses),5 cases of coincidental events(reported incidence 0. 92/100 000 doses) and 2 cases of undefined situation(reported incidence0. 37/100 000 doses). The general reactions mainly included fever,redness and swelling,and induration,all of which appeared 0 and 1 d after vaccination. However,the abnormal reactions mainly included allergic rash,angioedema,thrombocytopenic purpura and febrile convulsion,all of which appeared 0 d after vaccination. A total of 356 cases of AEFI were cured or improved. Conclusion IPV-Sabin showed high safety,which was suitable for the prevention of poliomyelitis in the population at appropriate ages.

    2019 12 v.32 [Abstract][OnlineView][Download 133K]

  • Development of fading spectrophotometric method for determination of catalase activity with rhodamine B

    DONG Na;DAI Xing-de;ZHANG Ai-ju;ZHANG Xiao-lin;Gansu Medical College;

    Objective To develop a fading spectrophotometric method for determination of catalase activity with rhodamine B. Methods In sulfuric acid medium,rhodamine B was oxidized by hydrogen peroxide,and trivalent iron ion was added to accelerate the reaction process. Catalase activity was determined according to the absorbance changes before and after the enzymatic reaction (ΔA). The condition for fading reaction of rhodamine B,such as catalyzer,dosage of rhodamine B,time and temperature and dosage of sulfuric acid,as well as that for enzymatic reaction,such as dosage of hydrogen peroxide and time,were optimized. The developed method was determined for linear range. Interference test was performed by addition of coexisting reducing substances at concentrations of less than 1/2 of hydrogen peroxide substance,such as glucose,fructose,lactose and galactose. The catalase activity in fish liver was determined by the developed method. Results Serving 1 mL trivalent iron ion as catalyzer,and 5 mL rhodamine B as the base solution,the absorbance of the system was controlled within 0. 8. In 0. 002 mol/L sulfuric acid medium,the fading degree of rhodamine B tended to be the greatest after reaction at 40 ℃ for 60 min. The enzymatic reaction was complete within 15 min when the volume of hydrogen peroxide was 1. 00 mL. Under the optimal reaction condition,the activity of catalase at a concentration range of 0. 02 ～ 0. 3 U/mL showed good linear relationship to the ΔA,with a r value of 0. 999 2. With a detection limit of 0. 011 U/mL,the developed method may be used for determination of catalase activity in fish liver. Conclusion The developed rhodamine B fading spectrophotometric method showed high selectivity and high sensitivity,which was simple in equipment and handling,and was suitable for the analysis of trace catalase activity in biological samples.

    2019 12 v.32 [Abstract][OnlineView][Download 232K]

  • Optimization and verification of purification process of pneumococcal capsular polysaccharide type 5 based on quality by design

    REN Ke-ming;SONG Xian-ming;ZHANG Yi;WANG Xi;ZHANG Li-zhi;ZHANG Xin-zhuang;REN Zhen-yun;BAI Gui-jie;WANG Jian-hong;SHEN Rong;LUO Shu-quan;Lanzhou Institute of Biological Products Co.,Ltd.,Center for Gansu Provincial Vaccine Engineering Research;

    Objective To optimize and verify the purification process of capsular polysaccharide of pneumococcus type5 based on the idea of"quality by design(QbD)". Methods The sample handling capacity(SHC) and polysaccharide recovery rate(PRR) were set as the critical quality attributes(CQAs). Based on the fractional factorial experiment,the pH value,sodium chloride concentration and crude polysaccharide concentration were identified as the critical process parameters(CPPs). The Box-Behnken design was used for experimental arrangements,and the design space of purification process was established based on the quadratic polynomial regression model. Results The results of ANOVA showed that all the P values of the model were less than 0. 000 1,while the P values of lack of fit were more than 0. 05,which indicated that the models described the relationship between CQAs and CPPS effectively. The recommended operation space of parameters was as follows:p H 7. 7 ～ 7. 9,sodium chloride concentration 220 ～ 280 mmol/L,and polysaccharide concentration 1 ～ 1. 6 mg/mL. Conclusion The robustness and flexibility of purification process for capsular polysaccharide of pneumococcus type 5 were enhanced via the establishment of design space based on the idea of Qb D.

    2019 12 v.32 [Abstract][OnlineView][Download 338K]

  • Determination of amino acid content in human coagulation factor Ⅷ by HPLC with pre-column derivation

    WANG Yue;YU Jian-hong;LIU Ying;LUO Jing;ZHENG Xiao-bei;ZHANG Xue;GONG Qin;Sinopharm Wuhan Plasma-derived Biotherapies Co.,Ltd.;

    Objective To establish a method for determination of amino acid content in human coagulation factor Ⅷ (FⅧ)by HPLC with pre-column derivation. Methods A stable derivative(pre-column derivatization)was prepared with amino acids under a certain condition by 6-aminoquinoline-N-hydroxysuccinimide carbamate(AQC). HPLC was performed by using amino acid analysis column(150 mm × 3. 9 mm,4 μm),and subjected to gradient elution with mobile phases A,B and C,which had been adjusted according to the requirements in Chinese Pharmacopoeia(Volume Ⅲ,2015 edition),at a column temperature of 37 ℃,a flow rate of 1. 0 mL/min and a detection wavelength of 248 nm. The developed method was verified for systemic suitability,specificity,linear range,precision and accuracy. Results Under the given chromatographic condition,glycine and lysine hydrochloride were separated well within 25 min,while the separation degree of the adjacent chromatographic peaks was more than 1. 5,with a tailing factor of 0. 95 ～ 1. 40. The kinds of amino acids in various samples were identified correctly. The time of appearance of peak of mixed samples was consistent with that of simplex samples. The relative deviation between the retention time of main peaks of sample solution and control solution was less than 2%. The linear ranges of glycine and lysine hydrochloride were 12. 5 ～ 62. 5 μg/mL and 4. 0 ～ 20. 0 μg/mL,with correlation coefficients(r) of 0. 999 9 and 0. 999 8,respectively. The RSDs of reproducibility of glycine and lysine hydrochloride were 0. 4% and 0. 5%,while those of intermediate precision were 1. 3%and 1. 0%,respectively. The recoveries of the two kinds of amino acids were 95% ～ 105%,with RSDs of not more than2%. Conclusion The developed method was specific,precise and accurate,which might be used for determination of amino acid content in FⅧ.

    2019 12 v.32 [Abstract][OnlineView][Download 272K]

  • Comparison of digestion of Vero cells with different types of digestive solutions

    ZHANG Ying;ZHANG Le;ZHAO Lan-ying;MA Xuan;DENG Li-qiang;JIANG Nan;DAI Hui-hui;ZHANG Jin;LIANG Hong-yang;No.5 Vaccine Workshop,Beijing Institute of Biological Products Co.,Ltd.;

    Objective To compare the effects of trypsin and TrypLE in digestion of Vero cells,and optimize the digestion procedure of Vero cells cultured in cell factory. Methods After the Vero cells in 10-layer cell factory grew into a dense monolayer,0. 25% trypsin and Tryple,pre-warmed at(37 ± 1)℃,and TrypLE stored at room temperature(18 ～ 26)℃were used for digestion the cells respectively. The cell suspension digested was collected for cell count,and the cell viability was calculated. Afterwards,the cell suspension was inoculated into the bioreactor for further culture,and the glucose metabolism was detected daily. The reproducibility of cell digestion with TrypLE was verified at room temperature.Results The densities of cells digested with 0. 25% trypsin at(37 ± 1)℃,TrypLE at(37 ± 1)℃ and TrypLE at room temperature were 1. 70 × 10~6,1. 68 × 10~6 and 1. 65 × 10~6 cells/mL respectively,while all the cell viability was more than 95%. The glucose consumption in further culture of cells digested with TrypLE was higher than that with 0. 25%trypsin. The coefficient of variation(CV) of number of cells obtained by cell digestion in the 10-layer factory was about2. 47%,while that of glucose metabolism in Vero cell culture stage after digestion and passage was less than 10%.Conclusion TrypLE had mild effects,less cell damage and good reproducibility during digestion. In the process of largescale cell culture in cell factory,TrypLE may be used instead of trypsin for cell passage.

    2019 12 v.32 [Abstract][OnlineView][Download 158K]

  • Verification of viral inactivation/removal process for recombinant human coagulation factor Ⅷ

    HUANG Juan;LI De-kuan;WU Zhi-qiang;SONG Chun-lei;LI Cheng;RONG Xin-zong;KOU Peng;LEI Tao;Key Laboratory of Bio-Resource and Eco-Environment of Ministry of Education,College of Life Sciences,Sichuan University;

    Objective To verify the viral inactivation/removal process for recombinant human coagulation factor Ⅷ(rhFⅧ).Methods The solvent/detergen(S/D)method was used as virus inactivation process,while the nanofiltration(20 nm pore size) as virus removal process for rhFⅧ to analyze the effect of the two-step virus inactivation/removal process on the biological activity of the product. Pseudorabies virus(PRV),vesicular stomatitis virus(VSV),xenotropic murine leukemia virus(X-MuLV)and murine minute virus(MMV)were used as indicators to verify the inactivation/removal effect of the above process,respectively. Results In the S/D inactivation process,the recovery rates of activities of the three batches of the products were 96. 2%,100. 4% and 103. 2%,respectively. After treatment at(24 ± 2)℃ for 180 min,the titers of PRV,VSV and X-MuLV decreased by less than 4 Log10,while no virus was detected after three blind passages. However,in the nanofiltration process,all the protein recoveries of three batches were more than 98%,while the specific activities were more than 90%. After nanofiltration for 120 min,the titer of MMV decreased by more than 4 Log_(10). Conclusion The developed processes were effective in inactivation/removal of viruses in rhFⅧ to ensure the quality and safety of products.

    2019 12 v.32 [Abstract][OnlineView][Download 142K]

  • Selection of calibrator for direct bilirubin enzymatic assay

    TU Ying;SUN Wen-hao;JIN Yu-lan;Shanghai Institute of Biological Products Co.,Ltd.;

    Objective To select the calibrator for direct bilirubin enzymatic assay. Methods Based on direct bilirubin enzymatic method,calibration was carried out with Roche Clinical Chemistry Multiconstituent Calibrator,Randox Clinical Chemistry Multiconstituent Calibrator and bilirubin pure calibrator,and the results were compared. Results There was no significant difference in the results of direct bilirubin enzymatic assay with different calibrators(each r > 0. 975,each P < 0. 01). All the systemic errors(SE%) were less than 1/2 of total error accepted(TEa) in CLIA'88,indicating that the test results were comparable. Conclusion Either pure bilirubin or Clinical Chemistry Multiconstituent Calibrator may be used for direct bilirubin enzymatic assay.

    2019 12 v.32 [Abstract][OnlineView][Download 128K]

  • Influence of different kinds of pre-filtration membranes on filtering efficacy of human immunoglobulin by nano film(DV20 nm)

    PENG Yan;LI Tao-jing;ZOU Hao-yong;CHEN Ke-jin;HU Yong;Department of Research and Development,Sinopharm Wuhan Plasma-derived Biotherapies Co.,Ltd.;

    Objective To compare the influence of different kinds of pre-filtration membranes on the filtering efficacy of human immunoglobulin by nano film(DV20 nm). Methods Flux screening experiments of nano film(DV20 nm)and prefiltration membranes,from manufacturers A,B and C,were performed on 1 000 mL 5. 3% IgG,and the protein recovery rates and fluxes were compared. Verification for virus removal was carried out on the porcine parvovirus(PPV)at a titer of 6. 50 LgTCID50/0. 1 mL in samples by using the screened pre-filtration membrane and nano film. Results The optimal prefiltration membranes from manufacturer A was minisart,of which the flux was 7. 68 L/(m~2·h),while the protein recovery was 96. 1%. The flux of pre-filtration membrane from manufacturer B,FTKDJL,was 9. 87 L/(m~2·h)for120 min,3. 93 L/(m~2·h) for 300 min and 1. 85 L/(m~2·h) for 400 min,while the protein recovery was 97. 6%.However,the fluxes of nano film from manufacturers A and B were 2. 39 and 3. 74 L/(m~2·h)for about 1 200 min,by which the titers of PPV as an indicator decreased by not less than 4. 63 and not less than 4. 25 LgTCID50/0. 1 mL,respectively. The PPV was removed completely within the effective flux,while the protein recoveries were more than 96%.Conclusion The different kinds of pre-filtration membranes showed significant influence on the filtration flux of nano film filters. Both the screened pre-filtration membrane and nano film removed the PPV effectively,which might be used for the large-scale production of human immunoglobulin.

    2019 12 v.32 [Abstract][OnlineView][Download 157K]

  • Optimization of condition for fermentation culture of group W135 meningococcus

    ZHOU Hui-guo;REN Jing;XU Shi-you;WU Yuan-yuan;ZHANG Sheng-yan;YANG Qian-wei;YU Fei-fei;Lanzhou Institute of Biological Products Co.,Ltd.,Center for Gansu Provincial Vaccine Engineering Research;

    Objective To optimize the condition for fermentation culture of group W135 meningococcus and increase the yield of capsular polysaccharide. Methods Group W135 meningococcus was cultured in 30 L fermentor. The culture condition was optimized by analysis of effects of glucose feeding strategy,fermentation temperature and inoculation amount on the biomass and polysaccharide yield in fermentation liquid. The optimized process was verified. Results The optimal glucose feed strategy was supplementation with 5 g/L glucose by fed batch,while the optimal fermentation temperature was 37 ℃,and the optimal inoculation amount was 16%. All the nucleic acid content,protein content,sialic acid content,O-acetyl content,KDvalue,recovery rate and polysaccharide content met the quality standard for group ACYW135 meningococcal polysaccharide vaccine. Conclusion The fermentation condition suitable for group W135 meningococcus was obtained, under which the yield of capsular polysaccharide increased. It provided an experimental basis for development of a complete fermentation process.

    2019 12 v.32 [Abstract][OnlineView][Download 189K]

  • Effect of serum-free culture of varicella zoster virus on microcarriers

    YAN Lei;WANG Yong-jun;ZHAO Hai-bo;WANG Wei;HE Liang;LI Chun-ming;SHEN Hong-jie;WANG Lei;Changchun Institute of Biological Products Co.,Ltd.;

    Objective To investigate the effect of serum-free culture of varicella zoster virus(VZV) on microcarriers in bioreactor. Methods The 2 BS cells were cultured on microcarriers in a bioreactor at a certain density to form a single layer,infected with VZV at a MOI of 0. 004 ～ 0. 008 and cultured in serum-free medium BD008,using the MEM containing 2% newborn calf serum as control. When 70% of the cytopathogenic effect(CPE) appeared,the sediment of cells cultured in serum-free medium BD008 was washed once with PBS,while that in serum-containing medium MEM for1 ～ 3 times,and harvested,each of which was prepared into three batches of bulk. The virus titer was determined by plaque assay,while the residual bovine serum albumin by ELISA. The prepared live attenuated varicella vaccine(VarV)was tested for heat stability. Results The titers of three batches of bulk prepared with the VZV cultured in serumcontaining medium decreased from 5. 00 to 4. 12 lgPFU/mL after being washed for 1 ～ 3 times,while the residual bovine serum albumin contents were 203. 2,120. 3 and 81. 2 ng/mL respectively,of which the former two were more than the standard for qualification (≤ 100 ng/mL). However,the titers of three batches of bulk prepared with the VZV culture in serum-free medium were 4. 92,5. 00 and 5. 30 lgPFU/mL respectively after being washed for one time.However,the residual bovine serum albumin content were 75. 2,82. 3 and 71. 9 ng/mL respectively,of which the mean was lower than those of bulk prepared with VZV cultured in serum-containing medium after being washed for 1 and2 times(P < 0. 05). All the heat stabilities of the three batches of VarV prepared with VZV cultured in serum-free medium met the relevant requirements. Conclusion The titer and residual bovine serum albumin content of vaccine prepared with VZV cultured in serum-free medium met the requirements in Chinese Pharmacopoeia(Vol Ⅲ,2015 edition). The study provided a reference for improvement of quality of VarV and microcarrier procedure in bioreactor.

    2019 12 v.32 [Abstract][OnlineView][Download 247K]

  • Progress in research on role of heat shock protein 90 in antiviral immunity

    BI Ying-jie;XIE Jing-ying;FENG Ruo-fei;Key Bio-engineering and Technology Laboratory,Biomedical Research Center of the Northwest Minzu University;

    Heat shock protein(HSP)are proteins that are synthesized to protect the body from heat stress,which possess the function of molecular chaperone. HSP is classified into small molecule HSP family,HSP70 family,HSP90 family and HSP110 family according to the relative molecular mass. Recently,more and more studies have shown that HSP90 was involved in viral infection. HSP90 plays a role in the viral replication through direct interaction with viral proteins or interaction with other proteins. This article reviews the role of HSP90 in viral immunity in recent years,and provides a theoretical basis for the development of related drugs and inhibitors.

    2019 12 v.32 [Abstract][OnlineView][Download 169K]


@2012 Editorial Department of "Chinese Journal of Biologicals"