2015年01期目次

  • Construction of recombinant baculovirus for co-expression of M1 and M2 genes of influenza virus

    YAO Li-hong;CHEN Ai-jun;XU Peng-wei;ZHANG Zhi-qing;GUO Jian-qiang;Key Laboratory for Medical Virology,Ministry of Health,National Institute for Viral Disease Control and Prevention,China CDC;

    Objective To construct recombinant baculovirus for co-expression of matrix protein 1(M1)and matrix protein 2(M2)genes of influenza virus. Methods Full lengths of M1 and M2 genes of influenza H1N1 virus(A / PR / 8 / 34)were amplified by PCR separately and inserted into the two polyclonal sites of vector p Fast Bacdual(p FBD)successively. Positive recombinant transposition vector p FBD-M1 / M2 was transformed to competent DH10 Bac cells containing shuttle vector Bacimd, based on which recombinant shuttle vector r Bacmid-M1 / M2 was obtained by homologous recombination, and transfected to sf9 cells in mediation of liposome for packaging. The obtained recombinant baculovirus r Bac-M1 / M2 was determined for titer by plaque formation test, for M1 and M2 genes in genome by PCR, and for expressions of M1 and M2 genes by indirect imunofluorescence assay(IFA)and Western blot. Results PCR proved that recombinant shuttle plasmid r Bacmid-M1 / M2 was constructed correctly. The titer of r Bac-M1 / M2 of passage 3 was 1 × 108 pfu / ml. A band at length of3 600 bp was amplified from sf9 cells infected with r Bac-M1 / M2 by PCR. Obviously specific yellowish-green fluorescence was observed in the infected cells. The lytic supernatant of cells infected with r Bac-M1 / M2 showed specific reaction bands,with relative molecular masses of about 26 000 and 11 000 respectively, showed specific reaction with mouse polyclonal antibody against influenza virus PR8 strain. Conclusion Recombinant baculovirus for co-expression of M1 and M2 genes of influenza H1N1 virus was construct successfully, which laid a foundation of construction of influenza virus-like particles and development of novel broad vaccine against influenza.

    2015 01 v.28 [Abstract][OnlineView][Download 295K]

  • Preparation and immunogenicity of inactivated hepatitis A vaccine(attenuated L-A-1 strain)

    LIU Ling-jiu;YUE Li-guang;XU Yan-ling;HUI Qi;XU Xiao-xia;HOU Li-juan;LI Shu-yan;LI Yong;XIA Qing-juan;ZHAO Xiao-lin;Changchun lnstititue of Biological Products Co.,Ltd.;

    Objective To prepare inactivated hepatitis A vaccine with attenuated L-A-1 strain and determine its immunogenicity. Methods Human embryo lung diploid cells(2BS strain) were cultured with cell factory and infected with attenuated L-A-1 strain. The cells containing propagated virus were harvested,lysed,precipitated with polyethylene glycol(PEG) 6000,extracted with chloroform,purified by gel filtration chromatography,inactivated with formaldehyde and adsorbed with aluminium hydroxide. Three batches of experimental vaccine were prepared and subjected to overall control tests according to the requirements in Chinese Pharmacopoeia(Volume Ⅲ,2010 edition). Potency test in mice was performed,based on which the median effective dilution was calculated. The vaccine was stored at 37 ℃ for 5,10,15,20,25 and 30 d to evaluate the accelerated stability. Results The antigen recovery rate of hepatitis A virus after extraction and purification was more than 80%,while the removal rate of foreign protein reached more than 95%. All the quality indexes of three batches of experimental vaccines met the requirements in Chinese Pharmacopoeia(Volume Ⅲ,2010 edition). The potency test in mice showed that the median effective dilutions were 1. 25 ~ 1. 40 and 2. 02 ~ 2. 26 times of those of imported and domestic vaccines respectively. The relative potency in vitro of vaccine after storage at 37 ℃for 30 d was still qualified. Conclusion Inactivated hepatitis A vaccine with high purity was prepared,which showed good immunogenicity in mice. It laid a foundation of further large-scale production of inactivated hepatitis A vaccine.

    2015 01 v.28 [Abstract][OnlineView][Download 257K]

  • Immune effect of Brucella DNA vaccine using CpG ODN1826 as adjuvant in mice

    ZHENG Yuan-qiang;ZHOU Yu-mei;LV Lu;LI Xin;HOU Li-qing;SUN Qi;BAO Yu-long;DING Feng;SHI Yan-chun;HAN Xin-rong;Research Center of Molecular Biology,Inner Mongolia Medical University;

    Objective To investigate the immune effect of Brucella DNA vaccine using CpG ODN1826 as adjuvant in mice.Methods C57 / BL6 mice were randomly divided into Vaccine + CpG,Vaccine and control groups,and injected i.m.twice with 20 μg pc DNA3. 1-L7 / L12 + 10 μg Cp G ODN1826,20 μg pc DNA3. 1-L7 / L12 and physiological saline respectively into back leg tibialis anterior(TA)muscle,at an interval of 2 weeks. The cytokines in sera were determined by ELISA 2 weeks after the second dose,while non-specific proliferation of splenocytes by MTT incorporation,and the histological changes in spleen by HE staining. Results The contents of Th1 type cytokines such as IL-2,IL-12 and IFNγin sera of mice in Vaccine + Cp G group increased significantly as compared with those in Vaccine and control groups(each P < 0. 01),while the proliferation of splenocytes was enhanced,the splenic germinal center was enlarged obviously,and the cell proliferation was active. Conclusion Brucella DNA vaccine using CpG ODN1826 as adjuvant induced Th1 immune response and enhanced the proliferation of splenocytes in C57 / BL6 mice.

    2015 01 v.28 [Abstract][OnlineView][Download 341K]

  • Preparation of inactivated enterovirus 71 vaccine by large-scale culture of Vero cells on microcarriers and its immunogenicity

    LUO Yong-bin;ZHAO Xian-chen;ZHANG Cheng-jian;HUANG Fen;ZENG Wei-kun;JING Shen-rong;Kunming Institute of Botany,Chinese Academy of Science;

    Objective To prepare inactivated enterovirus 71(EV71)vaccine by large-scale culture of Vero cells on microcarriers and determine its immunogenicity. Methods EV71 KC414134 strain was isolated from the fecal samples of infants with hand,foot and mouth disease(HFMD) complicated with severe encephalitis,and determined for specificity by immunofluorescent assay(IFA). KC414134 strain was prepared by large-scale culture of Vero cells on micro-carriers,filtered with 50 KD ultrafilltration membrane,purified by sucrose density gradient centrifugation,inactivated with β-propiolactone and mixed with aluminum hydroxide adjuvant. Kunming mice were immunized s. c. with the prepared inactivated vaccine for 3 times,and determined for the total antibody and neutralizing antibody titers in sera one week after the last immunization. Results Specific fluorescence was distributed in cytoplasma,indicating that the isolated KC414134 strain was EV71. A large quantity of KC414134 strain was prepared by large-scale culture of Vero cells on microcarriers.However,no virus particles with ideal purity were obtained after purification,though the virus was inactivated completely.The inactivated vaccine induced high total antibody and neutralizing antibody titers in mice. Conclusion Inactivated EV71 vaccine was successfully prepared by large-scale culture of Vero cells on microcarriers,which laid a foundation of development of inactivated vaccine.

    2015 01 v.28 [Abstract][OnlineView][Download 430K]

  • Inhibitory effect of herpes simplex virus type 1 glycoprotein B DNA vaccine on primary and latent infection with the virus

    MENG Xiang-jun;SU Yun;Department of ophthalmology,Affiliated Zhongshan Hospital of Dalian University;

    Objective To observe the role of herpes simplex virus type 1(HSV-1)glycoprotein B(pg B)DNA vaccine in the primary and latent HSV-1 infections. Methods BALB / c mice were randomly divided into test(A),positive control(B),vector control(C)and blank control(D)groups. The mice in group A were immunized i.m. with PBS containing plasmid pg B,while those in group B with inactivated HSV-1,those in group C with PBS containing plasmid pc DNA3,and those in group D with PBS. The mice in various groups were immunized for 3 times each at an interval of 3 weeks,and inoculated with HSV-1 by scarification in cornea 3 weeks after the last immunization. All the mice were killed on day 30 after inoculation,of which intrigeminal ganglia was inoculated to Vero cells,and the latent HSV-1 infection was observed.Results Only mild corneal epithelitis was observed in the mice in groups A and B after inoculation with HSV-1,which was recovered within 6 d and showed no significant difference in various groups(P > 0. 05). The corneal epithelitis was the most serious on the 2nd day in groups C and D,which disappeared gradually 7 d later and showed significant difference with that in group A(P < 0. 05). No interstitial keratitis or death was observed in groups A and B. However,inflammatory reactions were observed in groups C and D on day 5 after inoculation,which was exacerbated gradually and was most serious on day11 ~ 14 after inoculation,while 30% of mice died on day 30. Latent HSV-1 infection was observed in Vero cells. No CPEs were observed in groups A and B,while were observed on day 5 after inoculation in groups C and D. Conclusion HSV-1pg B effectively prevented primary infection and inhibited the latent infection with HSV-1 in mice.

    2015 01 v.28 [Abstract][OnlineView][Download 327K]

  • Fusion expression and verification of targeting of peptide Nts-1 with targeted binding to inflamed endothelial cells

    YANG Min;YANG Yu-tong;GUO Ming-yang;ZHANG Jun;LUO Yong;Department of Traditional Chinese Medicine,Centre of Integrated Medicine on Rheumatism,General Hospital of Chengdu Military Area Command PLA;

    Objective To construct the expression vector for peptide Nts-1 to express and purify the fusion protein and preliminarily verify its targeted binding to inflamed endothelial cells. Methods The original sequence of Nts-1 was processed with synonymous mutation according to the preference codon of E. coli,of which both C- and N-terminuses were added with a cysteine for cyclization. The sequence was inserted into the C-terminus of EGFP in vector p ET-14 bEGFP. The constructed prokaryotic expression vector p ET-14b-EGFP-Nts-1 was transformed to E. coli for expression under induction of IPTG. The expressed fusion protein his-EGFP-Nts-1 was purified and identified by SDS-PAGE,of which the affinity to LPS-inflamed human umbilical vein endothelial cells(HUVECs) was identified in vivo. Results Expression vector p ET-14b-EGFP-Nts-1 was successfully constructed,and the expressed fusion protein,with a relative molecular mass of about 32 000,reached a purity of more than 95%. The fusion protein was targeted to the surface of inflamed HUVECs specifically,of which the affinity was mediated by Nts-1. Conclusion Fusion protein his-EGFP-Nts-1 was expressed successfully,of which the affinity to inflamed HUVECs was verified preliminarily. It laid a foundation of study on biochemical activity and function of the fusion protein.

    2015 01 v.28 [Abstract][OnlineView][Download 466K]

  • Replication kinetics and antitumor activity in vitro of mammalian reovirus

    LIU Hui;YANG Hong-yu;DING Zhuang;TAO Dong;XU Zhi-qiang;ZHAO Hong;LI Ting-ting;BAI Cong;ZHANG Nan;ZUO Yi;CAO Yu;Changchun Institute of Biological Products Co.,Ltd.;

    Objective To investigate the replication kinetics of standard strain of mammalian reovirus type 3(Reovirus3)in various cells and its antitumor activity in vitro. Methods Reovirus 3 at low concentrations were inoculated to VeroE6,MRC-5 and MDBK cells for three blind passages,observed for CPE daily and determined for replication by direct IFA.Melanoma cells and MDBK cells were placed in a vessel for virus challenge,and observed for CPE daily. Results Obvious CPE was observed in MDBK cells 72 h after inoculation with Reovirus 3 at a low concentration,while no CPE in MRC-5 cells and only slight CPE in Vero-E6 cells,after three blind passages. Reovirus 3 was subcultured stably in MDBK cells. No virus was detected in MRC-5 cells after three blind passages. However,the virus was detected in VeroE6 cells after two blind passages,of which the fluorescence intensity was low. Melanoma cells were broken and died in a large quantity 30 h after inoculation with Reovirus 3,while local CPE in MDBK cells,and the pathogenic area was expanded. Conclusion MDBK cells were optimal for the culture of reovirus. However,the virus might be propagated in Vero-E6 cells,of which the replication kinetics were weak than that in MDBK cells. MRC-5 cells were unsuitable for culture of reovirus. Reovirus selectively killed cancer cells in vitro,of which the in vivo antitumor activity should be further studied in animal model because of more complicated immune system.

    2015 01 v.28 [Abstract][OnlineView][Download 407K]

  • Prediction of secondary structure and B cell epitope of glycoprotein of Ebolavirus

    ZHAO Yu-jiao;HUANG Xin-wei;LI Duo;QIU Li-juan;SUN Qiang-ming;Institute of Medical Biology,Chinese Academy of Medical Sciences and Peking Union Medical College,Yunnan Key Laboratory of Vaccine Research & Development on Severe Infectious Disease;

    Objective To predict and analyze the secondary structure and B cell epitope of glycoprotein(GP) of Ebolavirus strain of type Zaire isolated from patients in West Africa with an outbreak in 2014. Methods The genomes of Ebolavirus strain of type Zaire isolated from West Africa published recently were searched from Gen Bank to obtain the gene and amino acid sequences of GP1,GP2 and s GP,based on which the amino acid composition ratio and possible protein binding site were analyzed,and the possibly exposed and buried regions in amino acid sequence were judged by hydrophilicity / hydrophobicity parameters. Results Threonine occupied the maxiumum ratio of amino acids encoding GP1,GP2 and s GP. The protein binding sites of GP1 and s GP were abundant,while the exposed and buried regions were distributed alternatively. Compared with those of GP1 and s GP,GP2 showed a special structure with a large disorder region on protein surface. The sites 20 ~ 37,166 ~ 186 at N-terminus of GP1,sites 361 ~ 379 at N-terminus of GP2 as well as sites 20 ~ 37 and 166 ~ 185 at N-terminus of s GP might be transmembrane helical structure. There might be four disulfide bond regions in s GP. B cell epitope analysis showed that the hydrophobic region were mainly concentrated in AA 1 ~ 325 of N-terminus of GP1 and GP2,while the hydrophilic region in AA 326 ~ 676,with a distinct boundary but without obvious overlap. There were two potential linear epitopes at sites 324 ~ 450 and sites 461 ~ 676 at N-terminus respectively. A total of 25 amino acid sites or sequences which might form conformational epitopes were predicted,including 6 with possibilities of more than 90%. Conclusion The prediction of secondary structure and B cell epitope of GP of Ebolavirus strain of type Zaire provided a reference for study on the structure and function of GP,recognition of B cell epitope as well as development of prophylactic,therapy and diagnostic products against Ebolavirus.

    2015 01 v.28 [Abstract][OnlineView][Download 487K]

  • Prokaryotic expression and purification of truncated protein F212-489 of respiratory syncytial virus

    FENG Meng-die;FENG Jin;HONG Yu;XU Ze-yang;MAO Pu-jia;HUANG Fen;JING Shen-rong;ZENG Wei-kun;Medical Faculty,Kunming University of Science and Technology;

    Objective To construct the truncated F1 protein(F212-489) of human respiratory syncytial virus(RSV) in prokaryotic cells and purify the expressed product. Methods The f212-489 gene was amplified from plasmid p MD-18T-f by PCR and identified by TA cloning,then inserted into prokaryotic expression vector p ET-28 a. The constructed recombinat plasmid p ET-28a-f212-489 was transformed to E. coli BL21(DE3)for expression under induction of IPTG. The expressed recombinant protein was purified by nickel ion affinity chromatography and identified by Western blot. Results Restriction analysis showed that recombinant plasmid p ET-28a-f212-489 was constructed correctly. The expressed recombinant protein,with a relative molecualr mass of about 30 000,mainly existed in a form of inclusion body,and contained about 10% of total somatic protein. The purified recombinant protein reached a purity of 85%,and showed specific binding to goat anti-RSV polyclonal antibody. Conclusion The protein F212-489 of RSV was expressed successfully and showed good reactogenicity after purification,which laid a foundation of study on the immune mechanism of RSV and preparation of subunit or polypeptide vaccine by gene engineering technique.

    2015 01 v.28 [Abstract][OnlineView][Download 406K]

  • Analysis of physicochemical properties of three aluminum hydroxide adjuvant

    AI Xu-lu;SUN Xiao-yan;CHEN Shao-hua;XUE Hong-gang;HU Ye-qin;Wuhan Institute of Biological Products Co.,Ltd.;

    Objective To analyze the physicochemical properties of three aluminum hydroxide adjuvant. Methods The physicochemical properties of aluminum hydroxide adjuvant C prepared by the authors were analyzed by X-ray diffraction,transmission electron microscopy,determination of particle size and protein adsorptive capacity,sedimentation and the alteration of p H value after autoclaving,and compared with those of two commercial adjuvant A and B. Results All the three aluminum hydroxide adjuvant were poorly crystalline boehmite,of which the primary particles formed loose aggregates at sizes of 1 ~ 10 μm and the protein adsorptive capacity was more than 2 mg BSA / mg Al,while the sedimentation met the requirements in the European Pharmacopoeia 7. 0,and the alteration of p H value after autoclaving was less than0. 5. Conclusion Most of parameters of aluminum hydroxide adjuvant prepared by the authors were similar to,while the protein adsorptive capacity was significantly higher,than those of commercial adjuvant. The study provided a reference for the establishment of quality control standard of aluminum hydroxide adjuvant.

    2015 01 v.28 [Abstract][OnlineView][Download 470K]

  • Construction and identification of eukaryotic expression vector for human cyclin-dependent kinase 10

    YOU Yan-jie;YUAN Yi;LI Wen-mei;LIU Jia-jia;ZHANG Xiao-hui;Department of Pharmacy,Luohe Medical College;

    Objective To construct and identify a eukaryotic expression vector for human cyclin-dependent kinase 10(CDK10). Methods The full-length encoding region sequence of CDK10 gene was amplified by RT-PCR from human nasopharyngeal carcinoma NHE1 cells and cloned into eukaryotic expression vector pc DNA3. 1(+). The constructed recombinant plasmid pc DNA-CDK10 was transfected to CNE-2 cells,and the expression level of CDK10 was determined by Western blot. Results Both restriction analysis and sequencing proved that recombinant plasmid pc DNA-CDK10 was constructed correctly. Stable transfected CDK10c1 and CDK10c2 cells for over-expression of CDK10 were obtained 2weeks after screening with 800 μg / ml G418,in which the expression levels of CDK10 increased significantly as compared with those in the cells transfected with empty vector(each P < 0. 05). Conclusion The eukaryotic expression vector for human CDK10 was successfully constructed.

    2015 01 v.28 [Abstract][OnlineView][Download 353K]

  • Construction of eukaryotic expression vector for Smac-EGFP

    SHENG Si-chen;SUN Chao;JIN Ying-hua;LI Yang;College of Life Science,Key Laboratory of Ministry of Education for Molecular Enzymological Engineering;

    Objective To construct a eukaryotic expression vector for fusion gene of enhanced green fluorescent protein(EGFP)and second mitochondria-derived activator of caspases(Smac). Methods Total RNA was extracted from He La cells and reversely transcribed to c DNA which was used as a template for amplification of Smac gene by RT-PCR. The amplified Smac gene was inserted to vector p EGFP-N3,and the constructed recombinant plasmid p EGFP-N3-Smac was transfected to He La cells in mediation of lipofectamine,and observed for distribution and expression under inverted fluorescent microscope. The expressed fusion protein Smac-EGFP was identified by Western blot. Results Eukaryotic expression vector p EGFP-Smac was constructed successfully. Obvious and regular expression of GFP was observed in mitochondria of transfected He La cells. The relative molecular mass of expressed fusion protein Smac-EGFP was about49 000. Conclusion Recombinant eukaryotic expression vector p EGFP-N3-Smac was constructed successfully,and fusion protein was expressed in He La cells,which provided a reference for further study on the function of Smac.

    2015 01 v.28 [Abstract][OnlineView][Download 371K]

  • Characters of VP1 gene sequence of a Coxsackievirus B2 isolate

    SU Tingi;ZHU Yan-jui;YAN Shan-shani;CHEN Jun-yingi;YANG Shun-qiaoi;PAN Yuei;CHEN Weii;SHAO Cong-wen;MA Shao-hui;Institute of Medical Biology,Chinese Academy of Medical Sciences and Peking Union Medical College,Yunnan Key Laboratory of Vaccine Research & Development on Severe Infectious Disease;

    Objective To analyze the characters of complete VP1 gene of Coxsackievirus B2(CVB2) isolate. Methods Virus was isolated from 10 fecal samples of suspected children with hand-foot-mouth disease(HFMD)with RD cells,from which VP1 gene was amplified by RT-PCR and sequenced. The sequencing results were analyzed by using NCBI BLAST,Mega 6. 1 and Geneious software,based on which a phylogenetic tree was constructed. Results A CVB2 strain was isolated from 10 fecal samples of suspected children with HFMD and named as 509 / YN / CHN / 2010,of which the fulllength of VP1 nucleotide,846 bp,was consistent with those of other CVB2 strains. The homologies of nucleotide and amino acid sequences of the isolate were 81. 0% ~ 95. 3% and 97. 9% ~ 98. 9% to those of other strains isolated in China,and 81. 1% ~ 87. 8% and 97. 9% ~ 98. 6% to those isolated abroad,respectively. Phylogenetic analysis showed that the isolate belonged to two distinct clusters with other isolates from China. The evolutional relationship of Coxsackievirus B2 YN509 / CHN / 2010 strain was the closest to M10MG17 strain. Conclusion YN509 / CHN / 2010 strain was defined as Coxsackievirus B2,which belonged to one of the two clusters in China.

    2015 01 v.28 [Abstract][OnlineView][Download 636K]

  • Screening and identification of differential expression proteins in placenta tissue of cows with retained fetal membranes

    NIE Tian-tian;ZHENG Cheng-yuan;ZOU Xiao;LI Shi-ying;XU Qi-you;FU Shi-xin;College of Animal Science and Technology,Heilongjiang Bayi Agricultural University;

    Objective To screen and identify the differential expression proteins in placenta tissue of cows with retained fetal membranes(RFM),and provide a basis for study on the onset mechanism and treatment of retained fetal membranes in cows.Methods Placenta tissues were collected from five cows with RFM and five cows as normal control,from which proteins were separated by two-dimensional polyacrylamide gel electrophoresis(2D-PAGE)and silver staining. The obtained differential expression protein spots were analyzed by matrix-assisted laser desorption / ionization time of flight mass spectrometry(MALDI-TOF-MS). Results A total of 240 differential expression protein spots were observed in the placenta of fetal cows in RFM group compared with those in normal control group,of which 212 were up-regulated while 28 were down-regulated.However,214 differential expression protein spots were observed in the placenta of maternal cows in RFM group,of which134 were up-regulated and 80 were down-regulated. Five differential expression proteins were identified. Compared with those in normal control group,the expressions of bovine serum albumin(BSA)and alpha enolase in the placenta of fetal cows in RFM group were down-regulated,while that of glutathione transferase(GST)was up-regulated. However,the expressions of Annexin Ⅴ,alpha enolase and Annexin A2 were in maternal cows in RFM group were up-regulated. Conclusion The identified differential expression proteins may be involved in the development of RFM and still need further validation.

    2015 01 v.28 [Abstract][OnlineView][Download 418K]

  • Trend analysis of biological activity in quality control of anti-CD20 monoclonal antibody

    LIU Chun-yu;WANG Lan;GUO Wei;GAO Kai;National Institutes for Food and Drug Control;

    Objective To analyze the trend of biological activity in quality control of anti-CD20 monoclonal antibody.Methods The biological activity of anti-CD20 monoclonal antibody(m Ab) was monitored by complement dependent cytotoxicity(CDC) with Cyto Tox 96 Non-Radioactive Cytotoxicity Assay kit using human lymphoid tumor Raji cells as a target,while the relative percent potency was calculated with the reference of anti-CD20 m Ab. The warning limit(mean ±2 SD) and action limit(mean ± 3 SD) were defined according to the determination results of 99 batches of anti-CD20 m Ab by National Institutes for Food and Drug Control(NIFDC)and the quality control laboratory of manufacturer,based on which the trend graph was plotted,and the consistency and periodic trend of anti-CD20 m Ab were analyzed. Results The determination results of potencies of 99 batches of anti-CD20 m Ab from a certain manufacturer during 2009 ~ 2013 showed dose-dependent CDC activities of both the test samples and reference of anti-CD20 m Ab,which complied with the4-parameter equation: y =(A- D)/[1 +(X / C)B]+ D. The potencies determined by NIFDC and the manufacturer were(99. 95 ± 10. 83)% and(100. 71 ± 9. 29)% respectively. The consistency trend analysis of the above-mentioned results showed no result beyond the action limit. The one-way analysis of variance(ANOVA)showed no significant difference in the data from various years(P > 0. 05),indicating that the data were comparable. Periodic trend analysis showed that all the results were within the action limit,and no result was exorbitance,severe drift or over 4 SD,indicating that the total trend was relatively steady. Conclusion The trend analysis of anti-CD20 m Ab was carried out for the first time,of which the results revealed the batch consistency and production stability,and provided a reference for the trend analysis of critical quality attributes for other therapeutic m Abs.

    2015 01 v.28 [Abstract][OnlineView][Download 529K]

  • Analysis on result of confirmation assays and nucleic acid test for plasma samples proved as anti-HCV positive by ELISA

    ZHANG Rong;DENG E;KE Ling;WU Bing-ting;LIU Gui;XU Min;LIU Yu;LI Chang-qing;Institute of Blood Transfusion,Chinese Academy of Medical Science;

    Objective To investigate the correlation between the results of confirmation assays(Western blot,WB) and nucleic acid test(NAT)for plasma samples proved as anti-hepatitis C virus(anti-HCV)positive by ELISA. Methods A total of 172 anti-HCV positive serum samples proved by primary screening were retested by one HCV antibody confirmation kit and one NAT kit. Results Of the 172 samples,115 and 124 were confirmed as positive by WB and NAT respectively,while 100 as positive and 26 as negative by both tests. However,the confirmation results of 46 samples by the two tests were inconsistent. The stronger band intensity was,the higher NAT-positive rate was. Of the 46 samples with inconsistent results,15 were confirmed as positive by WB and negative by NAT,while 16 were negative by WB and positive by NAT,7 were indeterminate by WB and negative by NAT,and 8 were indeterminate by WB and positive by NAT. Conclusion There is some correlation and difference in the results of NAT and confirmation assay on plasma samples proved as anti-HCV positive by ELISA. WB and NAT should be used simultaneously in HCV test to ensure the safety of blood.

    2015 01 v.28 [Abstract][OnlineView][Download 403K]

  • Comparison of varicella vaccine effectiveness during outbreak in a primary school

    PANG Hong;WANG Chen;JIANG Yan;SHI Wei;Changning District of Shanghai Center for Disease Control and Prevention;

    Objective To analyze the incidence of breakthrough varicella(BV)and the effectiveness of various doses and kinds of varicella vaccine. Methods The duration of outbreak in a primary school in Shanghai was from October 2011 to January 2012. Medical record of cases as well as vaccination status and history of varicella of all the students in the school were collected,based on which vaccine effectiveness(VE) and incidence were calculated. Results A total of 29 cases were observed in 444 children included in the analysis,indicating an incidence of 6. 5%. The varicella vaccination coverage was 90. 8%. The attack rate of vaccinated children was lower than those unvaccinated(χ2 = 8. 222,P < 0. 05).Overall VE was 68. 0%[95% confidence interval(CI):55. 2% ~ 80. 9%]. The incidence rate of BV of children immunized with one dose of vaccine was 6. 7%,while the VE was 60. 8%(95% CI:45. 1% ~ 76. 6%). However,the incidence rate of BV and VE of children immunized with two doses of vaccine were 0 and 100% respectively. The incidence rate of BV of children immunized with two doses of vaccine was significantly lower than that with one dose(χ2 = 1. 701,P > 0. 05).Conclusion High vaccination coverage with a single dose of varicella vaccine is insufficient to prevent the outbreak in schools. However,two doses of varicella vaccine may be successful to decrease the BV. A second dose of varicella vaccine in children is recommended before entering primary school.

    2015 01 v.28 [Abstract][OnlineView][Download 343K]

  • Purification of rotavirus by using multimode media Capto core 700

    JIANG Ying;HAN Ping;HU Guang-hong;WANG Ming-qiang;BAO Hong;WANG Zhou;ZHOU Xu;Lanzhou Institute of Biological Products Co.,Ltd.;

    Objective To purify rotavirus(RV)with multimode media Capto core 700 so as to remove the residual host cell protein(HCP) and DNA in culture and increase the yield of virus. Methods The seeds of RV LH9 were inoculated to Vero cells,and the prepared bulk of RV was clarified,ultrafiltrated and purified by Capto core 700. LH9,LH9 +150 mmol / L Na Cl[using buffer A(20 mmol / L PB + 150 mmol / L Na Cl,p H 7. 0)],LH9 + 300 mmol / L Na Cl[using buffer B(20 mmol / L PB + 300 mmol / L Na Cl,p H 7. 0)],LH9 + 450 mmol / L Na Cl[using buffer C(20 mmol / L PB + 450 mmol / L Na Cl,p H 7. 0)]were served as samples for purification in groups A,B,C and D,respectively. The flowthrough peaks of various groups were collected and determined for the titer and yield of purified RV,RV RNA,RV antigenicity, residual HCP and DNA. Three batches of RV concentrated by ultrafiltration were purified by the preliminarily screened procedure,of which various indexes were tested and further verified. Results The yield of RV purified by using 20 mmol / L PB + 150 mmol / L Na Cl as buffer and addition of 150 mmol / L Na Cl into samples reached more than 80%,while the RV DNA was intact,the antigenicity showed no significant change,more than 94% of HCP was removed,and the residual DNA content met the requirements in Chinese Pharmacopoeia(Volume Ⅲ,2010 edition).Conclusion Both the yield and removal rate of residual HCP of RV purified by Capto core 700 were high. However,the procedure in group B was relatively simple and time-saving,which was more suitable for the purification of RV.

    2015 01 v.28 [Abstract][OnlineView][Download 723K]

  • Development of a Luminex-based multiplex immunoassay for rapid determination of serum antibody titer induced by multi-component pertussis vaccine

    ZHU Tao;LUO Peng;SONG Lin-lin;DENG Jie;MAO Hui-hua;YU Xue-feng;SHAO Zhong-qi;Tianjin University of Science and Technology;

    Objective To develop a Luminex-based multiplex immunofluorescence assay for rapid determination of serum antibody titer induced by multi-component pertussis vaccine. Methods Pertussis toxin(PT),filamentous haemagglutinin(FHA),pertactin(PRN),fimbriae-2 and-3(FIM 2,3)were conjugated to four kinds of micro-beads. The four antigens coupled microspheres were added into the same well and incubated with the serum sample to be tested, followed by addition of a biotin-labeled antibody and streptomycin labeled phycoerythrin, then antibody titers were calculated using the median fluorescence value(MFI)provided by the Luminex instrument, based on a standard curve by using standard serum against pertussis. The same set of serum samples were also analyzed by ELISA and the results obtained by the two methods were compared. Results Compared with that of ELISA method,the linear range of Luminex-based immunoassay was wide. Fifty serum samples from mice immunized with multi-component pertussis vaccine were analyzed by both Luminex-based immunoassay and ELISA. The result showed that the mean antibody titers by Luminex-based immunoassay were 65. 365 IU / ml for PT,15. 052 IU / ml for FIM,545. 236 IU / ml for FHA and 7. 876 IU / ml for PRN,while those by ELISA were 72. 640 IU / ml for PT,16. 480 IU / ml for FIM,589. 474 IU / ml for FHA and 7. 345 IU / ml for PRN,respectively,which showed good correlations,with R2 values of 0. 91,0. 98,0. 93 and 0. 99 for PT,FIM,FHA and PRN respectively. Conclusion Luminex-based immunoassay is an attractive alternative to ELISA in the determination of antibody titer induced by multi-component pertussis vaccine,which was sample-,cost- and time-saving,and more accurate for analysis of correlation of various target molecules.

    2015 01 v.28 [Abstract][OnlineView][Download 524K]

  • Advances in research on therapeutic antibodies against tumors

    CHEN Xue-jing;CHANG Liang;GAO Jian;New Drug Research and Development Company Limited,North China Pharmacentical Corporation,State Key Laboratory of Antibody Drug Development;

    As one of the most successful and the most important strategies,therapeutic antibodies against tumors have been developed for more than 30 years. This paper reviews the commercial therapeutic antibodies against tumors and their targets in clinic,including cluster of differentiation 20(CD20),epithelial growth factor receptor(EGFR),human epidermal growth factor receptor 2(HER2)and vascular endothelial growth factor(VEGF),and summarizes the advances in research on novel therapy of tumors,including antibody-drug conjugate and immunotherapy.

    2015 01 v.28 [Abstract][OnlineView][Download 573K]

  • Relationship between neutralizing antibody and cellular immunity in effect evaluation of viral vaccines

    ZHANG Zhi-xiao;ZHENG Ying;LI Qi-han;FAN Quan-shui;Center for Disease Control and Prevention,Chengdu Military Region;

    Neutralizing antibody and cellular immunity are two important aspects of the effect evaluation of viral vaccines.However,because of the differences in the vaccine category and the induction mechanisms of neutralizing antibody and cellular immunity to the viruses,uniform criterion has not been established to evaluate the effect of the viral vaccines.Thus,it is important to develop and assess the viral vaccines to establish effective standards and assays for the determination of neutralizing antibody and cellular immunity to various viruses.

    2015 01 v.28 [Abstract][OnlineView][Download 378K]

  • Progress in research on stability of human coagulation factor Ⅷ

    JIANG Gui-xiang;ZHANG Wei;HONG Hao-wu;LV Ying-nian;Guangdong Shuanglin Bio-pharmaceutical Co.,Ltd.,The Engineering Center of Blood Products in Guangdong;

    Human coagulation factor Ⅷ(FⅧ)is a glycoprotein with the function of clotting in the blood,which serves as a co-factor for factor IXa(FIXa)in the activation of factor X(FX)to factor Xa(FXa)during the complex blood clotting cascade. FⅧ is unstable whether in vitro or in vivo because of its complex structure. This article reviews the stability of FⅧ in the whole manufacture progress including collection of material plasma,purification,package,storage and clinic infusion.

    2015 01 v.28 [Abstract][OnlineView][Download 465K]


@2012 Editorial Department of "Chinese Journal of Biologicals"