2015年10期目次

  • Successive subculture and genetic stability of rubella virus vaccine strain BRD-Ⅱ

    XU Le-yan;YANG Wen-zhen;MAO Shu-bao;XU Wen-qing;Shanghai Institute of Biological Products Co., Ltd.;

    Objective To observe the genetic stability of BRD-Ⅱ strain for manufacturing rubella virus vaccine by Shanghai Institute of Biological Products Co., Ltd.(SIBP), i. e. S-BRD-Ⅱ, after subculture in MRC-5 cells, and compare the E1 genes of the strain to those of other strains for production of commercial vaccines. Methods S-BRD-Ⅱ strain was subcultured successively in MRC-5 cells for 10 passages, of which each passage was observed for culture characteristics and E1 gene sequence. The ORFs of passages 28, 29, 33 and 38 as well as 5′- and 3′-terminuses of passages 28 and 38 were sequenced. Meanwhile, the E genes of B-BRDⅡ strain used in Beijing Tiantan Biological Products Co., Ltd. and RA27 / 3 strain were determined, of which the nucleotide and the corresponding amino acid sequences were analyzed.Results The CPE and virus titer of S-BRD-Ⅱ strain of various passages were basically stable after subculture for 10 passages in MRC-5 cells, while the E1 gene showed no variation. However, a specific silence mutation was observed compared with that of BRD-Ⅱ strain in Gen Bank, and mutations at 122 sites, resulting in mutations of six amino acids,compared with those of RA 27 / 3 vaccine strain. Variations at five sites were observed in ORFⅠ and ORFⅡ regions of SBRD-Ⅱ strain of passages 28, 29, 33 and 38 compared with those of BRD-Ⅱ strain in Gen Bank, while a large quantity of genes in encoding region of non-structural protein P150 were deleted. The 5′-terminuses of S-BRD-Ⅱ strain of passages28 and 38 were consistent with those of BRD-Ⅱ strain in Gen Bank, while the variation of one amino acid at 3′-terminus.Conclusion BRD-Ⅱ strain showed good stability in subculture, and the amino acid sequences of E1 genes of rubella virus strains for production of various commercial vaccines showed no significant difference.

    2015 10 v.28 [Abstract][OnlineView][Download 411K]

  • Evaluation on stability of Haemophilus influenzae type b conjugate vaccine

    FANG Guo-liang;MA Bo;YANG Li;WU Xiu-li;LI Zi-cai;XU Dong-mei;QIAN Wen;HUANG Zhen;Yuxi Walvax Biotechnology Co., Ltd.;

    Objective To evaluate the stability of Haemophilus influenza type b(Hib)conjugate vaccine stored at 2 ~ 8 ℃.Methods A total of 11 batches of Hib conjugate vaccine put on the market after lot release, manufactured by Yuxi Walvax Biotechnology Co., Ltd., were stored at 2 ~ 8 ℃, from which samples were taken at months 0, 3, 6, 9, 12, 18, 24 and 30 after storage for identity test, inspection on final containers, determinations of polysaccharide content, p H value,sodium chloride content, high molecular conjugate content and polysaccharide molecular size, potency test, sterility test,pyrogen test, abnormal toxicity test and bacterial endotoxin test to evaluate the stability. Results All the indexes of Hib conjugate vaccine met the quality standard 30 months after storage at 2 ~ 8 ℃. Conclusion Hib conjugate vaccine showed high stability after storage at 2 ~ 8 ℃ for 30 months.

    2015 10 v.28 [Abstract][OnlineView][Download 144K]

  • Effect of catalase overexpression on DNA oxidative damages and phenotype of lung cancer cell model induced by radiation with α particles

    TONG Peng;WEI Zhi-quan;ZHANG Wei;SHAO Shuai;WANG Chun-yan;QI Xue-song;GOU Qiao;National Institute for Radiological Protection, Key Laboratory of Radiological Protection and Nuclear Emergency,Chinese Center for Disease Control and Prevention;

    Objective To investigate the effect of catalase(CAT) overexpression on DNA oxidative damage and malignant phenotype of lung cancer cell model BERP35T1 induced by radiation with α particles. Methods Eukaryotic expression vector p EGFP-CAT was constructed and identified by PCR, restriction analysis and sequencing, then transfected to BERP35T1 cells in mediation of liposome, using empty vector as control. G418-resistant cell strains were screened, in which the m RNA transcription and protein expression of CAT were identified by real-time PCR and Western blot. The effect of CAT overexpression on DNA oxidative damage(8-OHd G level), proliferation(cell surviva), migration ability(distance of cell migration)and anchoring independence(formation rate of soft agar colonies)in BERP35T1 cells were evaluated by immunocytochemical method, MTT assay, wound healing test and anchorage independent growth test respectively.Results PCR, restriction analysis and sequencing proved that recombinant plasmid p EGFP-CAT was constructed correctly. BERP35T1-CAT-7 cell strain for stable expression of CAT was obtained. The transcription levels of CAT m RNA in BERP35T1, BERP35T1-p EGFP and BERP35T1-CAT-7 cells were 0. 90 ± 0. 23, 1. 00 ± 0. 12 and 2. 91 ± 0. 41, while the relative expression levels of CAT protein were 0. 97 ± 0. 11, 1. 00 ± 0. 00 and 5. 72 ± 1. 28, and the expression levels of 8-OHd G were 1. 65 × 10-2, 1. 60 × 10-2and 8. 95 × 10-2, respectively. However, survival rates of BERP35T1,BERP35T1-p EGFP and BERP35T1-CAT-7 cells were(95. 33 ± 4. 26)%,(100. 00 ± 8. 29)% and(61. 63 ± 3. 08)%,while the migration distances were(150. 68 ± 9. 98),(115. 02 ± 30. 73) and(44. 25 ± 10. 53) μm, and the formation rates of soft agar colonies were(2. 50 ± 0. 02)‰,(2. 10 ± 0. 02)‰ and(0. 70 ± 0. 02) ‰, respectively. Conclusion CAT overexpression may inhibit the proliferation, migration and anchoring independence of BERP35T1 cells by decreasing the level of DNA oxidative damage.

    2015 10 v.28 [Abstract][OnlineView][Download 432K]

  • Lead ion-chelating function of metallothioneins from yeast origin in mimic gastrointestinal environment

    WANG Ying;XU Bing-zheng;ZHANG Gui-fang;ZHANG Dong-jie;Wang Xin-hui;Chen chun-qi;College of Food, Heilongjiang Bayi Agricultural University;

    Objective To investigate the lead ion-chelating function of metallothioneins(MTs)from yeast origin in mimic gastrointestinal environment and provide theoretical reference and data support for the further study on promoting effect of MTs from yeast origin on lead excretion as well as development of relevant products. Methods The lead ion-chelating effects of MTs of two configurations from yeast origin, MT-Ⅰand MT-Ⅱ, in mimic gastrointestinal environment were determined by graphite furnace atomic absorption spectroscopy, using Zn-MT, a metallothionein from animal origin, as control,based on which the chelation rate of lead ion by MT was calculated, the migration and transformation of heavy metal ions in the chelation system, as well as the stability of MTs from yeast origin in mimic gastrointestinal environment, were ana-lyzed. Results MTs showed significantly dose-dependent lead ion-chelating ability in mimic gastrointestinal environment.In mimic gastric juice, the chelation rates of lead ion by MT-Ⅰand MT-Ⅱwere significantly higher than that by Zn-MT(P < 0. 05). Pepsin weakened the lead ion-chelating abilities of MT-Ⅰand Zn-MT at a certain degree(P < 0. 05), while showed no significant effect on MT-Ⅱ(P > 0. 05). In mimic intestinal juice, the lead ion-chelating abilities of three MTs showed significant difference(P < 0. 05). In the turn of chelating abilities, the MTs were Zn-MT, MT-Ⅰ and MT-Ⅱ,which were significantly higher than those at the same concentration in mimic gastric juice(P < 0. 05). Trypsin showed little effect on the leading ion-chelating abilities of MTs(P > 0. 05). During chelation, the MTs released the bound heavy metal ions, of which the release rates were significantly higher than the chelation rate of lead ion(P < 0. 05). Both the degradation levels of MT-Ⅰand MT-Ⅱ were relatively low in mimic intestinal juice, indicating high stabilities. Conclusion MT-Ⅰand MT-Ⅱ from yeast origins chelated lead ion significantly in mimic gastrointestinal environment, and resisted the digestion within 12 h to maintain their chelating abilities.

    2015 10 v.28 [Abstract][OnlineView][Download 261K]

  • Construction and identification of recombinant human adenovirus 5 expressing rabies virus phosphoprotein

    JIN Hong-liang;WANG Shu-chao;GAO Shu-man;ZHANG Shou-feng;YAN Yan;WANG Dong-fang;LIU Ye;HU Rong-liang;College of Animal Science and Technology, Jilin Agricultural University;

    Objective To construct and identify a recombinant human adenovirus 5 expressing phosphoprotein(P) of rabies street virus BD06 strain(RABVP). Methods Entire P protein gene of rabies virus BD06 strain was obtained from plasmid p MD18-T-BD06 P by digestion with restriction endonuclease and cloned into shuttle plasmid pac Ad5CMVK-Np A.The constructed recombinant shuttle plasmid pac Ad5CMV-BD06 P and backbone plasmid pac Ad5 9. 2-100 were linearized with Pac Ⅰ, then cotransfected into HEK293 AD cells to obtain recombinant human adenovirus 5 expressing RABVP,named as r Ad5-BP, by homologous recombination. The titer of r Ad5-BP was calculated by K覿ber method, while the transcription and expression levels of P by RT-PCR, Western blot and direct immunofluorescence assay(d FA). Kunming mice were inoculated i.m. with 107TCID50 r Ad5-BP, of which serum samples were collected 14 d later, and determined for anti-RABVP antibody and its reactive activity by d FA. Results Restriction analysis and sequencing proved that recombinant shuttle plasmid pac Ad5CMV-P was constructed correctly. The obtained r Ad5-BP virus reached a titer of108TCID50/ ml, which mediated the transcription and expression of P protein in HEK293 AD cells elicited anti-RABVP antibody in mice, and showed good reactive activity with RABV. Conclusion Recombinant human adenovirus expressing phosphoprotein of RABV BD06 strain was constructed successfully, which laid a foundation of further study on the role of RABVP in viral infection.

    2015 10 v.28 [Abstract][OnlineView][Download 270K]

  • Expression of soluble tumor necrosis factor-related apoptosis-inducing ligand in E. coli as well as purification and biological activity of expressed product

    WANG Shao-hua;ZHENG Hai-feng;WANG Yu-tao;YANG Li-jun;YANG Tao;Department of Biochemistry and Molecular Biology, Key Laboratory of Cellular Physiology,Shanxi Medical University, Ministry of Education;

    Objective To express and purify soluble tumor necrosis factor(TNF)-related apoptosis-inducing ligand(TRAIL) and determine its biological activity. Methods Soluble TRAIL protein with 6 × His tag at C-terminus was expressed in E. coli, and the time for induction(4, 6 and 8 h) and IPTG concentration(0. 06, 0. 125, 0. 25, 0. 5 and1 mmol / L) were optimized. The expressed product was purified by Ni affinity and ion-exchange chromatography, of which the activities at various final concentrations(100, 200 and 400 ng / ml)were determined by MTT assay. The effects of two elution procedures(the eluant was maintained at 4 ℃ overnight in column before elution; the eluate was collected directly after the eluant was loaded to the column)and the time for dialysis(24 and 48 h) during purification on TRAIL activity were evaluated. Results The optimal time for induction was 6 h, while the optimal IPTG concentration was0. 5 mmol / L. The expression level of TRAIL protein was about 20% of total somatic protein, while the relative molecular mass was 20 000. The expressed product showed specific binding to rabbit anti-human TRAIL monoclonal antibody. The inhibiting rates of SW480 cells treated with TRAIL at final concentrations of 100, 200 and 400 ng / ml were about 36%,53% and 73% respectively. The elution procedure showed no significant effect on TRAIL activity(P > 0. 05). However,the TRAIL activity after dialysis for 48 h was significantly lower than that for 24 h(P < 0. 05). Conclusion Soluble TRAIL protein was successfully expressed in E. coli, which showed inhibiting effect on the growth of tumor cells. A large quantity of TRAIL may be produced under the optimized condition for induction by the optimized method for purification,which may meet the requirements for basic and clinical studies on TRAIL.

    2015 10 v.28 [Abstract][OnlineView][Download 294K]

  • Preparation and antigenicity of polyvalent EZH2 tumor associated antigen peptide

    YANG Wen;MA Xiao-meng;LI Qian;LV Ya-feng;YANG Jian-lin;WANG Yan-lin;China Three Gorges University Medical College, Hubei Key Laboratory of Tumor Microenvironment and Immunotherapy;

    Objective To artificially design and prepare polyvalent EZH2 tumor associated antigen peptide and identify its antigen characteristics. Methods The DNA sequence encoding polyvalent EZH2 tumor associated antigen peptide was designed and synthesized, then cloned into prokaryotic expression vector p ET-30a(+). The constructed recombinant plasmid p ET-30a(+)-EZH2-antigen containing four EZH2 tumor associated antigen peptides(Aa120-128, Aa165-174,Aa291-299 and As735-742)was transformed into E. coli BL21(DE3)and induced with IPTG. The expressed recombinant polyvalent EZH2 tumor associated antigen peptide was purified by affinity chromatography with Ni-NTA resin under denaturing condition, and analyzed for antigen characteristics by Western blot, with which rabbits were immunized. The obtained polyclonal antibody was determined for titer in sera by ELISA, and analyzed for specificity by Western blot.Results Restriction analysis and sequencing proved that recombinant prokaryotic expression plasmid p ET-30a(+)-EZH2-antigen was constructed successfully. The expressed recombinant polyvalent EZH2 tumor associated antigen peptide, with a apparent relative molecular mass of about 4 300, mainly existed in a form of inclusion body and reached a purity of not less than 90% and a concentration of 0. 5 mg / ml after purification, which was recognized and bound with rabbit antiEZH2 polyclonal antibody and rabbit anti-His tag polyclonal antibody effectively. The purified polyvalent EZH2 tumor associated antigen peptide induced antibody effectively in animals and recognized and bound the EZH2 protein expressed in human prostate cancer PC3 cells specifically, of which the antiserum titer was 1 ∶ 64 × 104. Conclusion A polyvalent EZH2 tumor associated antigen peptide with low relative molecular mass, high density and potentially high activity was successfully prepared, which was of good prospect in application.

    2015 10 v.28 [Abstract][OnlineView][Download 455K]

  • Preparation of monoclonal antibody against tetrapeptide repeated motif of malignant Plasmodium falciparum circumsporozoite protein

    LU Jian;LI Ping;MA Ya-ru;LI Gang;CHEN Yong;JIANG Lin;Lanzhou Institute of Biological Products Co., Ltd.;

    Objective To prepare the monoclonal antibody(Mc Ab) against Asn-Ala-Asn-Pro(NANP) repeated motif of circumsporozoite protein(CSP)of Plasmodium falciparum. Methods Maleimide-modified BSA was coupled with(NANP)5C, while ADH-EDC mediated BSA with(NANP)5. The synthetic NANP was coupled with carrier protein and immunized to BALB / c mice. Hybridoma cell strain secreting Mc Ab against(NANP)5 was obtained by hybridization of spleen cells of immunized mice with bone marrow hybridoma SP2 / 0 cells, determined for titer by indirect ELISA, and immunized to F1 mice. The obtained ascites was purified by precipitation with 50% ammonium sulphate, and identified for subclass and specificity. Results BSA-(NANP)5 coupling protein was prepared by two methods and used as an antigen to prepare ten hybridoma cell strains secreting Mc Abs against(NANP)5 peptide. All the Mc Abs were Ig G1 with Ⅱκ chain and titers of1 ∶ 80 000 ~ 1 ∶ 640 000, which were recognized by(NANP)5 and CSP antigen. Conclusion The Mc Ab against(NANP)5 peptide was successfully prepared, which played an important role in development of malignant malaria vaccine.

    2015 10 v.28 [Abstract][OnlineView][Download 262K]

  • Investigation of plasma protein concentration and distributions of ratio of albumin to immunoglobulins in plasma donors in Sichuan Province, China

    JIANG Yan-fang;DONG De-mei;ZOU Ling-ling;LIU Su-fang;ZHANG Yan;GAO Yang;SUN Li-hui;CHEN Pin-quan;ZHU Yue;Chengdu Rongsheng Pharmaceuticals Co., Ltd.;

    Objective To investigate the plasma protein concentration and distributions of ratio of albumin to immunoglobulins in plasma donors in Sichuan Province, China as well as the feasibility of extended age of plasma donors.Methods A total of 2 467 plasma samples from donors in Sichuan Province were grouped according to district(northeast,central and south parts of the province), age(18 ~ 35, 36 ~ 45 and 46 ~ 55 years), gender(male and female), time of donation(1 ~ 26, 27 ~ 52, 53 ~ 78 and 79 ~ 110 times)and annual frequency of donation(1 ~ 5, 6 ~ 10, 11 ~ 15 and16 ~ 23 times) separately, in which the plasma protein concentration was determined by quantitative biuret method specified in Chinese Pharmacopoeia(Volume Ⅲ, 2010 edition), and the ratio of albumin to immunoglobulins by agarose gel electrophoresis. Results The plasma protein concentrations based on age as well as time and annual frequency of donation showed no significant difference(each P > 0. 05). The ratio of albumin to immunoglobulins based on time and annual frequency of donation showed no significant difference(each P > 0. 05), while that based on age showed significant difference(P < 0. 01). However, all the plasma protein of the donors reached concentrations of more than 50 g / L, of which albumin accounted for more than 50%. All the ratios of albumin to immunoglobulins in protein were within the normal range. The test results of plasma by electrophoresis each year and protein content at each donation met the relevant national requirements. Conclusion Based on the Requirements for Physical Examination of Blood Donor and the result of this study, it is recommended that the upper limit of age of plasma donors may be extended from 55 to 60 years.

    2015 10 v.28 [Abstract][OnlineView][Download 131K]

  • Analysis of neutralizing activity in vivo of recombinant monoclonal antibody against tumor necrosis factor-α from human origin by using mouse model of liver necrosis

    ZHANG Ya-ting;PAN Yong-bing;ZHANG Yin-chuan;ZHANG Kun-ming;REN Bin;SONG Gui-zhi;GUI Fang;BI Lan;Wuhan Institute of Biological Products Co., Ltd.;

    Objective To establish a mouse model of liver necrosis and analyze the neutralizing activity in vivo of recombinant monoclonal antibody(Mc Ab)against tumor necrosis factor(TNF)-α from human origin. Methods The time for sensitization with D-galactosamine(D-Gal N)and lethal dose of TNF-α to C57 BL / 6 mice were determined. Mice were sensitized with D-Gal N and injected i. p. with TNF-α to establish liver necrosis model, with which the neutralizing activities in vivo of three batches of test samples(Mc Ab against TNF-α)and three batches of positive control(Humira誖)were determined, and the results were analyzed by Probit regression fitting. Results The time for sensitization with DGal N was 10 min, while the lethal dose of TNF-α was 100 μg / kg bodyweight. Probit regression fitting showed good fitting degree. The median effective doses(ED50)of three batches of test samples to C57 BL / 6 mice were 0. 505, 0. 434 and0. 609 mg / kg, while those of three batches of positive control were 0. 455, 0. 571 and 0. 484 mg / kg, respectively. The neutralizing activities in vivo of test samples and control in C57 BL mice showed no significant difference(P > 0. 05).Conclusion D-Gal N combined with TNF-α caused liver necrosis, resulting in the death of mice, which could be blocked by Mc Ab against TNF-α. The Mc Ab showed dose-dependent inhibitory effect on TNF-α.

    2015 10 v.28 [Abstract][OnlineView][Download 186K]

  • Determination of interferon β1b content in recombinant human interferon β1b for injection by reversed phase high performance liquid chromatography

    YANG Yun-kai;MA Yu;SUN Yu-jie;MA Xiao-yu;AN Huan-yu;Beijing Tiantan Biological Products Co.Ltd.;

    Objective To develop a reversed phase high performance liquid chromatography(RP-HPLC) method for determination of interferon β1b content in final product of recombinant human interferon(rh IFN) β1b. Methods The IFNβ1b content in final product of rh IFNβ1b was determined by RP-HPLC using ZORBAX 300SB-C18 column(4. 6 mm ×250 mm, 5 μm). The samples were eluted by gradient elution for 10 min using mobile phase A consisting of 95% water,5% acetonitrile and 0. 1% trifluoroacetic acid as well as mobile phase B consisting of 5% water, 95% acetonitrile and0. 1% trifluoroacetic acid, at a flow rate of 1. 5 ml / min, in which the proportion of mobile phase A decreased from 70%to 30%. The wavelength for detection was 214 nm, while the temperature of column was 35 ℃, and the sample loading was 20 μl. The method was verified for suitability, linearity, precision, reproducibility and accuracy. The IFN β1b contents in three batches of final products of rh IFNβ1b were determined by the method. Results The developed method showed good systemic suitability. The IFNβ1b content at a range of 20 ~ 100 μg showed good linear relationship to the peak area(r2 = 0. 998). The RSD of peak areas of samples in six tests was 0. 97%. The mean IFNβ1b contents of six samples of the same batch was 302. 83 μg / ml, with a RSD of 0. 99%. The mean spike recovery rates of IFNβ1b at concentrations of 40,60 and 80 μg / ml were 93. 1%, 94. 18% and 93. 67% respectively, of which the RSDs were less than 2%. The IFNβ1b contents in final products of rh IFNβ1b were 389. 8, 364. 5 and 304. 6 μg / ml respectively. Conclusion The developed RP-HPLC method was simple, precise, reproducible and accurate, which might be used for determination of IFN β1b content in final product of rh IFNβ1b.

    2015 10 v.28 [Abstract][OnlineView][Download 174K]

  • Development and verification of ELISA for typhoid Vi polysaccharide-specific Ig G content in human sera

    LIU Yue-ping;FAN Feng-feng;ZHOU Hai-fei;XU Bo;WANG Xin-ru;JIN Xin;ZHAO Zhi-qiang;ZHU Bing-dong;XIE Gui-lin;TAN Xiao-mei;Lanzhou Institute of Biological Products Co., Ltd., Vaccine Engineering Technology Center of Gansu;

    Objective To develop and verify an ELISA method for determination of typhoid Vi polysaccharide-specific Ig G content in human sera. Methods Using the ELISA method developed by the National Institute of Health(NIH)as a reference, an ELISA method for determination of typhoid Vi polysaccharide-specific Ig G content in human sera was developed based on the optimization of coating antigen, plates with different adsorptive capacities, antigen concentration for coating, reaction condition of samples and time for color development, and verified for specificity, linearity, accuracy, pre-cision and minimum detection limit. Results The optimal coating antigen, plate, antigen concentration for coating, reac-tion condition of samples and time for color development were typhoid Vi polysaccharide, costar92592 plate, 2. 0 μg / ml,20 ~ 25 ℃ overnight and 40 ~ 80 min(A405= 1. 8 ~ 2. 5) respectively. After absorption with various concentrations of polysaccharide, the antibody content in quality control sera was significantly dependent to polysaccharide concentration,while the inhibition rate of antibody by polysaccharide reached 92% at most. The dilution of sera showed linearly negative correlation to antibody content, with a R2 value of 0. 999. The antibody concentration of quality control sera determined by the developed method was 76. 80 EU / ml, with a CV of 8. 55%, which was consistent with that by NIH(75 EU / ml,CV ≤ 15%). Both the reproducibility(CV ≤ 15%)and intermediate precision(CV ≤ 20%)of the method met the relevant requirements, while the minimum detection limit was 0. 78 EU / ml. Conclusion An ELISA method was developed,which showed high specificity, linearity, accuracy and precision, and might be used for rapid and accurate determination of typhoid Vi polysaccharide-specific Ig G content in human sera.

    2015 10 v.28 [Abstract][OnlineView][Download 241K]

  • Verification of inactivation process for inactivated enterovirus 71 vaccine(human diploid cells)

    WANG Jing-jing;LIAO Yun;WANG Li-chun;LI Qi-han;LIU Long-ding;CHENG Chen;XIA Long-hui;YIN Xiao-xiao;ZHANG Ying;Yunnan Key Laboratory of Vaccine Research and Development on Severe Infectious Diseases, Institute of Medical Biology,Chinese Academy of Medicine Science & Peking Union Medical College;

    Objective To verify the inactivation procedure for inactivated enterovirus 71(EV71)vaccine(human diploid cells). Methods Samples were taken from three batches of harvests of EV71 inactivated with formaldehyde for four blind passages, using the harvests before inactivation as control. Each passage was observed for cytopathic effect(CPE), and determined for infectious titer by microtitration, for EV71-specific antigen content by ELISA, and for viral DNA level and production of virus negative strand by real-time quantitative PCR(q RT-PCR). Results After four blind passages, no obvious CPEs were observed in three batches of inactivated vaccine of various passages, and no infectious EV71 titers were detected by microtitration. After two blind passage, no negative strand of virus was detected. The test result for EV71 antigen was negative after three blind passages, while that for viral DNA was negative after four blind passages.However, obvious CPEs were observed in virus control of various passages after four blind passages, while the infectious titers were 106~ 107CCID50/ ml, the antigen content was 200 ~ 370 U / ml, and obvious propagation of viral DNA and negative strand were observed, after two blind passages. Conclusion The inactivated EV71 samples were inactivated with formaldehyde completely by typical three blind passages in cells combined with q RT-PCR.

    2015 10 v.28 [Abstract][OnlineView][Download 210K]

  • Comparison of three methods for determination of mycoplasmas

    LU Yong;JIA Ji-zong;TANG Jing;YE Xiang-zhong;Beijing Wantai Biological Pharmacy Enterprise Co., Ltd.;

    Objective To compare PCR, enzymic method and DNA fluorescent staining for determination of mycoplasmas.Methods Mycoplasma-positive BSR cell culture supernatant was 10-fold serially diluted(10-1 ~ 10-8) and inoculated to mycoplasma-negative Vero cells for five blind passages. The samples of various passages at each dilution were determined for mycoplasma by PCR, enzymic method and DNA fluorescent staining, using mycoplasma-negative Vero cells as control.Results After one blind passage, mycoplasma was detected in the positive samples at a dilution of 10-4by PCR, while in those at a dilution of 10-3by enzymic method and DNA fluorescent method. After two blind passages, mycoplasma was detected in samples at a dilution of 10-4by three methods. However, after three blind passages, mycoplasma was detected in samples at a dilution of 10-5by three methods, of which the titer showed no increase with the increasing passage.Conclusion After the samples were subjected to three blind passages, mycoplasma was detected by each of the three methods with consistent sensitivities. PCR and enzymic method were accurate, rapid and simple for determination of mycoplasma, which might be used as a supplement of DNA fluorescent staining.

    2015 10 v.28 [Abstract][OnlineView][Download 309K]

  • Determination of methyl pentose content in pneumococcal polysaccharide by microplate assay

    LIU Ai-ping;HU Hao;LI Xian-lin;KONG Su-juan;WANG Jing;YUAN Fei;WANG Xin-ru;LIU Fang-lei;WU Bing;ZHAO Zhi-qiang;XIE Gui-lin;Lanzhou Institute of Biological Products Co., Ltd.;

    Objective To determine the methyl pentose content in pneumococcal polysaccharide by microplate assay and verify the method. Methods Microplate was applied to the traditional method for determination of methyl pentose, and the concentration of L-cysteine hydrochloride was optimized. The developed method was verified for linearity, precision and accuracy, and used for determination of methyl pentose content and relative molecular mass distribution of polysac-charide derivative in pneumococcal polysaccharide. Results The optimal concentration of L-cysteine hydrochloride was15 mg / ml. The concentration of standard L-rhamnose at a range of 5 ~ 30 μg / ml showed good linear correlation with the value of(A396- A430)(R2 > 0. 999). The CVs of determination results of pneumococcal polysaccharide and its derivative in intra- and inter-assays were 0. 9 % ~ 1. 1% and 2. 2% ~ 3. 2% respectively. The recovery rates of pneumococcal polysaccharide 6B and its derivative, added with 2. 5, 5 and 10 μg / ml L-rhamnose, were 91. 73% ~ 100. 70%. The methyl pentose content in pneumococcal polysaccharide determined by microplate assay showed no significant difference with that by traditional method(P > 0. 05), while the relative molecular mass distributions by the two methods were in agreement. Conclusion Microplate assay was effective, accurate and stable for determination of methyl pentose content in pneumococcal polysaccharide and its derivative, which was highly sensitive, easy and rapid to handle, and material-saving,and was suitable for the quality control of vaccine during production.

    2015 10 v.28 [Abstract][OnlineView][Download 193K]

  • Optimization of culture medium for production of monoclonal antibody against human epidermal growth factor receptor 2 by using design of experiment

    ZHANG Hui-juan;XIANG Gang;LI Ruo-zhu;WU Ze-xuan;YU Yu-gen;Shenzhen Main Luck Pharmaceuticals Inc.;

    Objective To optimize the culture medium for production of monoclonal antibody(Mc Ab) against human epidermal growth factor receptor 2(HER2) by using design of experiments(DOE) so as to increase the expression level of Mc Ab. Methods The effects of various basal media and feeding media on cell density and expression of Mc Ab against HER2 in engineering cell were investigated by DOE with Minitab software. Results After optimization by DOE, the viable cell density of recombinant cell strain reached 296 × 105 cells / ml at most. However, the expression level of antibody titer reached 2. 07 g / L at most, which increased by 72% compared with that before optimization(1. 20 g / L).The quality of antibody was similar to that of antibody developed previously as control. Conclusion An optimized combination of basal media and feeding media was obtained, by which the cell density and antibody expression level increased. The optimized combination was suitable for production of Mc Ab against HER2, indicating DOE as an effective method for rapidly increasing the expression level of antibody within a short time period.

    2015 10 v.28 [Abstract][OnlineView][Download 347K]

  • Purification and quality control of plasmid TV-BIKDD for targeted therapy of lung cancer

    RAO Gui-rong;SHI Yan-fei;HUANG Bin;ZHANG Huan-jing;FAN Yu-min;MO Guo-yu;CHEN Guang-ming;WANG Hong-min;YANG Fu-qiang;Liver Disease Research Center, The 458th Hospital of PLA;

    Objective To extract and purify the plasmid TV-BIKDD for targeted therapy of lung cancer and control its quality. Methods Plasmid was crudely extracted from recombinant E. coli DH5α / TV-BIKDD by alkaline lysis, purified by two steps of chromatography with monolithic columns, i.e. CIM誖DEAE-8 Tube monolithic column and CIM誖C4 HLD-8 Tube Monolithic Column, and determined for plasmid content and proportion of supercoil DNA. Three consecutive batches of solutions mainly consisting of supercoil DNA were purified by the method and subjected to control tests.Results Most of RNA was removed from crude extract of plasmid by CIM 誖 DEAE-8 Tube monolithic column. After chromatography with CIM 誖 C4 HLD-8 Tube Monolithic Column, the proportion of supercoil DNA reached more than95. 21%, while the total recovery rate of plasmid was 78%. The results of overall control tests on three batches of final products of plasmids met the requirements of FDA of the USA and the Technical Guideline for Products for Gene Therapy in China. Conclusion A stable procedure for purification of recombinant plasmid TV-BIKDD was developed, which laid a foundation for further development of procedure for large-scale preparation of samples for clinical use.

    2015 10 v.28 [Abstract][OnlineView][Download 244K]

  • Optimization of condition for culture of influenza H5N1 virus in Vero cells

    XIE Ying-lin;ZHOU Qing-ling;WANG Wen-jun;YUE Zhen-qi;HAN Yun-fei;WANG Yi-ming;LI Chun-yan;LI Yang;ZHENG Chao;LI Guang-pu;Research and Development Center of Drugs, Jilin Yatai(Group)Co., Ltd.;

    Objective To optimize the condition for culture of influenza H5N1 virus in Vero cells. Methods Influenza virus was cultured in various maintenance media [M199, M199 + epidermal growth factor(EGF), MEM, MEM + EGF and serum-free medium(SFM)]and M199 medium containing various concentrations of trypsin(3, 5 and 7 μg / ml)respectively, of which the culture supernatants were collected and determined for hemagglutinin(HA) titer to optimize the concentrations of maintenance medium and trypsin. Vero cells were infected with influenza H5N1 virus-infected cell supernatant before and after freezing and thawing and subcultured, of which the supernatant was collected and determined for HA titer to evaluate the effect of freezing and thawing on virus propagation. Vero cells were inoculated with influenza H5N1 virus at a MOI of 0. 01 and cultured under the optimized condition, of which the culture supernatant was collected and determined for HA titer and infectious titer to evaluate the stability of H5N1 virus after continuous subculture in Vero cells. Results When the SFM containing 5 μg / ml of trypsin was used, and the harvest was infected to Vero cells directly and subcultured for two passages before the maintenance medium was substituted with M199 medium, the HA titer of influenza H5N1 virus reached the maximum. Both the HA and infectious titers of H5N1 virus were stable after subculture in Vero cells under the optimized condition. Conclusion Influenza H5N1 virus was subcultured continuously and stably in Vero cells under the optimized condition, which laid a foundation of establishment of working seed lot for the preparation of pandemic influenza vaccine.

    2015 10 v.28 [Abstract][OnlineView][Download 138K]

  • Determination of rabies virus titer by cell fluorescence focus unit assay

    ZHOU Lan-zhen;LIU Yong-di;DAI Mei-lan;RONG Wei-hua;WANG Hong-bing;TIAN Hua;ZHANG Jian-ping;Shenzhen Weiguang Biological Products Co., Ltd;

    Objective To determine the titer of rabies virus by cell fluorescence focus unit(FFU) assay. Methods Rabies virus was diluted, inoculated to BSR cells, cultured for 22 ~ 24 h, and stained with FITC-labeled specific antibody against rabies virus to count the fluorescence foci in infected cells, based on which the virus titer was calculated. The titers of 14 batches of rabies virus were determined by median lethal dose(LD50)assay in suckling mice and the developed method respectively, and the relationship between the two methods was analyzed. The same virus sample was determined at 3 time points, 6 times for each, to verify the reproducibility of the method. Three virus samples at different titers were determined for 3 times at various time points by 2 staff separately to verify the intermediate precision of the method. The same virus sample was 2-fold serially diluted to 9 dilutions, each of which was tested for titer for 3 times to determine the linear range of the method. A total of 38 batches of rabies virus CTNCEC strain were determined for titer by the developed method. Results The range of-Lg LD50 values of 14 batches of rabies virus determined by LD50 assay in suckling mice was 4. 20 ~ 8. 19, while the range of Lg FFU by FFU assay was 3. 76 ~ 7. 69. The-Lg LD50 and Lg FFU exhibited the same trends, which showed a significantly linear correlation(adjusted R2 = 96. 1). The CVs of samples determined for 6 times at the same time point and for 3 times at various time points were less than 2. 00%. However, the CVs of samples determined for 3 times at various time points by two staff were less than 8. 00%, which showed no significant difference(P > 0. 05). The sample concentration showed a good linear relationship to Lg FFU, with an adjusted R2 value of 99. 3%,of which the linear range was 2. 26 ~ 6. 12. The mean Lg FFU of 38 batches of CTNCEC strain was 3. 63 ~ 7. 69, with a standard deviation(SD)of 0. 021 ~ 0. 57 and a CV value of less than 10. 00%. Conclusion The developed FFU assay showed high accuracy, reproducibility and intermediate precision, which might be used for the determination of rabies virus titer.

    2015 10 v.28 [Abstract][OnlineView][Download 203K]

  • Evaluation on immunogenicity of Haemophilus influenzae type b(Hib)vaccine as well as novel and combined Hib vaccines

    ZHU Lang;LIU Yi-fei;LU Wei;LIN Ji-sheng;GAO Qiang;CAI Fang;R & D Center, Sinovac Biotech Co., Ltd.;

    Haemophilus influenzae type b(Hib) is an important pathogen causing serious pneumonia, meningitis and sepsisin in young children aged from 3 months to 5 years worldwide. Vaccination is the only effective public health mea-sure to prevent and control Hib diseases. Both the morbidity and carrier rate of Hib disease dramatically reduced in coun-tries where Hib conjugate vaccines were brought into routine childhood vaccine program. This review covers Hib vaccines,including the classification, characteristics of immune response and clinical efficacy, as well as evaluation on immuno-genicity of novel or combined Hib vaccine.

    2015 10 v.28 [Abstract][OnlineView][Download 269K]

  • Progress in research on methods for separation and purification of inactivated enterovirus 71 vaccine

    XIAO Hui;MING Ping-gang;CHEN Xiao-qi;XU Ge-lin;Wuhan Institute of Biological Products;

    In recent years, hand-foot-mouth disease(HFMD)which caused by enterovirus 71(EV71)keeps prevalence in China and becomes one of the major public health problems at this stage. Vaccines are the most cost-effective approaches to prevent the EV71 outbreak. To date, inactivated EV71 vaccines are developed rapidly, and a number of research institutions have completed phase Ⅲ clinical trials. The methods for separation and purification of virus harvest are critical to obtaining the vaccine products with high purity, yield and safety. Based on the production technology of inactivated EV71 vaccine, this article introduces the progress in research on methods for separation and purification of the vaccine.

    2015 10 v.28 [Abstract][OnlineView][Download 143K]


@2012 Editorial Department of "Chinese Journal of Biologicals"