2018年02期目次

  • Complete genome sequence analysis of China rabies vaccine strains CTN-1 and PV-2061

    LU Xue-xin;LI Yan-rong;YU Peng-cheng;TAO Xiao-yan;LIU Shu-qing;YAN Jiang-hong;ZHOU Lei;ZHU Wu-yang;National Institute for Viral Disease Control and Prevention,Chinese Center for Disease Control and Prevention;

    Objective To analyze the complete genome sequence of China rabies vaccine strains CTN-1 and PV-2061.Methods The whole genome sequences of master seed lots of CTN-1 and PV-2061 strains were amplified by RT-PCR,and compared with those of other strains isolated in China by using multiple sequence alignment,and the main antigen of rabies virus was analyzed. Results CTN-1 strain showed high homology to PV-2061 strain. However,CTN-1 strain was more closely related to the street virus strain isolated in China. PV-2061 strain showed some unique mutations in the amino acid sequence,which was speculated to be associated with the production of neutralizing antibody. Conclusion The multiple alignment of whole genome sequences of rabies virus vaccine strains CTN-1 and PV-2061 laid a foundation of preparation of safe and effective rabies vaccine.

    2018 02 v.31 [Abstract][OnlineView][Download 670K]

  • Immunogenicity of groups A,C,Y and W135 meningococcal polysaccharide conjugate vaccines prepared with various protein carriers

    TONG Wei;SUN Xiao-dong;LIU Jian-bin;YIN Shan-shan;LIU Jian-kai;Beijing Minhai Biotechnology Co.,Ltd;

    Objective To investigate the immunogenicity of groups A,C,Y and W_(135) meningococcal polysaccharide conjugate vaccines prepared with various protein carriers(tetanus toxoid and diphtheria toxoid). Methods Groups A,C, Y and W_(135) meningococcal polysaccharide were activated with cyanogens bromide(CNBr) and prepared into polysaccharide derivatives using 1,6-adipic acid dihydrazine(ADH) as a derivating agent,then combined with purified tetanus toxoid(TT) and diphtheria toxoid(DT) respectively in mediation of 1-ethyl-3(3-dimethylaminopropyl)-carbodiimide(EDAC),purified by Sepharose 4 FF chromatography and added with stabilizer to prepare Men Aps-TT,Men Cps-TT,Men Yps-TT,Men Wps-TT,Men Aps-DT,Men Cps-DT,Men Yps-DT and Men Wps-DT conjugate vaccines.Mice were immunized s.c. with the vaccines at dosages of 1 and 2. 5 μg(contents of polysaccharides of various types)separately on days 0 and 14,of which the serum samples were collected on day 28 after the first immunization and determined for antibody titer by ELISA to calculate the antibody positive conversion rate. Results The positive conversion rates induced by the vaccines at dosages of 1 and 2. 5 μg were more than 80%,which were higher in the mice immunized with 2. 5 μg of vaccines than in those with 1 μg. In general,the positive rates induced by the vaccines using TT as protein carrier were higher than those using DT,especially those by groups A and W_(135) meningococcal polysaccharide conjugate vaccines.

    2018 02 v.31 [Abstract][OnlineView][Download 268K]

  • Effect of presynaptic membrane antibody on replication capacity of dengue virus Ⅱ in THP-1 cells

    JIANG Li-ming;YANG Jia-jia;LUO Jia;YE Chao;WEN Song-jiao;SUN Qiang-ming;Institute of Medical Biology, Chinese Academy of Medical Science & Peking Union Medical College, Ynunan Key Laboratory of Vaccine Research & Development on Severe Infectious Disease;

    Objective To investigate the effect of presynaptic membrane(prM)antibody on replication capacity of dengue virusⅡ(DENVⅡ)in THP-1 cells. Methods THP-1 cells were co-infected with anti-prM monoclonal antibody of DENV-Ⅱ, at various dilutions(1/4 ~ 1/16 384)and DENVⅡat various MOIs(3, 0. 75 and 0. 19). The standard curve of real-time fluorescent quantitative PCR for DENVⅡwas plotted and used for determination of virus copy number in culture supernatant of THP-1 cells. Results High virus load was induced in THP-1 cells infected with DENV Ⅱat a MOI of 3 and prM dilutions of 1/16 and 1/16 384, while the virus growth was inhibited when the dilutions of prM were 1/64 and 1/256. Higher virus load was induced in the cells infected with DENV Ⅱ at a MOI of 0. 75 and a prM dilution of 1/16. However, the DENVⅡat a MOI of 0. 19 induced no high virus load. Conclusion The prM antibody at a moderate concentration inhibited the replication capacity of DENV Ⅱ in THP-1 cells, while those at high and low concentrations promoted the replication capacity.

    2018 02 v.31 [Abstract][OnlineView][Download 245K]

  • Screening of animal-derived component-free medium for Bordetella pertussis and effect on production of pertussis toxin and filamentous haemagglutinin

    CHEN Yuan-fen;MA Li-geng;WU Qiang;WANG Wen;WANG Yu;ZHANG Jian-jun;Chengdu Olymvax Biopharmaceuticals Inc.;

    Objective To optimize the formula of an animal-derived component-free medium for Bordetella pertussis, and investigate its effect on the production of pertussis toxin and filamentous haemagglutinin. Methods The animal-derived component(casein acid hydrolysate) in the culture medium was replaced by non-animal-derived Hy Pep5603 or/and DiffxoTMSelect Soytone as nitrogen source. Bacterial concentration and production of pertussis toxin(PT) and filamentous haemagglutinin(FHA)of B. pertussis in 8 kinds of culture media in shake flask were determined. The formula of animalderived component-free medium was optimized serving the highest productions of PT and FHA as a reference standard.Four batches of B. pertussis were cultured in the optimal medium by fermentation in 30 L fermenter, using the medium containing aminal-derived components as control. PT and FHA were isolated and purified by Capto SP Imp Res and Capto MMC chromatography from the above fermentation liquid, and determined for relative molecular mass by SDS-PAGE. Results By using the optimized medium consisting of 4. 0 g/L non-animal-derived nitrogen source Hy Pep5603,6. 0 g/L soybean peptone and modified SS medium, the maximum A550 of B. pertussis was 3. 8 in shake-flask and about 5. 8 in fermenter, while the bacteria grew well as compared with those in animal-derived component-containing or animalderived component-free medium alone. The production of PT of B. pertussis in the optimized medium by fermentation was about 5. 7 mg/L, while that of FHA was about 23. 0 mg/L, which were higher than those in the medium containing animal-derived components. The relative molecular masses of PT and FHA were in agreement. Conclusion Animalderived component-free medium containing 4. 0 g/L Hy Pep5603 and 6. 0 g/L DiffxoTMSelect Soytone for culture of Bordetella pertussis may be used for production of PT and FHA instead of the medium containing animal-derived components.

    2018 02 v.31 [Abstract][OnlineView][Download 271K]

  • Expression of recombinant human nerve growth factor in E. coli and determination of activity of expressed product

    SHEN Li;HUANG Zhi-li;JIANG Wei;LIN Yu-qiong;WANG Yan;The Applied Chemistry and Biology School of Shenzhen Polytechnic;

    Objective To express recombinant human nerve growth factor(rhNGF) in E.coli and determine its activity.Methods The gene for expression of rh NGF was designed by codon optimization, based on which recombinant plasmid pET-28 a-hNGF was constructed and transformed to E. coli pLyS(DE3) for expression under induction of IPTG. The expressed protein was purified by chromatography, then determined for purity by SDS-PAGE and HPLC, for concentration by Lowry method, and for biological activity by TF-1 cell proliferation assay. Results E. coli fusion expression system for hNGF was constructed. The purities of three batches of expressed protein were 100%, while the specific activities were 5. 5 × 10~5, 6. 8 × 10~5 and 7. 4 × 10~5 IU/mg respectively, which were higher than those of NGF standard extracted from mouse salivary gland. Conclusion The hNGF with high purity and specificity was expressed successfully, which provided a reference for large-scale production of hNGF.

    2018 02 v.31 [Abstract][OnlineView][Download 447K]

  • Immune intervention of Astragalus polysacchaside to dendritic cells in rat model of allergic asthma

    LIU Jing;YOU Jin;Chongqing Sanxia Medicine High Academy;

    Objective To investigate the immune intervention of Astragalus polysacchaside(APS) to the dendritic cells(DCs) in rat model of allergic asthma(AS). Methods Thirty rats were sensitized with ovalbumin by continuous nebulization to establish the model of AS. Of the model rats,20 were injected i. p. with APS at dosages of 100 and 200 μg/kg,10 for each,while the other 10 as model control were untreated. Another 10 healthy rats were served as normal control. The symptoms of rats were observed 4 h after injection,and the curative effect was evaluated according to the presupposed standard. The lung functions of rats in various groups were determined 24 h after injection,while the rats were killed,of which the pulmonary mucosa associated lymphoid tissues were collected and determined for expressions of CD80, CD86, IL-4, IL-10 and IL-12 by immunohistochemical technique. Results According to the presupposed standard,remarkable effectiveness was observed in 9 rats in 100 μg/kg APS intervention group,while remission in one rat. However,remarkable effectiveness was observed in all the 10 rats in 200 μg/kg APS intervention group. Compared with those in normal control group,the forced expiratory volume in one second(FEV1)/forced vital capacity(FVC)ratio,peak expiratory flow rate(PEF) as well as expression levels of IL-10 and IL-12 in model control group decreased significantly(P < 0. 05),while the expression levels of CD80,CD86 and IL-4 increased significantly(P < 0. 05).However,compared with those in model control group,the FEV1/FVC ratio,PEF and as well as expression levels of IL-10 and IL-12 in 100 and 200 μg/kg APS intervention groups increased significantly(P < 0. 05),while the expression levels of CD80 and IL-4 decreased significantly(P < 0. 05),which showed no significant difference in two intervention groups(P > 0. 05). Conclusion APS may relieve AS in rat model by adjusting the phenotype of DCs.

    2018 02 v.31 [Abstract][OnlineView][Download 279K]

  • Immune intervention of porphyra polysaccharide to T cell subsets and cell factor in suckling mouse model of enterovirus 71 infection

    TIAN Bing;LIU Jing;Chongqing Sanxia Central Hospital;

    Objective To investigate the immune intervention of porphyra polysaccharide in suckling mouse model of enterovirus 71(EV71) infection. Methods Forty healthy suckling mice at age of 7 d were selected, of which 30 were injected i.p. with international standard EV71 VR-1432 strain, 200 μL(about 300 TCID50)for each, to establish the model of EV71 infection, while the other 10 were untreated and served as normal control. Twenty of the model mice were served as test group, which were treated with porphyra polysaccharide at a dosage of 40 mg/(kg · d) by gavage 24 h after the establishment of model, while the other 10 were untreated and served as model control. The clinical manifestations of suckling mice in three groups were observed on days 3 and 7 after inoculation of EV71. The serum samples were collected and determined for T cell subsets by flow cytometry, and for the expression levels of interferon α(IFNα), tumor necrosis factor-α(TNF-α), interleukin-6(IL-6) and IL-10 by ELISA. Results After immune intervention by porphyra polysaccharide, significant efficiency, efficiency, inefficiency and death occurred in 5, 13, 1 and 1 mice respectively. The proportions of CD4~+ and CD8~+ T cells in test group on day 7 after injection with EV71 increased significantly(P < 0. 01),while the CD4~+/CD8~+ decreased significantly(P < 0. 05), the IL-6 and TNF-α levels decreased significantly(P < 0. 001),and IL-10 and IFNα levels increased significantly(P < 0. 001). Conclusion Porphyra polysaccharide may induce T cell activation, promote the secretion of anti-inflammatory cytokines such as IFNα and regulate the immune function in suckling mice infected with EV71, which plays an role of immune intervention in EV71 infection.

    2018 02 v.31 [Abstract][OnlineView][Download 249K]

  • Effect of tetramethylpyrazine on cognitive impairment and expression of glial fibrillary acidic protein in hippocampal astrocytes of mice with chronic cerebral ischemia

    HOU Liang-juan;HUANG Xiao-huan;Chongqing Three Gorges Medical College;

    Objective To investigate the effect of tetramethylpyrazine(TMP) on cognitive impairment and expression of glial fibrillary acidic protein(GFAP) in hippocampal astrocytes of mice with chronic cerebral ischemia. Methods The mouse model of chronic cerebral ischemia was established by modified bilateral common carotid artery ligation(2 VO).The mice in two test groups were injected i. p. with TMP at low(30 mg/kg)and high(80 mg/kg)dosages respectively,10 for each, while those in model control and normal(sham operation)control groups with an equal volume of physiological saline, 24 h after operation, once a day for 8 weeks. The spatial learning capacity and cognitive function of mice were evaluated by Morris water maze. The superoxidedismutase(SOD) content in hippocampus of mice was determined by xanthine oxidase test, while the malonaldehyde(MDA) content by thiobarbituric acid test, and the tumor necrosis factor(TNF)-α and interleukin-1β(IL-1β)contents by double antibody ELISA. The expression level of GFAP in hippocampal CA1 region of mice was determined by immunohistochemical staining and Western blot. Results Compared with those in model control group, the escape latency and swimming distance of mice injected with TMP at low and high dosages in water maze localization experiment were shortened significantly(P < 0. 05), the times of crossing the original platform in space exploration experiment increased significantly(P < 0. 05), and the SOD content in hippocampus increased significantly(P < 0. 05). However, the MDA, TNF-α and IL-1β contents, the IOD value and the GFAP expression level decreased significantly(each P < 0. 05). Conclusion TMP improved the cognitive impairment of mice with chronic cerebral ischemia by a possible mechanism of enhancing the anti-lipid oxidation ability, inhibiting the proliferation and activation of astrocytes, decreasing the expression of GFAP, relieving the inflammatory response and reducing the hippocampus damage.

    2018 02 v.31 [Abstract][OnlineView][Download 290K]

  • Preparation and identification of monoclonal antibody against gE protein of bovine infectious rhinotracheitis virus

    ZHAO Wei;BI Ying;WEN Xiao-bo;NI Hong-bo;College of Animal Science and Veterinary Medicine, Heilongjiang Bayi Agricultural University;

    Objective To prepare and identify the monoclonal antibody(McAb) against gE protein of bovine infectious rhinotracheitis virus(IBRV). Methods IBRV gE gene was amplified and inserted into vector pET-32 a(+), and the constructed recombinant plasmid was transformed to E. coli BL21(DE3)and induced with IPTG. The expressed recombinant protein IBRV g E was purified with Ni-NTA Agarous Kit, with which BALB/c mice were immunized. The splenocytes of immunized mice were fused with SP2/0 cells, and the positive hybridoma cells were screened by indirect immunofluorescence assay(IFA). Mice were injected i. p. with the hybridoma cells, 3 × 10~6 cells for each, of which the ascites were collected. McAb was purified from the ascites by precipitation with ammonium sulphate and subclassed, the determined for concentration and chromosome number, and identified by Western blot and IFA. Results The recombinant protein IBRV gE, with a relative molecular mass of about 35 000, reached a purity of 1. 2 mg/mL after purification. A hybridoma cell strain stably secreting McAb was screened and named as 3 G9, and the concentration of obtained McAb was 1. 46 mg/mL. The McAb belonged to IgG1 subgroup with kappa light chain and 95 ~ 105 chro-mosomes, which showed specific reaction with IBRV. The McAb showed specific binding to the MDBK cells inoculated with IBRV, producing specific fluorescence. Conclusion IBDV gE protein was expressed by prokaryotic expression system and purified, and the McAb was prepared, which laid a foundation of development of specific diagnostic method and study on pathogenic mechanism of IBRV.

    2018 02 v.31 [Abstract][OnlineView][Download 270K]

  • Monitoring of specific functional antibody levels in elderly individuals inoculated with 23-valent pneumococcal polysaccharide vaccine

    QIAN Xiao-hua;SHEN Hong-bo;PAN Rong;QIAO Rui-jie;TAN Xiao-mei;CHEN Wei-zheng;The Center for Disease Control and Prevention in Hongkou District,Shanghai City;

    Objective To monitor the level of specific functional antibodies in elderly individuals inoculated with 23-valent pneumococcal polysaccharide vaccine(PPV-23). Methods Healthy individuals and those recovered from tuberculosis, with registered permanent residence in Shanghai and at age of more than 60 years, were vaccinated with PPV-23. The OPA antibody levels against 13 serotypes(1, 3, 4, 5, 6 A, 6 B, 7 F, 9 V, 14, 18 C, 19 A, 19 F, 23 F) of pneumococcus before as well as 1 and 6 months after vaccination were determined by multi-specificity opsonophagocytosis killing assay(MOPA), and the GMTs and increasing rates of antibodies against the 13 serotypes 1 and 6 months after vaccination were analyzed statistically. Results The GMTs of OPA antibodies against 13 serotypes of pneumococcus in most of the elderly individuals 1 and 6 months after vaccination were significantly higher than those before vaccination(P < 0. 05). However, the GMTs of antibodies against serotypes 1 and 3 six months after vaccination showed no significant difference with those before vaccination(P > 0. 05). The GMTs of antibodies against serotypes 6 A, 6 B, 7 F,18 C, 19 F and 23 F 6 months after vaccination showed no significant difference with those 1 month after vaccination(P >0. 05). Except that against serotype 1 in the individuals recovered from tuberculosis(37. 5%), the 2-fold increasing rates of GMTs of antibodies against all the 13 serotypes 1 month after vaccination were not less than 60%, which showed no significant difference in the healthy and recovered individuals(P > 0. 05). Except those against serotypes 1 and 3 in the individuals recovered from tuberculosis, the 4-fold increasing rates of GMTs of OPA antibodies against various serotypes showed no significant difference in the healthy and recovered individuals(P > 0. 05). However, the 2-fold increasing rates of GMTs of OPA antibodies against the 13 serotypes were more than 45% and, except that against serotype 1, showed no significant difference in healthy and recovered individuals(P > 0. 05). The 4-fold increasing rates of OPA antibody levels,except those against serotypes 1 and 18 C, showed no significant difference in the healthy and recovered individuals(P >0. 05). Conclusion Specific functional antibodies against 13 serotypes of pneumococcus were induced by PPV-23 in healthy elderly individuals and the individuals recovered from tuberculosis, which last for 6 months. However, the levels and persistence of OPA antibodies against serotypes 1 and 3 in the recovered individuals were significantly lower than those in healthy individuals.

    2018 02 v.31 [Abstract][OnlineView][Download 265K]

  • Analysis on application of vaccines of class Ⅱ in Minhang District,Shanghai City during 2011～2015

    WANG Xi;WANG Ye;JIN Bao-fang;LI Jie;ZHANG Li-ping;MEI Ke-wen;Center for Disease Control and Prevention of Minhang District,Shanghai City;

    Objective To analyze the application of vaccines of class Ⅱ in Minhang District, Shanghai City, China during2011 ~ 2015, and to provide a basis for strengthening the vaccination service management of the vaccines. Methods Data on application of vaccines of class Ⅱ in Minhang District from 2011 to 2015 were derived from the China Immunization Information System and the Biological Product Management Information System for descriptive analysis.Results In 2011 ~ 2015, the kinds of vaccines of class Ⅱ used in Minhang District remained stable. However, the amounts used and the proportion in total vaccines used increased from 516 000 to 578 200 doses and from 36. 67% to42. 48% respectively. The amount used of vaccines of class Ⅱ in floating population was higher than that in local population. The amount used of vaccines of class Ⅱ as substitutes of those of class Ⅰ increased year by year, while the substitution rate increased from 17. 92% to 36. 68%. However, the substitution rate of groups A + C to group A meningococcal conjugate vaccine was the highest. Conclusion Varieties of vaccines of class Ⅱ were used in a large quantity in Minhang District during 2011 ~ 2015, which prevented and controlled the vaccine preventable diseases effectively.

    2018 02 v.31 [Abstract][OnlineView][Download 192K]

  • Analysis of suspected adverse events following immunization during 2011～2016 in Dalian City,Liaoning Province,China

    HAN Yi-nan;YANG Yue;LIN Qian;Dalian Center for Disease Prevention and Control;

    Objective To analyze the monitoring result of adverse events following immunization(AEFI) during 2011 ~2016 in Dalian City,Liaoning Province,China and evaluate the operation of AEFI monitoring system. Methods The cases of AEFI during 2011 ~ 2016 in Dalian were collected from AEFI monitoring system. Descriptive statistical analysis was performed on the characteristics and relevant indexes of AEFI by using Excel 2007 software. Results A total of 467 cases of AEFI were reported during the period,and the estimated annual incidence of AEFI was 4. 87 per 100 000 doses.The 48 hour report rate of AEFI was 97. 43%,while the timely investigation rate was 98. 46%. A total of 209 cases were reported from June to August,accounting for 44. 75% of the total cases. A total of 378 cases occurred in the infants at ages of less than 2 years,accounting for 80. 94% of total cases. A total of 142 cases were caused by DTa P,accounting for 30. 41% of the total cases. A total of 401 cases of general reactions were reported,accounting for 85. 87% of the total cases,while 43 cases of adverse reactions reported,accounting for 9. 21%. A total of 379 cases occurred within 48 hours after vaccination,accounting for 81. 16% of the total cases. Most of the general reactions were caused by Da TP(133 cases),accounting for 33. 17% of total cases. However,most of adverse reactions were caused by MR vaccine(12 cases),accounting for 27. 91%. The most frequent symptoms of AEFI included fever, redness and swelling and induration,accounting for 83. 30% of total cases. Conclusion The monitoring system of AEFI in Dalian during 2011 ~2016 run well. However,the retention observation and follow-up of vaccination with key vaccines should be strengthened to improve the quality of monitoring and the ability in emergency handling of AEFI.

    2018 02 v.31 [Abstract][OnlineView][Download 221K]

  • Paired case-control research of risk factors for measles incidence in Minhang District, Shanghai City, China

    CHEN Hong-hong;JIN Bao-bang;WANG Ye;WANG Xi;LU Jia;DU Yan;ZHANG Li-ping;MEI Ke-wen;Center for Disease Prevention and Control of Minhang District,Shanghai City;

    Objective To analyze the relevant risk factors for measles incidence in Minhang District, Shanghai City,China and provide a basis for prevention and control of measles. Methods A 1 ∶ 1 paired case control study was carried out among measles cases in Minhang District during 2014 ~ 2016. The information including social and demographic characteristics, the history of measles immunization and population mobility were collected, and the relevant risk factors for measles incidence were subjected to single factor and paired multi factor Logistic regressions. Results The factors for measles incidence included exposure to hospital, taking the subway, contact with the suspected patient with measles and travelling 7 ~ 21 d before incidence(P < 0. 05). In the children at ages of less than 8 months and of 8 months to 15 years, the history of measles vaccination of children or their mothers as well as high degree of education of mothers were protective factors of incidence of measles. However, in the age group of more than 15 years, the history of measles vaccination was the protective factor(P < 0. 05). Conclusion Absence of history of measles vaccination, exposure to hospital, taking the subway and travelling were the relevant risk factors for incidence of measles in Minghan District in recent years, targeting which the measure should be taken to prevent and control the epidemic.

    2018 02 v.31 [Abstract][OnlineView][Download 182K]

  • Development of an improved method for determination of hepatitis C virus titer using a fluorescence protein relocalization system

    BI Yan-wei;LI Zhi-hua;LONG Qiong;ZHANG Hui;CHEN Chen;LI Ya-dong;LI Yu-zhong;CUN Wei;Institute of Medical Biology, Chinese Academy of Medical Sciences & Peking Union Medical College;

    Objective To develop a simple and rapid fluorescent reporter system for determination of hepatitis C virus(HCV)infection. Methods The amino acid residues 462 ~ 540 at C-terminus of interferon beta promoter stimulator-1(IPS-1) gene sequence, IPS-1(C462-540)-2 A-EGFP-puro, and an SV40 nuclear localization signal fused to residues462 ~ 540 of IPS-1 gene, IPS-1(C462-540)-IRES-EGFP-puro, were amplified by RT-PCR or PCR, then inserted into lentiviral vectors LV-CAG--EGFP-WPRE and LV-CAG—m Cherry(RFP)-WPRE to construct recombinant plasmids for expressions of EGFP-IPS-1(C462 ~ 540) and RFP-NLS-IPS-1(C462 ~ 540), based on which the recombinant plasmids containing IPS-1(C462-540)-2 A-EGFP-puro and IPS-1(C462-540)-IRES-EGFP-puro were constructed, respectively.The constructed recombinant lentiviral plasmids containing IPS-1(C462-540)were packaged in 293 T cells. Huh7. 5 cells were infected with the obtained recombinant lentiviruses, from which the clones expressing enhanced green fluorescent protein(EGFP)or red fluorescent protein(RFP)were screened by flow cytometry. Alternatively, the Huh7. 5 cells stably expressing EGFP were screened with puromycin. The cells were infected with 10-fold serially diluted HCV, and determined for virus titer. Results Sequencing proved that four recombinant lentiviral plasmids were constructed correctly. EGFP or RFP were observed in Huh7. 5 cells 48 h after infection with the obtained lentivirus. The cell lines stably expressing EGFP-IPS-1(C462-540) and RFP-IPS-1(C462-540) were obtained by screening with flow cytometry, while those stably expressing EGFP-IPS-1(C462-540) and RFP-IPS-1 by screening with puromycin. After infection with 10-fold serially diluted HCV, EGFP showed diffuse fluorescence in cells while RFP showed nuclear translocation of fluorescence, which formed obvious fluorescence spots in the presence of methyl cellulose. The HCV titers were obtained by calculating the spot number, which were(5. 6 ± 0. 2) × 10~5 and(5. 5 ± 0. 2) × 10~5 PFU/m L respectively. Conclusion A fluorescent cell-based reporter system was successfully constructed, based on which a simple and rapid method for dteremination of HCV titer was developed.

    2018 02 v.31 [Abstract][OnlineView][Download 507K]

  • A modified high-throughput screening method for cephalosporin C acylase

    TANG Cun-duo;SHI Hong-ling;ZHANG Mei;MA Ying;TANG Qing-hai;ZHOU Jun-shi;YAO Lun-guang;KAN Yun-chao;Henan Provincial Engineering Laboratory of Insect Bio-reactor, Nanyang Normal University;

    Objective To modify a high-throughput screening method for cephalosporin C(CPC) acylase to obtain a mutant with higher catalytic activity. Methods Based on the principle that the compound of p-dimethylaminobenzaldehyde(p-DAB) and 7-amino cephalosporanic acid(7-ACA) had the maximum absorption at 405 nm, the recombinant strains were induced and expressed at low temperature and low concentration using sterile deep 96-well plate, and lyzed with the buffer containing lysozyme and DNase, then the substrate hydrolysis and reaction termination were carried out on 96-well plate. The products were subjected to rapid quantitative analysis by ELISA after p-DAB colour development reaction to realize the high-throughput screening of CPC acetlase mutants. Results Two mutants, 1A10 and 2G8, were screened from mutant library of CPC acylase by the modified p-DAB colorimetry. The specific activities of the two mutants were 1. 7 and 2. 4 U/mg at 13 ℃ respectivly, which were significantly higher than that of the original caylase(0. 8 U/mg).Under the optimized condition for screening, the change of A405 reflected the enzyme activity accurately, which guaranteed the reliability of the method. Conclusion The modified p-DAB colorimetry was rapid, simple and of high throughput and high accuracy, which increased the efficacy of modifcation and screening of CPC acylase, and provided a technical support for the development of cold CPC acylase.

    2018 02 v.31 [Abstract][OnlineView][Download 192K]

  • Optimization of condition for ultracentrifugation for verification of inactivation effect of inactivated enterovirus 71 vaccine

    ZHAO Heng;YUAN Mei;YAN Jiao;WANG Yun-fei;YANG Jia;JIANG Guo-run;SHI Lei;FENG Kai;YANG Xiao-lei;HONG Chao;Institute of Medical Biology, Chinese Academy of Medical Sciences and Peking Union Medical College;

    Objective To optimize the condition for ultracentrifugation for verification of inactivation effect of inactivated enterovirus 71(EV71) vaccine, and provide a basis for treatment of samples of inactivation verification test of the vac-cine. Methods The harvested liquid EV71 was ultracentrifuged at various rotation rates(45 000, 40 000, 35 000, 30 000 and 25 000 r/min)for various(120, 90, 60 and 30)minutes, of which the titers before and after ultracentrifugation were determined, and recovery rates were calculated to compare the recovery effects under various conditions. Results The precipitation effects of EV71 at the same rotation rate were positively related to the time for ultracentrifugation(R~2 increased from 0. 77 to 0. 99), while those at the same time point after ultracentrifugation were positively related to the rotation rate(R~2 increased from 0. 88 to 0. 99). When the rotation rate was more than 40 000 r/min, the recovery rates of EV71 after ultracentrifugation for various minutes were more than 90%. When the time for ultracentrifugation was 120 min, and the rotation rate was 45 000, the precipitation effect of EV71 was satisfactory, while the recovery rate reached a maximum of 98%. The recovery rate of EV71 after ultracentrifugation at 45 000 r/min for 60 min was 97%,which showed no significant difference with that after ultracentrifugation for 120 min. However, the recovery rate after ul-tracentrifugation for 30 min showed significant difference with those after ultracentrifugation for 120, 90 and 60 min(P <0. 05). Conclusion The precipitation effect of EV71 was positively related to rotation rate and time for ultracentrifuga-tion. The effect was improved, while the recovery rate increased, with the increasing rotation rate and time.

    2018 02 v.31 [Abstract][OnlineView][Download 154K]

  • Development of a method for preparation of apolipoprotein A1 from human plasma by fumed silicon dioxide

    LI Guang-fei;TAO Ling;WANG Wei;Hualan Biological Engineering Inc.;

    Objective To prepare apolipoprotein A1(apoA1) from human plasma by using fumed silicon dioxide and to evaluate the effect of this method. Methods Fumed silicon dioxide was added into human plasma to adsorb apoA1,and the effects of proportion(m/V)of fumed silicon dioxide added into human plasma and incubation time on the adsorption efficiency were investigated to optimize the adsorption condition. After adsorption,the fumed silicon dioxide was separated from the plasma by centrifugation,on which the adsorbed apoA1 was washed by alcohol solution at various concentrations,and the alcohol concentration in the elution solution was optimized. The eluted product was determined for protein concentration by BCA method,from which the proteins were separated by SDS-PAGE. The purity of apoA1 was analyzed by gel imaging analysis software to evaluate the efficiency of this method. Results Addition of 1%(m/V) of fumed silicon dioxide into human plasma was optimal to extraction of apoA1. The saturated adsorption of apoA1 was completed by incubation of the mixture of fumed silicon dioxide and human plasma for 10 min. The centrifugation at a centrifugal force of more than 12 000 × g for 10 min was optimal for the solid-liquid separation of the mixture. The optimal concentration of ethanol in the elution buffer was 20%(V/V)and,under this condition,0. 92 mg of apoA1 with a purity of 79% was prepared from 1 mL of human plasma. Conclusion A method for preparation of apoA1 from human plasma by fumed silicon dioxide was developed,which was simple and effective.

    2018 02 v.31 [Abstract][OnlineView][Download 264K]

  • Analysis of Th1/Th2 balance in bronchoalveolar lavage fluid of children with Mycoplasma pneumoniae pneumonia by next-generation sequencing

    ZHUANG Hua-dong;LI Wei;WANG Chun-yan;LU Rui-hua;LIU Jun-hui;MENG Fan-zheng;Department of the Second Pediatric Respiratory, The First Hospital of Jilin University;

    Objective To analyze the expression levels of interferon-gamma(IFNγ) and interleukin-4(IL-4) in the bronchoalveolar lavage fluid(BALF) of children with Mycoplasma pneumonia(MPP) as well as the change trend of the immune response balance by next-generation sequencing(NGS). Methods The BALF specimens of children with bronchial foreign matters, mild MPP and severe MPP were collected and sequenced by transcriptome sequencing technique in NGS. The expression levels of IFNγ and IL-4 were determined, based on which the IL-4/IFNγ ratio was calculated.The change trends of IFNγ, IL-4 and IL-4/IFNγ were analyzed by real-time q PCR. Results The IL-4 and IFNγ levels as well as IL-4/IFNγ ratio in the children with mild MPP were higher than those with bronchial foreign matters.However, the IL-4 and IFNγ levels of children with severe MPP were higher than those with mild MPP and with bronchial foreign matters, while the IL-4/IFNγ ratio was higher than that with bronchial foreign matters but lower than that with mild MPP. Conclusion Both IL-4 and IFNγ levels increased in the BALF of children with mild and severe MPP, and the IL-4/IFNγ ratio indicated the imbalance of Th1/Th2 responses. Th2 response was predominant in the children with mild and severe MPP.

    2018 02 v.31 [Abstract][OnlineView][Download 269K]

  • Application and progress in research of live attenuated vaccines

    LI Zheng;LIU Ye;LI Chun-yang;Department of Recombinant Pharmaceuticals and Novel Vaccine, Chengdu Institute of Biological Products Co., Ltd.;

    Live attenuated vaccines may induce immune response and stimulate the production of memory B and T lymphocytes, resulting in long-term or life-lasting protection, which shows high and persistent immunity as compared with inactivated vaccines. Live attenuated vaccines play a key role in the early stage of vaccine history, such as vaccinia for pro-phylaxis of smallpox. The live attenuated vaccines developed subsequently, such as meales, mumps and rubella combined vaccine, Japanese encephalitis vaccine, varicella vaccine, rotavirus vaccine, Dengue fever vaccine and BCG, still take an irreplaceable role in prevention of diseases. The novel live attenuated vaccines may also be used for prevention and treat-ment of tumor, diabetes, pneumonia, and novel tuberculosis, which show broad prospects of development and application.This paper reviews the application of live attenuated vaccines in infectious diseases, tumor and other diseases as well as progress in research of the vaccines.

    2018 02 v.31 [Abstract][OnlineView][Download 192K]

  • Progress in development of rotavirus vaccine

    SI Lu;WEI Zhi-dong;Lanzhou Institute of Biological Products Co., Ltd.;

    Rotavirus(RV)is one of the most important causes of severe acute diarrhea in children younger than 5 years,with the highest burden of disease observed in low-income countries in Africa and Asia. About 500 000 to 600 000 children die from RV-induced diarrhea each year, of whom more than 90% come from developing countries. Regardless of socioeconomic status and regional differences, almost all children are infected with RV before the age of five. There is no specific therapy for RV-induced diarrhea. The study found that the rational use of RV vaccine is appropriate to reduce diarrhea. This paper reviews the progress, challenge and prospect of development of RV vaccine.

    2018 02 v.31 [Abstract][OnlineView][Download 190K]

  • Advances in research of pertussis toxin

    LI Xin;ZHANG Xin-chuang;XIE Gui-lin;Lanzhou Institute of Biological Products Co., Ltd, Center for Gansu Provincial Vaccine Engineering Research;

    Pertussis toxin is a major virulence factor produced by Bordetella pertussis and has a variety of biological activities. It is the cause of many clinical symptoms of pertussis and the only uncontested antigenic component of acellular pertussis vaccine. This paper summarizes the molecular structure and biological activity of pertussis toxin as well as its application in vaccines.

    2018 02 v.31 [Abstract][OnlineView][Download 261K]


@2012 Editorial Department of "Chinese Journal of Biologicals"