2016年11期目次

  • Preparation of group W135 meningococcal conjugate and its immunogenicity in mice

    WANG Jing;ZHANG Hai-hong;KONG Su-juan;YUAN Fei;LI A-ni;LIU Ai-ping;LIU Fang-lei;WU Bing;XIE Gui-lin;Lanzhou Institute of Biological Products Co., Ltd., Center for Gansu Provincial Vaccine Engineering Research;

    Objective To compare the influences of various activating reagents, carrier proteins and immune schedules on immunogenicity of group W135 meningococcal conjugate in mice. Methods Group W135 meningococcal polysaccharide derivative was prepared using CNBr and CDAP as activating agents respectively and ADH as linking arm, and conjugated to tetanus toxoid(TT), diphtheria toxoid(DT)and recombinant Pseudomonas aeruginosa exotoxin A(r EPA)using EDAC as condensation reagent respectively to obtain six kinds of conjugates, of which the immunogenicity in mice by four(at weeks 0, 2, 4 and 6) and three(at weeks 0, 4 and 8) dose immune schedules were compared. Results Both the activating agents effectively activated the polysaccharide without any damage to antigenicity. All the six kinds of conjugates showed good antigenicity in mice in a dose-dependent manner. Various activating agents and carrier proteins showed no significant influence on the immunogenicity of conjugates inoculated at a dosage of 2. 5 μg by three-dose schedule. However, the enhance effect of conjugate using DT as carrier protein was weaker than those using TT and r EPA.The increase of time interval of inoculation enhanced the immunogenicity of conjugates in mice significantly. Conclusion The conjugation procedure for group W135 meningococcal conjugate was preliminarily developed, which provided a novel alternative immune schedule for study on immunogenicity of the conjugates in mice.

    2016 11 v.29 [Abstract][OnlineView][Download 228K]

  • Characterization analysis of recombinant hepatitis E vaccine antigen P179

    CHI Xiang;LI Zheng;JIA Yuan;TANG Jian-guang;SONG Hao;CHEN Zi-yang;CHANG Dong-ying;CHI Chun-ping;Changchun Institute of Biological Products Co., Ltd.;

    Objective To perform characterization analysis on recombinant hepatitis E vaccine(HEV)antigen. Methods Three batches of bulk of r HEV antigen P179 with both HPLC and electrophoretic purities of 100% were determined for relative molecular mass and dimer content by SDS-PAGE, for amino acid component by OPA-FMOC automatic precolumn derivative amino acid-Amino Quant analysis, for amino acid sequence at C-terminus by electrospray ionization-quadrupole time-of-flight-mass spectrometry(ESI-Q-TOF-MS)method, and that at N-terminus by Edman degradation method, and for secondary structure by circular dichroism spectrometer. The protein particles were observed by transmission electron microscopy(TEM)with negative staining, while the antigen activity was determined by double antibody sandwich ELISA.Results The relative molecular masses of three batches of r HEV antigen P179 were 17 500 ~ 21 300, while dimer accounted for more than 85 % of total protein. The amino acid component, amino acid sequences at C- and N-terminuses, peptide map and isoelectric point were consistent with those expected. The α-helix accounted for 4. 8% ~5. 6% of the secondary structure, while β-sheet for 34. 8% ~ 36. 4%, β-turn for 20. 8% ~ 22. 2%, and the irregular coiling for 37. 7% ~ 38. 0%. Uniform protein particles at sizes of about 20 nm were observed by TEM. The antigen activity ratios were 5. 0 × 105~ 2. 0 × 10~6 CCU / mg. Conclusion The three batches of r HEV antigen P179 showed homogeneous structural characters, which were consistent with that designed. It laid a foundation of industrialization of the vaccine.

    2016 11 v.29 [Abstract][OnlineView][Download 403K]

  • Immunogenicity and immunoprotection of trivalent influenza virus split vaccine for nasal spray in mice

    NIU Yan;YU Shu;XING Li;LI Heng-bin;BAO Li-li;REN Xiang-yu;ZHANG Ming-yu;SUN Xiao-lin;ZHAO Fang-xin;SUN Peng;ZHAO Peng-wei;WANG Xi-liang;Department of Immunology and Microbiology, Inner Mongolia Medical University;

    Objective To evaluate the immunogenicity and immunoprotection of trivalent influenza virus split vaccine for nasal spray in mice. Methods Three kinds of split antigens, H1N1, H3N2 and B, were combined and mixed with HTCC to prepare trivalent influenza virus split vaccine. BALB / c mice were immunized twice with the vaccine by nasal spray at a dosage of 2 μg of each antigen, on days 0 and 14 respectively. The sera as well as nasal, lung and vaginal washes mice were collected on day 14 after the last immunization. The hemagglutination inhibition(HI) antibody titer in sera was determined by HI test, while those of Ig G and its subtype antibodies in sera as well as sIgA titer in nasal, lung and vaginal washes by ELISA. The mice were challenged with influenza H1N1 virus 14 d after the last immunization, and observed for bodyweight and mortality rate. Results High titer HI antibody was induced with the trivalent vaccine by nasal spray in sera of mice. The IgG subtypes were mainly IgG1 and IgG2 a. Specific s IgA were detected in nasal, lung and vaginal wash-es of mice. All the mice survived 14 d after challenge, of which the bodyweight showed no significant change. Conclusion Immunization with trivalent influenza virus split vaccine for nasal spray induced humoral, cellular and mucosal im-mune responses in mice, which showed good immunoprotection.

    2016 11 v.29 [Abstract][OnlineView][Download 332K]

  • Genetic characteristics of varicella-zoster virus 84-7 strain

    ZOU Wei-ran;LIU Ya-yu;HE Yun-song;LIU Ye;MA Guang;LIAO Hai-tang;LI Yong-zhong;Chengdu Institute of Biological Products Co., Ltd.;

    Objective To analyze the genetic characteristics of varicella-zoster virus(VZV) 84-7 strain. Methods The ORF38(PstⅠ), ORF54(BglⅠ) and ORF62(SmaⅠ, Bss HⅡ and Nae Ⅰ) of VZV84-7 strain were amplified by PCR respectively, and analyzed for restriction site by bioinformatics software. The fragment of origin of virus replication was amplified by PCR and analyzed for charcteristics. The g E gene of VZV84-7 strain was amplified by PCR and sequenced.Results The restriction characteristrics of VZV84-7 strain was PstⅠ~+ BglⅠ~+ SmaⅠ~-Bss HⅡ~- NaeⅠ~-. The origin of virus replication included the structure of 13 TA + 19 GA differentiated from other strain of VZV. The g E gene sequence showed difference of C→A in one base at site 1 170 compared with that of Oka strain, which was a nonsense mutation. However,the homology of amino acid sequences in encoding regions of the two strains was 100%. Conclusion Both the restriction site and origin of replication of VZV84-7 strain were specific. Meanwhile, the g E gene of VZV84-7 strain showed only mutation in one base compared with that of Oka strain, while the homology of amino acid sequences of the two strains was100%.

    2016 11 v.29 [Abstract][OnlineView][Download 218K]

  • Construction of recombinant adenovirus vector pAd-4-hMASP-2

    ZHANG Guo-chao;LUO Yan-ping;LU Xiao-ling;WANG Qian;DONG Xin-fang;CAO Ming-qiang;YU Hong-juan;MA Xing-ming;School of Basic Medical Sciences, Lanzhou University;

    Objective To construct recombinant adenovirus vector p Ad-4-h MASP-2 by using p Yr-adshuttle-4. Methods Human mannan-binding lectin associated serine protease-2(h MASP-2)gene was amplified by PCR, digested with Bam HⅡand Eco RⅡ, and inserted into plasmid p Yr-adshuttle-4. The constructed shuttle plasmid p Yr-ads-4-h MASP-2 and p Ad /PL-DEST adenovirus backbone vector was reconstructed through homologous recombination in mediation of LR enzyme.The obtained plasmid p Ad-4-h MASP-2 was transfected to HEK293 cells for packing. The obtained recombinant adenovirus r Ad-h MASP-2 was identified by restriction analysis, PCR and gene sequencing, and determined for titer. BALB / c mice were infected with the recombinant adenovirus, and the expression of h MASP-2 m RNA in lung was determined by realtime fluorescent quantitative PCR. Results The shuttle plasmid p Yr-4-h MASP-2 was constructed correctly as proved by restriction analysis and sequencing. Recombinant adenovirus vector p Ad-4-h MASP-2 was obtained by homologous recombination, based on which the recombinant adenovirus particles with a titer of 1. 5 × 109 PFU / ml was amplified, and expressed in the lung tissues of mice. Conclusion Both recombinant adenovirus vector p Ad-4-h MASP-2 and recombinant adenovirus r Ad-h MASP-2 were successfully obtained, which provided an effective transgenic vector for in vitro and in vivo studies on h MASP-2.

    2016 11 v.29 [Abstract][OnlineView][Download 242K]

  • Construction and knockout efficiency of shRNA lentiviral vector for filamin A

    HUANG Wen-yu;LONG Fei-yan;YANG Chen-chen;ZHU Ying-rou;HUANG Fen;CHU Jia-rui;Changchun Institute of Biological Products Co., Ltd.;

    Objective To construct the sh RNA lentiviral vector for filamin A and determine its knockout efficiency in HepG2 and Huh7 cells. Methods The knockout gene fragment was synthesized and inserted into the sh RNA lentiviral vector GV298 with a cherry protein reporter. The constructed recombinant plasmids sh RNA-Filamin1 and sh RNA-Filamin2 were transfected into HepG2 and Huh7 cells respectively. The expression of cherry protein was observed by fluorescent microscopy, and the knockout efficiency of filamin A was determined by Western blot. Results Sequencing results proved that the FLNa sh RNA lentiviral vectors for filamin A were constructed correctly. Strong cherry fluorescence was observed in HepG2 and Huh7 cells 24 h after transfection. However, the knockout efficiencies of sh RNA-Filamin1 and sh RNAFilamin2 were 45. 4% and 63. 7% in HepG2 cells, and 76. 5% and 78. 3% in Huh7 cells, 48 h after transfection,respectively. Conclusion The FLNa sh RNA lentiviral vectors for filamin A were successfully constructed, of which the knockout efficiencies were high in HepG2 and Huh7 cells. Filamin A was able to interact with the receptor proteins of various viruses, thus participate in the replication cycle of the virus. It provided a new approach for study on the virus replication and the pathogenic mechanism.

    2016 11 v.29 [Abstract][OnlineView][Download 249K]

  • Culture and biological characters of keratinocytes of patients with condyloma acuminatum

    ZHU Wei;CAO Ping;LI Yong-fu;Kunming University of Science and Technology;

    Objective To culture and identify the keratinocytes in skin lesion sites of patients with condyloma acuminatum(CA), determine the expression of β-catenin m RNA and analyze the cell cycle. Methods The cells in lesion sites were digested by two-step digestion method, and the obtained primary keratinocytes were cultured in vitro in serum-free medium, observed for morphology under microscope and identified by immunohistochemical staining, based on which the cell growth curve was plotted, the cell proliferation ability was determined by MTT assay, and cell cycle by flow cytometry. Results The cells under microscope were mainly in polygonal or irregular shape and in flagstone-like array,which grew well with clear outline and good refractivity, and the cytoplasm was stained red, indicating the characters of keratinocytes. The cells entered to the logarithmic growth phase on the 3rd day and to the platform phase on days 7 and 8after culture. The obtained keratinocytes were proliferated stably, in which the β-catenin was expressed abnormally during pathogenesis of CA, while the cell cycle changed. Conclusion Abnormal expression of β-catenin m RNA and change of cell cycle during pathogenesis of CA provided a theoretical basis for the clinical treatment of CA.

    2016 11 v.29 [Abstract][OnlineView][Download 293K]

  • Screening and comparison of methods for preservation of Koi herpesvirus

    LI Ying-ying;ZENG Wei-wei;WANG Ying-ying;PAN Hou-jun;LIU Chun;SHI Cun-bin;WU Shu-qin;WANG Qing;Key Laboratory of Aquatic Animal Immune Technology of Guangdong Province, Key Laboratory of Fishery Drug Development of Ministry of Agriculture, Pearl River Fisheries Research Institute, Chinese Academy of Fishery Sciences;

    Objective To screen and compare the methods for preservation of Koi herpesvirus(KHV). Methods The tissues of Koi fish with KHV disease(KHVD) were preserved at-80 ℃, in liquid nitrogen, in 50% glycerol phosphate buffer, in ethanol solution and in isopropyl alcohol for 90 d respectively, and identified by PCR, based on which the effect of tissues preserved by various methods on infected fish and CCB cells were evaluated. CCB cells were infected with KHV,and virus liquid was harvested, stored at 4,-20 and-80 ℃ and in liquid nitrogen for 90 d respectively, and determined for titer(TCID50). Results The tissues of Koi fish with KHVD preserved by five methods were proved as positive for KHV by PCR. The tissues preserved at-80 ℃ and in liquid nitrogen showed the ability to infect healthy Koi fish. However, the tissues preserved in 50% glycerol phosphate buffer, ethanol and isopropyl alcohol failed to infect Koi fish. The tissues preserved in 50% glycerol phosphate buffer only infected CCB cells. The titers of virus harvested from infected CCB cells decreased by 61% and 50% after storage at 4 and-20 ℃ respectively, while decreased by only 7% after storage at-80 ℃and in liquid nitrogen. Conclusion The cryopreservation at-80 ℃ and in liquid nitrogen were suitable for long-term preservation of infected tissues and virus liquid, while preservation in 50% glycerol phosphate buffer were suitable for the separation of KHV from cells.

    2016 11 v.29 [Abstract][OnlineView][Download 216K]

  • Eukaryotic expression and identification of extracellular domain of glycoprotein E of varicella-zoster virus

    LI Chun-ming;ZHU Xiao-wen;HE Bao-shuang;ZHU Lin;YU Tie-nan;ZHANG Yu;Changchun Keygen Biological Products Co., Ltd.;

    Objective To express the extracellular domain of glycoprotein E(gE_(537))of varicella-zoster virus(VZV)in eukaryotic cells and identify the expressed product. Methods Human diploid cell 2BS strain was infected with VZV-Oka,from which genomic DNA was extracted and used as a template for amplification of gE_(537) fragment by PCR. The PCR product was cloned into vector pCI-neo, and the constructed recombinant plasmid pCI-neo-gE_(537)-His was transfected to293 FT cells for transient expression. The expressed product was purified by nickel ion affinity chromatography and identified by Western blot and ELISA. Results Restriction analysis and sequencing proved that recombinant plasmid pCI-neo-gE_(537)-His was constructed correctly. The optimal concentration of imidazole as an eluant was 200 mmol / L. The purified target protein, with a relative molecular mass of about 90 000, showed specific binding with mouse anti-gE monoclonal antibody as well as reactions with m Ab-10 and mAb-12 against gE. Conclusion The gE_(537) of VZV was successfully expressed in 293 FT cells, which laid a foundation of study on its immunogenicity.

    2016 11 v.29 [Abstract][OnlineView][Download 200K]

  • Preparation and screening of monoclonal antibodies targeting human epidermal growth factor receptor 3

    LI Ao-xiang;LIANG Hong-yuan;ZHU Jing-ye;WU Li-na;HUANG Hai-wu;PENG Bo;GUO Jin-lin;ZHAO Xin;QIU Jian-hua;LU Jin;XU Fan-hong;QU Ai-dong;LI Wei-min;Shanghai Institute of Biological Products, Co., Ltd.;

    Objective To p repare and screen the monoclonal antibodies(Mc Abs) targeting human epidermal growth factor receptor 3(Her3). Methods The extracellular domain of Her3 protein was expressed by using CHO cell system.BALB / c mice were immunized by the obtained recombinant protein h GH-Her3-ECD, based on which Mc Abs were prepared by using traditional hybridoma technology and screed by blocking Her3-HRG binding assay, affinity assay FACS assay and A431 cell proliferation inhibition assay, identified for subtype, and determined for antigen binding domain.Results Of the more than 100 kinds of Mc Abs obtained, those secreted by strains 927, 1002, 1050, 993, 1044 and 1056 showed strong inhibiting activities to binding of Her3 to HRG, all of which were of subtype Ig G1 and showed good affinities. The Mc Abs secreted by strains 927, 1002 and 1050 showed binding to domain Ⅰof Her3, while those by strains993, 1044 and 1056 to domain Ⅲ, all of which showed binding to the Her3 molecules on surface of A431 and Bx Pc3 cells, and inhibited the proliferation of A431 cells significantly. Conclusion The obtained Mc Abs targeting Her3 basically met the need for development of therapeutic antibodies of cancers.

    2016 11 v.29 [Abstract][OnlineView][Download 384K]

  • Establishment of national reference panel for anti-hepatitis E virus IgG

    LAN Hai-yun;GU Wen-jie;LI Ke-jian;ZHOU Cheng;Division of Hepatitis Virus Vaccines, National Institutes for Food and Drug Control;

    Objective To prepare the national reference panel for anti-hepatitis E virus(HEV)IgG. Methods A total of300 plasma or serum samples were collected from blood centers and plasma collection station in various provinces in China, and screened with domestic and imported HEV IgG diagnostics kits, based on which the national reference panel for anti-HEV IgG was prepared, and evaluated for stability and suitability. Results The national reference panel for antiHEV IgG was prepared, including 30 negative, 10 positive reference panels, one sensitivity and one precision reference panels. The HEV IgG content in sensitivity reference panel was defined as 3 U / ml by calibration with international standard for HEV IgG. The determination results of reference panels after storage at 4 ℃ or room temperature for 10 d,37 ℃ for 7 d and after three cycles of freezing and thawing were consistent with that after storage at-20 ℃, indicating a high stability. The test results by kits from five manufacturers showed good suitability of the reference panel. Conclusion The national reference panel for anti-HEV IgG was established, which laid a foundation of improving the quality of HEV IgG diagnostic kit.

    2016 11 v.29 [Abstract][OnlineView][Download 165K]

  • Assay of C-kit and PDGFRA gene mutations in gastrointestinal stromal tumors

    LI Ming-li;CHENG Hong-xia;ZHOU Ce-fan;WANG Wei-xu;YAN Xiao-feng;CHENG Peng-fei;Hospital of Wuhan University;

    Objective To analyze the mutation status of C-kit and platelet derived growth factor receptor A(PDGFRA)genes in gastrointestinal stromal tumor(GIST), and investigate the relationship between the mutation and the pathogenesis of GIST. Methods Genomic DNA was extracted from the paraffin sections of tumor tissues of 102 patients with GIST from Renmin Hosptital of Wuhan University and Zhongnan Hospital of Wuhan University, based on which the specific target gene fragments were amplified by PCR, identified by sequencing, and analyzed for mutations of C-kit and PDGFRA genes. Results C-kit mutations were detected in 76 of the 102 samples, of which 65 occurred in exon 11, including deletion(43 cases, 66. 15%), point mutation(12 cases, 18. 46%), insertion(4 cases, 6. 15%), and point mutation accompanied by deletion(6 cases, 9. 23%). The mutations in exons 9, 13 and 17 were detected in 7, 3 and 2 cases respectively. PDGFRA mutations were detected in 4 cases, of which 4 in exon 12 and only one in exon 18. Conclusion C-kit mutations are common in the majority of GIST patients, most of which are located in exon 11. However, PDGFRA mutation exists in the patients without C-kit mutation.

    2016 11 v.29 [Abstract][OnlineView][Download 507K]

  • Investigation on antibody levels against Haemophilus influenzae type b polysaccharide in children in various regions of China

    LI Ya-nan;LI Hong;SHI Gang;HE Peng-fei;TANG Jing;LI Mao-guang;LIANG Li;YE Qiang;National Institutes for Food and Drug Control;

    Objecti ve To investigate the antibody levels against polyribosylribitol phosphate(PRP) of Haemophilus influenzae type b(Hib)in children in various regions of China. Methods A total of 13 799 serum samples were collected from children at ages of 2 months to 5 years in Guangxi Zhuang Autonomous Region as well as Jiangsu and Henan Provinces in China during 2007 ~ 2014, and determined for naturally acquired antibody level against PRP by ELISA.Results During 2007 ~ 2014, the positive rate of naturally acquired antibody against PRP, the percentage with long-term protective antibody level and the GMC of antibody in children at ages of 2 months to 5 years increased year by year.However, the antibody level against PRP in the children at ages of 2 ~ 11 months were relatively low. The antibody level showed no significant difference in the children in various regions, and was distributed evenly in two genders. Conclusion The children at ages of 2 ~ 11 months in China remain the population susceptible to Hib, and hence are the target population of vaccination with Hib conjugate vaccine.

    2016 11 v.29 [Abstract][OnlineView][Download 151K]

  • Post-marketing study on safety of recombinant hepatitis B vaccine(Saccharomyces cerevisiae)

    ZHANG Min;KANG Guo-dong;MA Yan-li;ZHANG Zhi-lan;ZHAI Li-Jun;LIAN Li-hua;CHEN Hai-ping;XU Bin;YANG Yun-kai;MA Fu-bao;China National Biotec Group Company Limited;

    Objective To perform a post-marketing study on the clinical safety of recombinant hepatitis B(r HB)vaccine(Saccharomyces cerevisiae) in newborns. Methods A cohort study on r HB vaccine(S. cerevisiae) was conducted.Newborns from April 1 to August 30, 2015 in Nantong City, Jiangsu Province, China were immunized with the vaccine according to the schedule in national EPI. The data on vaccination and the safety after vaccination were collected through the Individual Information System of Vaccination in Children in Jiangsu Province and the AEFI Information System to evaluate the safety of the vaccine. Results A total of 37 cases of AEFI were reported during the period, indicating an incidence rate of 40. 93 / 100 000. No severe adverse events or severe unexpected events were observed. Of the 37 cases,32 were general reactions, while 5 were abnormal events, and no accidental or psychogenic reactions were reported. A portion of 67. 57% of the general reactions were fever, especially those occurred after the 3rd dose, with an incidence rate of 69. 84 / 100 000. The abnormal reactions included two cases of allergic rash, one case of urticaria and one case of febrile convulsion. The 37 cases of AEFI events mainly occurred within 30 min to 24 h after vaccination, with good prognosis. Conclusion The r HB vaccine(S. cerevisiae)showed good safety in newborns.

    2016 11 v.29 [Abstract][OnlineView][Download 143K]

  • Analysis on surveillance of adverse events following immunization with hepatitis B vaccine in Jilin Province during 2009～2014

    CAO Feng-rui;TIAN Xin;CHENG Tao;FU Si-mei;CHEN Chao;LIN Lin;LV Shu-mei;LI Da-qiang;ZHOU Jian-hui;HUANG Biao;Immunization Program Directing Department,Center for Disease Control and Prevention of Jilin Province;

    Objective To analyze the characteristics of adverse events following immunization(AEFI) with hepatitis B(Hep B) vaccine in Jilin Province and evaluate the safety of Hep B vaccines and the quality of AEFI surveillance.Methods The data on AEFI of Hep B vaccine reported by AEFI Monitoring Information Management System of China from 2009 to 2014 in Jilin Province were collected, and analyzed by descriptively epidemiological method. Results A total of 897 cases of AEFI of Hep B were reported during 2009 ~ 2014, of the sex ratio was 1. 09 ∶ 1, indicating an incidence of 159. 11 / million doses. A portion of 88. 85% of the cases occurred in the infants at ages of less than one year. The reported incidence rates of general and abnormal reactions were 154. 50 / million doses and 3. 37 / million doses respectively. The AEFI mainly appeared within 1 d after vaccination. Conclusion The Hep B vaccine showed high safety,of which the AEFI were mainly general reactions and allergic reactions.

    2016 11 v.29 [Abstract][OnlineView][Download 131K]

  • Analysis of protein composition of purified poliovirus Sabin strain

    LI Ai-ling;ZHAO Yu-xiu;LIU Xiao-juan;DONG Yuan;LI Wan-li;LIANG Hong-yang;YU Shou-zhi;YUE Zhuo;ZHAO Shuo;GAO Bo;ZHANG Da-lun;WANG Hui;Beijing Tiantan Biological Products Co., Ltd.;

    Objective To analyze and identify the protein composition of purified poliovirus Sabin strain of types Ⅰ, Ⅱand Ⅲ. Methods Monovalent bulk of poliovirus was prepared by using NBS basket bioreactor according to the production procedure of vaccine, concentrated by ultrafiltration and purified by molecular sieve and ion exchange column chromatography. The protein peaks were collected and inactivated, then observed for morphology and size of virus by transmission microscopy, determined for host cell protein(HCP) and residual bovine serum albumin contents by ELISA,for purity by HPLC, and for protein composition by LC / ESI-MI technique. Results Spherical virus particles at diameters of 25 ~ 30 nm were observed in the purified poliovirus Sabin strains of types Ⅰ, Ⅱ and Ⅲ, while the HCP and residual bovine serum albumin contents were less than 50 ng / ml and less than 2. 5 μg / ml respectively, and the purity was more than 95%. The monovalent poliovirus samples contained the genome-encoded protein of the corresponding types, of which the relative scores in protein database were the highest. Conclusion The foreign proteins were effectively removed from the purified poliovirus Sabin strain of typesⅠ, Ⅱ and Ⅲ, while the viral protein compositions were retained. The purified monovalent virus showed clear protein composition and high purity, which provided a reference for subsequent development and production of the vaccine.

    2016 11 v.29 [Abstract][OnlineView][Download 329K]

  • Subculture of varicella-zoster virus in Vero cells and preparation of antiserum

    WANG Yuan-zheng;SHI Xiao-li;FAN Shu-lan;Beijing Tiantan Biological Product Co.Ltd.;

    Objective To subculture varicella-zoster virus(VZV) in Vero cells and prepare the antiserum. Methods VZV 84-2 strain was subcultured in Vero cells, from which antigen was extracted after stable CPE appeared. Rabbits and guinea pigs were immunized with the prepared antigen for 4 times, each at an interval of 14 d. The rabbits were divided into two groups. Adjuvant-free antigen was immunized s.c. to the rabbits in group 1 for the first and the second doses,while the antigen containing complete Freund adjuvant was immunized i.p. to those in the same groups for the third and the forth doses. However, the rabbits in group 2 and the guinea pigs were immunized s.c. with the antigen containing complete Freund adjuvant. The serum samples of animals in various groups were collected 2 weeks after the last immunization for sterility test, determination of neutralizing antibody titer and specific interference test. Results CPE appeared in 80% ~ 90% of Vero cells in which VZV 84-2 strain was subcultured to passage 5, and was stable after passage 10. The protein contents of antigens prepared by using the media containing rabbit and guinea pig sera were 2. 1and 1. 7 mg / ml respectively. The antisera were qualified in sterility. The serum neutralizing antibody titers of rabbits in groups 1 and 2 were 1 ∶ 128 and 1 ∶ 256 respectively, while that of guinea pigs was 1 ∶ 64. The antiserum against VZV showed no interference to the titration of measles(Shanghai 191 strain), mumps(S79 strain)and rubella(BRDⅡ strain)viruses. Conclusion VZV was subcultured in Vero cells successfully, and antiserum against VZV was prepared, of which the tier was relatively low.

    2016 11 v.29 [Abstract][OnlineView][Download 167K]

  • Development of a HPLC method for determination of free protein content in groups A & C meningococcal polysaccharide conjugate vaccine

    LIU Lu;CAI Feng;WANG Gong-xiao;WANG Xue-lin;XU Rong-hua;XIONG Hui-ling;Chengdu Institute of Biological Products Co., Ltd.;

    Objective To develop and verify a HPLC method for determination of free protein content in groups A & C meningococcal polysaccharide conjugate vaccine. Methods The free protein content in groups A & C meningococcal polysaccharide conjugate vaccine was determined by HPLC on TSKgel G3000 SW column(300 mm × 7. 5 mm, 10 μm),using 0. 01 mol / L phosphate buffered saline as mobile phase at a flow rate of 0. 5 ml / min. The sample load, detection wavelength and column temperature were 100 μl, 280 nm and room temperature respectively. The method was verified for specificity, separation degree, linearity, precision and accuracy, and determined for quantitative and detection limits. The free protein content in three batches of bulks and three batches of final products of groups A & C meningococcal polysaccharide conjugate vaccine were determined by the developed method. Results The method showed high specificity and separation degree in determination of free protein content in polysaccharide conjugate vaccine. The standard curve showed good linear relationship to the concentration of standard protein at a range of 3 ~ 18 μg / ml(R~2 =0. 998). The recovery rates of samples in three batches of bulk and final product were 82. 9% ~ 119. 33%, with RSDs of less than 5%. The detection limit and quantitative limit of the method were 1. 0 and 2. 6 μg / ml respectively.Conclusion The developed HPLC method was simple, sensitive and accurate, which might be used for determination of free protein content in groups A & C meningococcal polysaccharide conjugate vaccine.

    2016 11 v.29 [Abstract][OnlineView][Download 226K]

  • Establishment and validation of an indirect ELISA method for determination of Haemophilus influenzae type a capsular polysaccharide specific IgG content in mouse sera

    MIAO Xin;WANG Xin-ru;ZHAO Zhi-qiang;LIU Jia;LIU Yue-ping;LIU Fang-lei;LI Yan-ting;GE Yu-wen;XIE Gui-lin;TAN Xiao-mei;Lanzhou Institute of Biological Products Co., Ltd.,Vaccine Engineering Technology Center of Gansu Province;

    Objective To establish and validate an indirect ELISA method for determination of Haemophilus influenzae type a(Hia) capsular polysaccharide(CPS)-specific IgG content in mouse sera. Methods Capsular polysaccharidetyramined(CPS-Tyr) was prepared by activation of Hia CPS with 1-cyano-4-dimethylaminopyridiniumtetrafluoroborate(CDAP). The distributions of relative molecular masses of CPS-Tyr and Hia CPS were analyzed by Sepharose CL-4B chromatography. The conditions for coating and blocking as well as time for color development were optimized, based on which an indirect ELISA method was established using alkaline phosphatase-labeled goat anti-mouse IgG as the second antibody, and 4-nitrophenyl phosphate disodium salt hexahydrate as color development reagent, and validated for specificity, linearity and precision. Results The distribution of relative molecular mass of CPS-Tyr showed no significant change as compared with that of CPS. The condition for determination was optimized as follows: CPS-Tyr was used as coating antigen, of which the concentration for coating was 5 μg / ml. Costar 42592 was used as microplate, which was blocked with blocking solution containing 1% bovine serum albumin(BSA) at 37 ℃ for 2 h. The time for color development was 2 h. After absorption with various concentrations of Hia CPS or CPS-Tyr, the inhibition rate of Hia positive serum was significantly concentration-dependent, which was 0 ~ 100%. The antigenicity of Hia CPS-Tyr was consistent with that of CPS. However, the inhibition rate of Hia positive serum by Haemophilus influenzae type b(Hib)CPS at the same concentration was 0. The dilution of sera was negatively related to the antibody titer, with a k approximate value of-1 and a R2 value of 0. 998. Both the CVs in intra- and inter-assays were 8. 8% and 10. 9% respectively.Conclusion The developed indirect ELISA method showed high specificity, linearity and precision, which was suitable for the determination of Hia CPS-specific IgG content in mouse sera.

    2016 11 v.29 [Abstract][OnlineView][Download 335K]

  • Development of reverse high performance liquid chromatography method for determination of free trace formaldehyde in influenza virus split vaccine

    WANG Xiao;GU Li-juan;LI Jing-tao;WANG Jia-feng;Jilin Institute for Quality Control of Foods;

    Objective To develop a reverse high performance liquid chromatography(HPLC)method for determination of free trace formaldehyde in influenza virus split vaccine. Methods The condition for derivation was optimized, based on which the free formaldehyde in 2, 4-dinitrophenylhydrazine(DNPH)-derived influenza virus split vaccine was determined by revere HPLC. TC-C18 column(250 mm × 4. 6 mm, 5 μm) was served, using acetonitrile-water(45 ∶ 55) as mobile phase. The sample load, flow rate, detection wavelength and column temperature were 10 μl, 1. 0 ml / min, 360 nm and35 ℃ respectively. The wavelength for detection of DNPH derivative was determined, based on which the developed method was verified for linearity, accuracy, precision, stability and reproducibility, and determined for quantitative and detection limits. Four batches of influenza virus split vaccine from various manufacturers were determined for trace formaldehyde by the method, of which two batches were derived at first, and the other two batches were determined directly. Results The optimal derivation solution, derivation temperature and reaction time were 5 mol / L phosphoric acid, 20 ℃ and 30 min respectively. The detection wavelength for DNPH derivative was 360 nm. The formaldehyde concentration within a range of 10. 02 ~ 250. 5 ng / ml showed good linear relationship to the peak area(r = 0. 999 8).The mean recovery rate of six samples of DNPH derivatives was 99. 93%, with a RSD of 0. 34%. The mean peak area of DNPH derivative samples loaded for six times was 724. 15, with a RSD of 0. 74%. The mean content of trace formaldehyde in six tests at various time points was 8. 04 μg / ml, with a RSD of 0. 98%, which was stable within 24 h. However, the mean content of trace formaldehyde in six samples of the same batch was 8. 06 μg / ml, with a RSD of 0. 68%. The minimum detection limit of trace formaldehyde was 0. 01 μg / ml, while the quantitative limit was 0. 03 μg / ml. All the contents of trace formaldehyde in four batches of influenza virus split vaccine were less than the standard of 50 μg / ml in Chinese Pharmacopoeia(Volume Ⅲ, 2010 edition). Conclusion The developed reverse HPLC method was simple,effective and sensitive, which was suitable for determination of free trace formaldehyde in influenza virus split vaccine.

    2016 11 v.29 [Abstract][OnlineView][Download 225K]

  • Analysis of ability of cold ethanol fractionation process in removal of human parvovirus B19

    ZENG Fei-xiang;ZHANG Yan;YU Ding;MA Yi-yun;ZENG Lang-xia;WANG Bing;LIU Li;HE Pei-de;YANG Hui-chuan;Chengdu Rongsheng Pharmaceuticals Co., Ltd.;

    Objective To analyze the abilities of cold ethanol fractionation and pressure filtration in removal of human parvovirus B19 during the production of plasma-derived proteins. Methods Samples were taken in the major process of cold ethanol fractionation and pressure filtration. The pooled plasma was determined for B19 content, based on which the process samples produced from pooled plasma at B19 content of not less than 103 IU / ml were determined for B19 content, to analyze the ability of major process in removal of B19. Results In the processes from pooled plasma to F_(Ⅰ+Ⅱ+Ⅲ)supernatant and from the solution of FⅠ+ Ⅱ + Ⅲprecipitate to F_(Ⅰ+ Ⅲ)supernatant, more than 4 log of B19 virus was removed.However, in the process from F _(Ⅰ+ Ⅲ)supernatant to IVIG product, more than 2. 86 log of B19 virus was removed.Conclusion B19 virus can be removed efficiently by cold ethanol fractionation with pressure filtration used for separation of human albumin and immunoglobulin. The B19 safety of human albumin and immunoglobulin can be guaranteed by cold ethanol fractionation process together with virus removal and inactivation techniques, however, for coagulation factor products, the load of B19 virus in pooled plasma should be controlled.

    2016 11 v.29 [Abstract][OnlineView][Download 178K]

  • Application of variance analysis to random-effect model in assessment of intermediate precision of Kjeldahl method

    LI Li;WU Fan;TU Jing;YANG Bang-ling;The Division of Quality Control, Wuhan Institute of Biological Products Co.Ltd.;

    Objective To assess the source and proportion of variability in Kjeldahl method by using random-effect model in analysis of variance(ANOVN). Methods Three variables during determination of protein content by Kjeldahl method,date, measurement instrument and operators, were included in ANOVN, based on which the intermediate precision(IP)and intra-class correlation coefficient(ICC)of the method were calculated. Results During determination of protein content by Kjeldahl method, 49. 20% of total variability was due to operators, followed by measurement instrument and date.The IP and ICC of the method were 2. 58% and 0. 59 respectively. Conclusion In the process of determination of protein by Kjeldahl method, a majority of the variability might be due to the operators. The determination result of protein content was reliable with high accuracy and good precision in this laboratory. The random-effect of variance analysis may be applied to the analysis of randomly influencing factors during determination.

    2016 11 v.29 [Abstract][OnlineView][Download 116K]

  • Biology of Mycoplasma pneumoniae and research on vaccine

    LI Ze-hua;LIAO Guo-yang;Institute of Medical Biology, Chinese Academy of Medical Science & Peking Union Medical College, Yunnan Key Laboratory of Vaccine Research & Development on Severe Infection Disease;

    The mortality of acute respiratory infections is only below those of cardiovascular diseases, which has become a serious public health problem. There are several kinds of vaccines against the pneumonia associated pathogens in market or in development. However, effective vaccines against Mycoplasma pneumonia are rarely reported. The special structure of M. pneumoniae makes it is difficult to be treated effectively by conventional drugs, resulting in that the incidence increased year by year. The research on the related mechanism as well as methods for prevention and treatment of pneumoniais caused by M. pneumonia has become a hot spot, and it is more and more important to decrease the risk of disease by development of the relevant vaccines.

    2016 11 v.29 [Abstract][OnlineView][Download 131K]

  • Current situation of research on relationship of complement activation to replication of hepatitis virus

    WANG Jue;YU Wen-hai;JIANG Dian-cai;HUANG Fen;Medical Faculty, Kunming University of Science and Technology;

    Complement system is an important part of immune system, which participates in the recognition and defense of pathogen and is served as the first line of defense against pathogenic infection. After being activated by the classical pathway, lectin pathway and alternative pathway, complement selectively recognizes foreign pathogens and damaged self-cells, which plays an important role in the host immune and a variety of physiogical and pathological processes. This paper reviews the research progress of relationship of complement activation to replication of hepatitis virus.

    2016 11 v.29 [Abstract][OnlineView][Download 134K]


@2012 Editorial Department of "Chinese Journal of Biologicals"