2018年05期目次

  • Preparation of inactivated tick-borne encephalitis vaccine by different cultivation methods

    LIU Shuang-jun;LIU Yan-song;SUN Hong-liang;SUN Xiao-ran;SUN Chao;XIE Ji-ying;WANG Lin-lin;WANG Xuan-ying;LIU Zhi-hui;Changchun Institute of Biological Products Co.,Ltd.;

    Objective To compare the yields and qualities of tick-borne encephalitis vaccines(TBEV)prepared by using3 L rolling bottle and 14 L basket bioreactor. Methods Vero cells were cultured in 3 L rolling bottle and 14 L basket bioreactor respectively. The Vero cells in 3 L rolling bottle were infected with TBE virus SZV strain 96 h after culture at MOIs of 0. 01,0. 04,0. 1 and 0. 4,and the virus was harvested for the first time 80 ~ 96 h after further culture,then harvested every 48 h,500 m L for each. However,the Vero cells in 14 L bioreactor were infected with TBE virus after culture for 7 d at MOIs of 0. 01,0. 04,0. 1 and 0. 4,and the virus was harvested for the first time 96 h after further culture the harvested every 24 h,10 ~ 15 L for each. The virus liquids harvested by two methods were concentrated by ultrafiltration,inactivated with β-propiolactone and purified by column chromatography. The obtained bulk of vaccine was subjected to control tests. Results The virus in Vero cells cultured in 3 L rolling bottle could be harvested for 6 ~ 7 times,of which the yield was 3 ~ 3. 5 L and the mean titer was 7. 4 lgLD_(50)/m L. However,the virus in Vero cells cultured in 14 L basket bioreactor could be harvested for 14 d,of which the yield was 140 ~ 160 L and the mean titer was 8. 4 lgLD_(50)/m L.All the quality indexes of inactivated tick-borne encephalitis vaccines prepared by two methods met the relevant requirements. However,the potency and protein content of vaccine prepared by using 14 L basket bioreactor were higher than those using 3 L rolling bottle. Conclusion The production efficiency of preparation of inactivated TBEV by culture in bioreactor was higher than that in rolling bottle. The study lays a foundation of large-scale production and technological improvement of TBEV prepared by culture of Vero cells in bioreactor.

    2018 05 v.31 [Abstract][OnlineView][Download 272K]

  • Immunogenicity of live attenuated yellow fever vaccine at different dosages

    ZHAO Dan-hua;NA Rui;LI Yu-hua;National Institutes for Food and Drug Control;

    Objective To analyze the immunogenicity of live attenuated yellow fever vaccine at different dosages in mice.Methods NIH mice were immunized with live attenuated yellow fever vaccine at a single,1/2,1/3 and 1/5 dosage for human use respectively,of which the serum samples were collected 1,2,3,4,5 and 6 weeks after immunization and determined for antibody level by plaque reduction neutralization test(PRNT). Results No significant differences were observed in the serum antibody titers of mice at various time points within 1 ~ 6 weeks after immunization with yellow fever vaccine at various dosages(each P > 0. 05),while all the antibody positive conversion rates were 100%,indicating that the immunogenicities of vaccine at 1/2,1/3 and 1/5 dosages for human use were basically consistent with that at a single dosage. Conclusion The immunogenicity of live attenuated yellow fever vaccine showed no significant change even at 1/5 of dosage for human use.

    2018 05 v.31 [Abstract][OnlineView][Download 170K]

  • Identification and analysis of NS1 gene of typeⅠdengue virus isolated in Yunnan Province,China in 2017

    WEN Song-jiao;LIN Yao;XI Jue-min;WANG Xiao-dan;CHEN Jun-ying;PAN Yue;YE Chao;GUAN Jiao-qiong;HONG Shan;SUN Qiang-ming;Institute of Medical Biology,Chinese Academy of Medical Science & Peking Union Medical College,Yunnan Key Laboratory of Vaccine Research & Development on Severe Infectious Diseases;

    Objective To identify and analyze the NS1 gene of dengue virus isolated in Yunnan Province,China in2017. Methods RNA was extracted directly from the serum of a patient with dengue fever(DF)by using the viral RNA extraction kit,of which the serotype was identified by RT-PCR,and NS1 gene was amplified and sequenced. The nucleotide and amino acid sequences of the NS1 were compared with those of standard DENVⅠ strain(DQ672562. 1)by using BIOEDIT software to analyze the mutation,based on which a phylogenetic tree was plotted by the NJ method(1 000 boot strap replicate) in MEGA 5. 1 software. Results NS1 gene was amplified from 16 DENV Ⅰ strains.Nucleotide mutation of C to T at site 21 of NS1 gene were found in isolates 201703,201704,201708,201709 and201713,all of which were nonsense mutation. However,nucleotide mutation of T to A at site 289 of NS1 gene were found in isolates 201705,201706 and 201712,which were sense mutation resulting in the amino acid mutation of leucine(L)to methionine(M). The mutations at other sites of NS1 gene of the 16 strains were identical. A total of 85 nucleotide mutations were found,including five sense mutations. Phylogenetic analysis showed close relationship of the 16 strains to2011-China Dongguan(JQ048541. 1),2008-Singapore(GU370049. 2),2014-Taiwan(KU365900. 1),2005-China Fujian(DQ-193572. 1),2016-Malaysia(KX452065. 1),2007-Indonesia(KC762646. 1),2015-South Korea(KP406802. 1),2006-Japan(AB178040. 1),2007-Thailand(HM469966. 1)and 2013-singapore(KJ806945. 2)strains. Conclusion All the 16 isolates in Yunnan in 2017 were DENVⅠ,which might belong to the imported strains from the countries in Southeast Asia.

    2018 05 v.31 [Abstract][OnlineView][Download 345K]

  • Soluble expression of natural mutant of HPV 16 L1 in Pichia pastoris and assembly of virus-like particles

    YANG Xu;HUANG Wei-wei;YAO Yu-feng;LIU Cun-bao;SUN Wen-jia;BAI Hong-mei;MA Yan-bing;Institute of Medical Biology,Chinese Academy of Medical Sciences & Peking Union Medical College;

    Objective To express two natural mutants of human papilomavirus (HPV) type 16 L1 protein in Pichia pastoris,and analyze the solubility of expressed products as well as the difference in assembly of virus-like particles (VLPs). Methods Full-length gene sequences M16 and Y16,encoding HPV 16 L1 protein,were inserted into plasmid p Pink LC respectively,and the constructed recombinant plasmids Y16 LC and M16 LC were transformed to p Pink strain 1.After induction with methonol,the expression level and solubility of L1 protein were analyzed by Western blot,while the assembly of VLPs by density gradient centrifugation combined with transmission electron microscopy. The amino acid arrangement of the two gene sequences were analyzed by online Alignment in pubmed software. Results Restriction analysis and sequencing proved that the two recombinant plasmids were constructed correctly. The protein expression levels of two sequences were similar,while Y16 protein showed low solubility,and few of VLPs were formed. However,M16 showed high solubility and high ability in assembly of VLPs. Variations of amino acids were observed in five sites of Y16 and M16. Conclusion Slight variation of amino acids of HPV 16 L1 showed no significant effect on expression level of recombinant protein while influence the obtainment of expected functional protein,indicating that,in the development of recombinant viral vaccines,the selection of virus strains even activate variation of specific amino acids of antigen should be taken into account.

    2018 05 v.31 [Abstract][OnlineView][Download 373K]

  • Construction of recombinant nuclease prokaryotic expression vector

    ZHAO Yu;CHEN Zhong-min;College of Pharmacy and Bioengineering, Chongqing University of Technology;

    Objective To construct recombinant nuclease prokaryotic expression vector and determine the expressed protein. Methods Ben-p GH gene was designed and synthesized by removal of N-terminal signal peptide sequence of Serratia marcescens(SM)nuclease gene in Gen Bank and usage of E. coli-preferred codon. The target gene was amplified by PCR using the full length nuclease gene as template, and inserted into vector A10-p ET-32 a-c(+)digested with NcoⅠand XhoⅠ. The constructed recombinant plasmid was transformed to E. coli Top10, and the positive clones were screened by PCR and sequenced. Recombinant plasmid Ben-p ET-32 a-c(+) was transformed to E. coli BL21(DE3) for expression under induction of IPTG. The expressed product was identified by SDS-PAGE. Results The homology of nuclease gene in Ben-p ET-32 a-c(+)was 82% to that of SM nuclease genes reported in Gen Bank. The expressed recombinant nuclease,with a relative molecular mass of 30 000, existed in a form of inclusion body, and contained about 84. 45% of total somatic protein. Conclusion The recombinant nuclease prokaryotic expression vector was successfully constructed, and target protein was stably expressed.

    2018 05 v.31 [Abstract][OnlineView][Download 658K]

  • Cloning,bioinformatic analysis and preliminary functional identification of MFS transporter gene from Halomonas YH-I

    WANG Yan-hong;YIN Sheng-xiang;WANG Ji-hong;WANG Yu;QIAO Zhi-gang;JI Si-yu;JIA Gui-yan;College of Life Science and Technology,Heilongjiang Bayi Agricultural University;

    Objective To clone the MFS transporter gene from Halomonas YH-I and analyze its bioinformatics and function. Methods Total genomic DNA was extracted from Halomonas YH-I cells and partially digested with Sau3 A Ⅰ.The recovered DNA fragments at lengths of 4 000 ~ 10 000 bp were inserted into vector p UC18. The constructed recombinant plasmids were transformed to competent E. coli EP432 by functional complementation,and a novel MFS transporter gene was screened,analyzed for bioinformatics and used as the template for PCR. The PCR product was inserted into plasmid p ET19(b),and the constructed recombinant plasmid was transformed to E. coli BL21. The positive clones were screened,inoculated to liquid LBK medium in which the sodium ion content was controlled at 0 ~ 0. 8 mol/L,and induced with IPTG. Results A fragment at full length of 4 219 bp was cloned from Halomonas YH-I,which contained four open reading frames as proved by DNAMAN analysis. Each ORF was submitted to NCBI for blast comparison,and the result showed that the ORF2 at a full length of 1 209 bp shared 86% sequence identity with the corresponding gene encoding MFS from Halomonas campaniensis(WP_038484815. 1)and encoded a protein consisting of402 amino acid residues,of which the predicted relative molecular mass was 43 000 and the isoelectric point was 9. 63.This MFS transporter was a hydrophobic transmembrane protein with 12 membrane-spanning domains located in the cell membrane. Phylogenetic tree showed that MFS transporters from Halomonas YH-I were belonged to an independent branch,which might be a new member of MFS transporter. LBK transport capacity test indicated no ability of the protein in sodium ion transport. Conclusion The cloned novel MFS transporter gene was subjected to bioinformatic analysis and preliminary functional identification,which laid a foundation of further study on the protein function of MFS transporter from Halomonas YH-I.

    2018 05 v.31 [Abstract][OnlineView][Download 1184K]

  • Effect of osthole on radiotherapy of transplanted human nasopharyngeal carcinoma in nude mice and relevant mechanism

    CHEN Jiong-yu;ZHUANG Yi-xuan;CHEN Jian;PENG Lin;Central Laboratory,Cancer Hospital of Medical College,Shantou University;

    Objective To investigate the effect of osthole on therapy of transplanted nasopharyngeal carcinoma(NPC) in nude mice as well as the possible mechanisms. Methods CNE2 cells were inoculated s. c. to 20 NU/NU nude mice in the right axillas,2 × 10~8 cells for each. When the tumor size reached 150 mm~3,the mice were randomly divided into control, osthole,radiotherapy and osthole + radiotherapy groups. The mice in control group were injected i. p. with0. 2 m L of physiological saline every other day,while those in osthole group with 1. 5 μg/(g·10 μL)osthole,and those in radiotherapy received radiation with X-ray at a dosage of 5 Gy every 3 d. However,the mice in osthole + radiotherapy group received radiation at the same dosage 2 h after injection i.p. with 1. 5 μg/(g·10 μL) osthole. The tumor size was measured every 3 d,while the tumors were taken out and weighed 21 d after the first injection. The apoptosis rate of CNE2 cells was determined by flow cytometry,while the m RNA transcription and protein expression levels of vascular endothelial growth factor(VEGF)and hypoxia inducible factor-1α(HIF-1α)by fluorescent quantitative PCR and Western blot. Results Both size and weight of tumors in osthole,radiotherapy and osthole + radiotherapy groups,especially in osthole + radiotherapy group,decreased significantly,while the cell apoptosis rate increased significantly,as compared with those in control group(each P < 0. 05). However,both the m RNA transcription and protein expression levels of VEGF and HIF-1α in osthole + radiotherapy were significantly lower than those in osthole and radiotherapy groups(each P < 0. 05). Conclusion Osthole may increase the sensitivity of NPC in nude mice to radiotherapy by a possible mechanism of down-regulation of expressions of VEGF and HIF-1α and inhibition of angiogenesis.

    2018 05 v.31 [Abstract][OnlineView][Download 346K]

  • Expression of core-streptavidin in Pichia pastoris and purification of expressed product

    ZI Jing;ZHANG Yue-juan;WAN Yi;Research Center of Molecular Biology,Shaanxi Institute of Microbiology;

    Objective To construct the vector for expression of core-streptavidin(CSA) in Pichia pastoris,purify the expressed product and determine its activity. Methods The CSA gene was obtained by genetic engineering approach and cloned into expression vector p PIC9 K. The constructed recombinant plasmid p PIC9 K-CSA was transformed to competent P. pastoris KM71 by electrotransforamtion for expression under induction of methanol. The expressed CSA was purified by ammonium sulfate precipitation and 2-iminobiotin agarose gel chromatography,of which the binding activity to biotin was determined by ultraviolet spectrophotometry. Results CSA was effectively expressed in the constructed recombinant P. pastoris,which reached a purity of about 95% after purification by ammonium sulfate precipitation and 2-iminobiotin agarose gel chromatography and showed a binding activity of 13. 6 U/mg to biotin. Conclusion Recombinant protein CSA was effectively expressed in a secretory form in P. pastoris,which showed high biological activity. It provided a basis for the large scale purification of CSA.

    2018 05 v.31 [Abstract][OnlineView][Download 231K]

  • Effect of amino nitrogen/total nitrogen value in medium on production of tetanus toxin

    QU Ming-xia;YANG Hai-yan;XU Jiang-feng;ZHAN Yi-xiong;Wuhan Institute of Biological Products Co.,Ltd.;

    Objective To analyze the effect of amino nitrogen(AN)/total nitrogen(TN) value in medium on the production of tetanus toxin. Methods Based on the same TN and other conditions,media were formulated with animalderived nitrogen sources prepared by various methods at AN/TN values of 0. 25,0. 35 and 0. 55 respectively and used for the culture of Clostridium tetani to analyze the relationship between AN/TN value and tetanus toxin production.Results The tetanus toxin production in media at various AN/TN values showed significant difference(P < 0. 001),while the former was negatively correlated to the latter(r =-0. 85,P < 0. 001). The tetanus toxin production decreased at AN/TN values of 0. 25 ~ 0. 55. When the AN/TN value was 0. 25,the tetanus toxin production reached a peak valule of 105. 5 Lf/m L. Conclusion The decrease of AN/TN value within a certain range is beneficial to the tetanus toxin production.

    2018 05 v.31 [Abstract][OnlineView][Download 178K]

  • Prokaryotic expression and purification of α-6 Giardin of Giardia duodenalis

    LI Xian-he;LIANG Jin-long;WU Na;GONG Peng-tao;LI Jian-hua;YANG Ju;LI He;ZHANG Xi-chen;College of Veterinary Medicine,Jilin University;

    Objective To express the α-6 Giardin of Giardia duodenalis in prokaryotic cells and purify the expressed product. Methods Using the c DNA of G. duodenalis c DNA as a template,α-6 Giardin gene fragment was amplified by PCR and cloned into vector p MD18-T. The constructed recombinant plasmid p MD18-T-α-6 was digested with Eco RⅠand Hind Ⅲ,and the obtained target fragment was cloned into expression vector p ET-28 a. The obtained recombinant plasmid p ET-28 a-α-6 was transformed to competent E. coli Rosetta cells and induced with IPTG. The expressed α-6 Giardin was identified by SDS-PAGE and Western blot,and purified by Ni-NTA chromatography. Results Restriction analysis showed that recombinant plasmid p ET-28 a-α-6 was constructed correctly. The expressed α-6 Giardin was a His-tagged inclusion body protein with a relative molecular mass of about 40 000,which showed specific binding to anti-His monoclonal antibody,indicating a good reactogenicity. The purified α-6 Giardin fusion protein,with a relative molecular mass of about 40 000,reached a purity of 80%. Conclusion The α-6 Giardin of G. duodenalis was successfully cloned,expressed and purified,which laid a foundation of further study on the function of α-6 Giardin.

    2018 05 v.31 [Abstract][OnlineView][Download 296K]

  • Effect of interferon γ on immunoregulatory properties of human umblical cord mesenchymal stem cells cultivated in platelet lysate

    MA Zhi-jie;ZHAO Kui-jun;Beijing Friendship Hospital, Capital Medical University;

    Objective To investigate the effect of interferon(IFN)γ on immunoregulatory properties of human umblical cord mesenchymal stem cells(UCMSCs) cultivated in platelet lysate. Methods UCMSCs were cultivated in platelet lysate, and analyzed for morphology as well as phenotypic and differentiation characteristics to evaluate the effect of IFN γ(10 ng/m L). Meanwhile, the effect of IFNγ on secretion of PEG-2, TGF-β1 and IL-6 was analyzed by ELISA, while that on proliferation capacity of peripheral blood mononuclear cells(PBMCs) by mixed lymphocyte culture assay. Results Pre-treatment with IFNγ stimulation showed no significant effect on the morphology, immunophenotype and differentiation potential of UCMSCs(P > 0. 05), while promoted the secretions of TGF-β1 and IL-6(P < 0. 05) but showed no significant effect on that of PGE-2(P > 0. 05). However, the pre-treatment enhanced the inhibitory effect of UCMSCs on proli-feration of PBMCs(P < 0. 05). Conclusion Pre-treatment with IFNγ may enhance the immunoregulatory properties of UCMSCs.

    2018 05 v.31 [Abstract][OnlineView][Download 321K]

  • Pharmacodynamic of recombinant human granulocyte-macrophage colony stimulating factor oncolytic herpes simplex virus type 2 (Vero cells) injection

    JIN Jing;WANG Yang;HU Han;MAO Ze-yong;SHI Xiao-tai;FANG Zhi-zheng;LIU Bin-lei;Hubei University of Technology;

    Objective To analyze the in vivo and in vitro pharmacodynamics of recombinant human granulocyte-macrophage colony stimulating factor(GM-CSF)onxolytic herpes simplex virus type 2(OH2). Methods Human colon cancer Lo Vo cells and human laryngeal carcinoma Hep2 cells were infected with OH2 at various MOIs(0. 002,0. 01,0. 05,0. 25 and 1. 25). The oncolytic effect of OH2 in vitro was determined by MTT assay. BALB/c-nu mice were injected s. c. with Lo Vo and Hep2 cells respectively to establish the tumor models. After the sizes of tumors were more than100 mm~3,the OH2 were injected into the tumors at high(1 × 10~7 CCID_(50)/m L),moderate(1 × 10~6 CCID_(50)/m L)and low(1 × 10~5 CCID_(50)/m L)respectively,each on days 0,3 and 6,using 5 mg/m L 5-FU as positive control and DMEM/F12 medium as negative control. The longitudinal(a)and transverse(b)diameters of tumors were measured on days 0,3,6,9,12,15 and 18 after the first injection,based on which the tumor volume(TV) and relative tumor proliferation rate were calculated. Results OH2,even at a low concentration,killed Lo Vo and Hep2 cells(at MOIs of 0. 168 and0. 063) effectively. Compared with that in negative control group,obvious tumor-inhibiting effects were observed in the high,moderate and low dose OH2 as well as positive control groups in the two tumor models(P < 0. 05). In the mice injected with Lo Vo cells,the tumor-inhibiting effect in high dose OH2 group was superior to those in moderate and low dose OH2 groups(P < 0. 05). However,in the mice injected with Hep2 cells,the tumor-inhibiting effects of OH2 at three dosages showed no significant difference(P > 0. 05). Conclusion OH2 showed good oncolytic activity in both in vitro and in vivo tests,which was of prospect of being developed as a novel biopharmaceuticals. It provided an experimental basis for preclinical study of OH2.

    2018 05 v.31 [Abstract][OnlineView][Download 216K]

  • Preparation and application of internal quality control for HIV-1 antibody in urine

    MA Zhong-hui;CHEN Bing;CHANG Hao;PEI Li-jian;XING Wen-ge;National Center for AIDS/STD Control and Prevention,China CDC,National HIV/AIDS Reference Laboratory;

    Objective To prepare the internal quality control for HIV-1 antibody in urine and evaluate its homogeneity,stability and application so as to assurance the precision of laboratory test. Methods A HIV-1 positive urine sample was2-fold serially diluted with a HIV-1 negative urine sample and determined by ELISA kit for HIV-1 antibody in urine,and the dilution ratio of sample with low-reactivity was determined according to the S/CO value of test result,based on which the internal quality control for HIV-1 antibody in urine was prepared. The homogeneity and stability of the internal quality control in urine were evaluated by the detection kit for HIV-1 antibody in urine. The internal quality control was tested for20 times,and the results were analyzed by Levy-Jennings quality control chart. Results The dilution ratio of internal quality control in urine was 1∶4. In homogeneity test,the CV of results of 20 internal quality control was 15. 89%.However,in stability test,the CVs of results of the control after storage at 37 ℃,indoor temperature,2 ~ 8 ℃ and-20 ℃ were 8. 69%,17. 47%,18. 08% and 23. 04% respectively. The mean S/CO value in Levy-Jennings quality control chart was 5. 54,with a standard deviation of 0. 48. When the tests were carried out according to the instructions for use,the results were in the control. However,when some conditions were changed,partial results were in alarm status or out of control. Conclusion The prepared internal quality control in urine showed high stability and homogeneity,which might be used for the control of quality of laboratory test for HIV-1 in urine.

    2018 05 v.31 [Abstract][OnlineView][Download 289K]

  • Immunogenicity and safety of freeze-dried live attenuated varicella vaccine by a two-dose schedule in healthy children

    CHU Yan;CHAI Wen-qing;CHEN Dan-dan;Shanghai Institute of Biological Products Co.,Ltd.;

    Objective To evaluate the immunogenicity and safety of live attenuated varicella vaccine by a two-dose schedule in healthy children. Methods Healthy children at ages of 2 ~ 7 years,who have been inoculated with one dose of varicella vaccine,were inoculated with freeze-dried live attenuated varicella vaccine 1,3 and 5 years after primary immunization respectively. Serum samples were collected before as well as 4 weeks and one year after immunization and determined for antibody by ELISA,based on which the geometric mean concentration(GMC)and positive rate of antibody were calculated. Adverse reactions were observed after immunization,of which the incidence was calculated. Results In the children immunized with two doses of live attenuated varicella vaccine at an interval of one year,the antibody positive rates before,one month and one year after the second immunization were 73. 68%,98. 25% and 94. 74%,while the GMCs were 211. 27,201. 30 and 500. 15 m Iu/m L,respectively. In the children immunized at an interval of3 years,the antibody positive before,one month and one year after the second immunization were 65. 22%,98. 55% and89. 86%,while the GMCs were 182. 28,1 642. 35 and 365. 77 m Iu/m L,respectively. However,in the children immunized at an interval of 5 years,the antibody positive rates before,one month and one year after the second immunization were 71. 70%,100. 00% and 92. 45%,while the GMCs were 288. 99,1 923. 73 and 504. 93 m Iu/m L,respectively. In general,the GMCs and positive rates of antibody one month and one year after the second immunization in children immunized with two doses at various intervals showed significant difference with those before the second immunization(P < 0. 05). However,in the children immunized with two doses at an interval of 5 years,the GMC of antibody one year after the second immunization showed no significant difference with that before the second immunization(P > 0. 05). A total of 431 children were immunized,in whom systemic adverse reactions were observed in 74 ones,indicating a adverse reaction rate of 17. 17%,most of which were reactions of grade Ⅰ. Only one case of local reaction appeared,which was expressed as transiently local pain. Conclusion The two-dose immunization strategy of freeze-dried live attenuated varicella vaccine may enhance the protective effect of vaccine significantly,by which the protective rate one year after the second immunization reached about 90%. A booster immunization with varicella vaccine one year after primary immunization may be served as a optimal strategy.

    2018 05 v.31 [Abstract][OnlineView][Download 271K]

  • Evaluation on effect of sequential immunization with inactivated poliovirus vaccine and oral live attenuated poliomyelitis vaccine

    SHI Xiao-juan;LIU Lan;ZHOU Li-wei;ZHOU Lu-ping;Ningxia Hui Autonomous Region Center for Disease Control and Prevention;

    Objective To evaluate the effect of sequential immunization with domestic inactivated poliovirus vaccine and oral live attenuated poliomyelitis vaccine. Methods A non-randomized controlled experimental study was carried out.During the period from September to December 2015,180 healthy children at age of 2 months in Tongxin County,Ningxia Hui Autonomous Region,China were selected,and immunized with poliovirus vaccine at ages of 2,3 and 4 months according to the national immunization schedule. The children were divided into two groups,one for a immunization schedule of t OPV + t OPV + t OPV(O-O-O) while another one for IPV(Sabin) + t OPV + t OPV(I-O-O). The serum samples were collected before the first dose and 30 d after the third dose,and determined for antibody titers against poliovirus of types Ⅰ,Ⅱ and Ⅲ,based on which the GMT and protection rate were calculated. Results After 3 doses of vaccines were given,the antibody titers against poliovirus of typesⅡand Ⅲ in I-O-O group were higher than those in O-O-O group. However,high protective rates against the three types of poliovirus were observed in the two groups,which showed no significant difference(P > 0. 05). Conclusion Compared with full course of immunization with t OPV,the sequential immunization with domestic IPV(Sabin)and t OPV provided similar or even higher immune protection against poliovirus.

    2018 05 v.31 [Abstract][OnlineView][Download 162K]

  • Development of chemiluminescent microparticle immunoassay for cytokeratin 19 fragment 21-1 content

    WANG Ying-xue;GUO Xiao-yi;LIU Jiang-wu;WENG Zu-xing;GE Sheng-xiang;CHEN Yi-xin;State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics,School of Public Health,Xiamen University;

    Objective To develop a chemiluminescent microparticle immunoassay(CMIA) for cytokeratin 19 fragment21-1(CYFRA21-1,CY21-1 for short) content. Methods Recombinant CY21-1 antigen was prepared by prokaryotic expression and used for immunization of BALB/c mice. The splenocytes of mice were collected and fused with Sp2/0 cells by routine hybridoma technique. The hybridoma cells stably secreting the monoclonal antibody(m Ab)against CY21-1 were screened by indirect ELISA and labeled with magnetic beads(MB) and acridinium ester(AE) respectively. The m Ab pair for specific detection of CY21-1 were screened,based on which CY21-1 CMIA was developed,verified for linearity and sensitivity,and compared with the kits of same kind. Results An antibody pair MB*26 B5-3 E4*AE was obtained,with which CY21-1 CMIA was developed. The linear range of the developed method for determination of CY21-1 calibrator was 0. 1 ~ 1 000 ng/m L,with a R~2 value of 0. 992 3,while the sensitivity was 0. 076 ng/m L. The determination results of 250 human serum samples by the developed method showed good correlation with that by commercial Abbott CY21-1 CMIA kit(R~2 = 0. 961 6). Conclusion CY21-1 CMIA was developed successfully,which showed high linearity and sensitivity. It laid a foundation of clinical screening and early diagnosis of lung cancer in China.

    2018 05 v.31 [Abstract][OnlineView][Download 251K]

  • Identification and treatment of cell contamination caused by Pseudomonas luteola

    ZHU Jie;YE Meng-ling;LIU Ming-Jia;LI Ming-xuan;LIU Xu;School of Basic Medical Sciences,Wuhan University;

    Objective To identify the pathogens causing cell contamination in cell culture and screen antibiotics to eliminate the pathogen. Methods The supernatant of contaminated cells was collected and precipitated by centrifugation,from which nucleic acid was extracted and identified by PCR using universal primers of 16 S r DNA. The PCR products were inserted into vector p MD-18 T,and the positive colonies were identified by sequencing and classified by sequence alignment. According to the classification of cell pollutants,the corresponding antibiotics were further screened,of which the concentration was optimized so as to eliminate the pathogen without affecting the normal growth of the cells. Results The 16 s r DNA sequencing result showed that the cells were infected with Mycoplasma and Pseudomonas luteola. Antibiotic treatment result indicated that the contamination with Pseudomonas luteola was eliminated by 10 μg/m L ciprofloxacin,10 μg/m L meropenem and 10 μg/m L sulfamethoxazole. However,the growth function of cells after treatment was damaged,which was recovered to normality after culture and subculture for more than two passages under normal culture condition. Conclusion The combined use of ciprofloxacin,meropenem and sulfamethoxazole may eradicate the contamination of experimental cells with Pseudomonas luteola.

    2018 05 v.31 [Abstract][OnlineView][Download 349K]

  • Procedure for removal of endotoxin from pertussis toxin and filamentous hemagglutinin

    JI Qiu-yan;GAO Na;LIANG Jiang-li;HU Wen-zhu;GU Qin;MA Yan;DAI Yong-juan;LI Jing-yan;SHI Li;SUN Ming-bo;HENG Xie;YANG Hui-juan;Institute of Medical Biology, Chinese Academy of Medical Sciences and Peking Union Medical College;

    Objective To investigate the removal of endotoxin from pertussis toxin(PT) and filamentous hemagglutinin(FHA). Methods The endotoxin in fermentation supernatant and fermentation liquid purified by Capto SP Imp Res column chromatography were removed by using Triton and IEX separately. According to the requirements in Chinese Pharmacopoeia(Volume Ⅲ, 2015 edition), the purified samples were determined for the contents of protein, endotoxin and antigen, and analyzed by SDS-PAGE. Results Both Triton and IEX methods decreased the endotoxin content in FHA and PT before the stage of detoxification to 200 EU/mg below. However, under the suitable condition for purification, the ability of IEX in removal of endotoxin was superior to that of Triton, while the ability of Triton method in recovery of antigen was superior to that of IEX. Conclusion Both the methods may solve the problem of endotoxin removal from PT and FHA effectively. However, the effect of two procedures on protein activity needs to be further studied.

    2018 05 v.31 [Abstract][OnlineView][Download 221K]

  • Purification of rabies vaccine by using complex purification medium Capto Core700

    LI Xu;XU Wei;YU Hai;CHEN Zhi-cheng;Liaoning Chengda Biological Co., Ltd.;

    Objective To remove the foreign matters such as residual Vero cell host protein and host cell DNA from rabies vaccine by using Capto Core700. Methods Vero cells were inoculated onto microcarriers and incubated in celligen plus bioreactor, to which rabies virus was inoculated at MOI of 0. 002. Ten consecutive batches of bulk of rabies vaccine were prepared, filtrated, concentrated, and inactivated with β-propiolactone at 2 ~ 8 ℃ for 24 h, then purified with complex media Capto Core700. The purified virus liquid was determined for removal rates of host cell protein and host cell DNA,recovery rate of rabies virus glycoprotein and protein removal rate according to the requirements in Chinese Pharmacopeia(VolumeⅢ, 2015 edition). Results The removal rates of host cell protein in ten batches of purified vaccine were 58. 3% ~63. 2%, while those of host cell DNA were more than 90%, the recovery rates of glycoprotein were 52. 3% ~ 74. 5%, and the protein removal rates were 7. 22% ~ 11. 76%, which showed no significant difference with those of vaccine purified by traditional Sepharose gel chromatography. However, the sample load for purification with Capto Core700 was 3 times of column volume, which was larger than that by Sepharose gel chromatography(15%). Conclusion The foreign matters in rabies vaccine was effectively removed by using Capto Core700 gel, while the time for purification was shortened.

    2018 05 v.31 [Abstract][OnlineView][Download 235K]

  • Development and verification of a PCR assay for porcine circovirus in vaccines for human use and cells used for production

    QIAN Xing-li;REN Fang-fang;SONG Cai-hua;DUAN Nan;LI Ya-xian;CHEN Wei;ZHAO Yong;YUAN Mei;LI Ya-dong;YU Jian-kun;YANG Xiao-lei;HONG Chao;Laboratory of Quality Control, Institute of Medical Biology, Chinese Academy of Medical Science and Peking Union Medical College, Yunnan Key Laboratory of Vaccine Research & Development on Severe Infectious Disease;

    Objective To develop and verify a PCR assay for porcine circovirus types 1(PCV1) and 2(PCV2) in vaccines for human use as well as in the cell lines used for production. Methods PCV1 and PCV2 gene sequences were synthesized according to the PCV1(KC447455)and PCV2(AY578327)sequences in Gen Bank and used as the templates for PCR. The temperature for annealing in PCR assay was optimized. The optimized PCR assay was verified for sensitivity and specificity, and used for tests for PCV1 and PCV2 in KMB_(17) cells for vaccine production, working cell banks of Vero cells, virus harvests of oral poliomyelitis vaccine(OPV), final product of freeze-dried hepatitis A vaccine(f HAV), harvest and final product of enterovirus 71(EV71)vaccine as well as harvest and final product of inactivated poliomyelitis vaccine prepared with Sabin strain(SIPV). Results The optimal temperature for annealing was 63 ℃. The minimum detection limit of the developed PCR assay was 10~(-1) fg/μL. Specific DNA bands were amplified by the developed PCR assay only from the genomes of PCV1 and PCV2. No PCV1 and PCV2 DNAs were found in the KMB_(17) cells for vaccine production,working cell banks of Vero cells, virus harvests of OPV, final product of f HAV, harvest and final product of EV71 vaccine as well as harvest and final product of SIPV. Conclusion A highly sensitive and specific PCR assay for PCV1 and PCV2 in vaccines for human use and cell lines for production was developed, which might be used for test for PCV1 and PCV2 in the working cell banks, virus harvest and final product and beneficial to the improvement of safety of biological products.

    2018 05 v.31 [Abstract][OnlineView][Download 293K]

  • Culture of Vero cells and influenza H1N1 virus in bioreactor

    ZHOU Jian;LI Guo-liang;SONG Shao-hui;OUYANG Sheng-jie;LIU Ze;DAI Yong-juan;LIAO Guo-yang;LI Wei-dong;Institute of Medical Biology,Chinese Academy of Medical Science & Peking Union Medical College;

    Objective To develop a new process for preparation of inactivated influenza H1 N1 vaccine by culture of influenza H1 N1 virus in Vero cells using a bioreactor. Methods Vero cells as a cellular matrix for proliferation of influenza virus were cultured in serum-free medium Pro VERO and serum-containing DMEM respectively in a 3 L bioreactor using5 g/L Cytodex-1 microcarriers at a temperature of(37 ± 0. 5) ℃,a dissolved oxygen concentration of(50 ± 20) %,a p H value of(7. 2 ± 0. 2)and a stirring rate of(50 ± 20)r/min. Vero cells-adapted high-producing influenza virus H1 N1/JD/Va strain was inoculated onto Vero cells at a MOI of 0. 1 after a monolayer was formed on microcarriers. The harvested virus liquid was purified by Sepharose 4 FF and Capto Q chromatography and inactivated to prepare the bulk and final product of vaccine which was subjected to control tests according to the requirements in Chinese Pharmacopoeia(Volume Ⅲ,2015 edition). Results The adherence rate of Vero cells cultured in serum-free medium for 24 h was about83%,while that in serum-containing medium was 112%. All the cells on day 6 after perfusion culture formed a monolayer. The final counts of cells in serum-free medium was(133. 4 ± 2. 0) × 10~4 cells/m L,while that in serumcontaining medium was(353. 4 ± 5. 9)× 10~4 cells/m L. The hemagglutination titer and virus production ratio in unit cells of virus after culture in serum-free maintenance medium for 66 h were 388 and 2. 9,while those in serum-containing maintenance medium were 891 and 2. 5,respectively. All the quality indexes of bulk and final product of vaccine prepared with the virus liquid from Vero cells culture in serum-containing medium met the requirements in Chinese Pharmacopoeia(Volume Ⅲ,2015 edition). Conclusion A new process for culture of Vero cells and influenza H1 N1 virus in serum-containing medium by using 3 L bioreactor was developed,which laid a foundation of further scale-up of production scale.

    2018 05 v.31 [Abstract][OnlineView][Download 217K]

  • Development of purification process for recombinant oncolytic herpes simplex virus type Ⅱ

    WU Zhen;ZHANG Zi-yi;WANG Yang;HU Han;SHI Xiao-tai;MAO Ze-yong;FANG Zhi-zheng;LIU Bin-lei;Hubei University of Technology;Wuhan Binhui Bioscience and Technology Co.,Ltd.;

    Objective To develop a purification process for recombinant herpes simplex virus type Ⅱ(oncolytic herpes simplex virus typeⅡ,OH2)and prepare the virus with high purity. Methods Filters of different materials were applied,including PDH4 cystic filter,Bio20 cystic filter,Ultipor GF U6-40,polypropylene(PP) filter membrane and cellulose acetate(CA)filter membrane. The filtered liquid was purified by different chromatography columns,including Mustang Q column,Sartobind Q nested ion exchange column and Capto~(TM) Core 700 column. The volume of chromatographic column applied was further increased according to the purification process. The condition for purification was optimized based on the change of virus titer. Results The virus titers after purification by PP and CA membranes showed slight change,indicating low virus adsorption ability of the two materials. The recovery rate of OH2 virus purified by Mustang Q column and Sartobind Q capsule type ion exchange column was 13% at most. However,the application of Capto~(TM) Core 700 in AKTA instrument almost caused no loss of virus during the purification process. The optimal diameter of Capto~(TM)Core 700 column was 26 mm. Under the conditions mentioned above,the titer and recovery rate of virus were satisfactory.Conclusion The OH2 at high purity was obtained by filter of cell lysate by CA filter membrane and purification by Capto~(TM)Core 700 column chromatography.

    2018 05 v.31 [Abstract][OnlineView][Download 166K]

  • Progress in research on technique for analysis of epitopes of monoclonal antibody

    ZHAO Xiao-rui;MAO Xiao-yan;Lanzhou Institute of Biological Products Co.,Ltd.;

    With high homogeneity,affinity and specificity,monoclonal antibodies have been widely used for the therapy of autoimmune disorders,inflammatory diseases and cancer. The antigenic epitope recognized by the monoclonal antibody determines its reactivity with the corresponding antigen,so the determination of location of epitopes on the antigen structure is the key to the screening of monoclonal antibodies. This paper reviews the principle,characters as well as progress in research of several methods for analysis of epitopes of monoclonal antibody,including ELISA,enzymatic hydrolysis,chemical cleavage,phage random peptide library,X-ray crystallography,alanine scanning,hydrogen deuterium exchange mass spectrometry(HDX-MS)and bioinformatics tools for epitope prediction.

    2018 05 v.31 [Abstract][OnlineView][Download 283K]

  • Application of nanobodies in infectious diseases

    WANG Zhi-ming;YANG Li-xia;NCPC New Drug Research and Development Co.,Ltd.,State Key Laboratory of Antibody Drug Development;

    Given the unique molecular characteristics,nanobodies are amenable to many applicability as diagnostic or therapeutic tools. These nanobodies have been exploited in the field of infectious diseases:the nanobodies of anti-virus,anti-bacteria,anti-parasites and detoxifying agents are in different stages of development. The key activities of the nanobodies targeting the pathogen surface targets are engagement of the host immune system through complement fixation and opsonophagocytic killing(OPK),and neutralizing the toxin produced by the pathogen,which significantly reduce the damage to the structure and function of host cells,and reduce the severity of infection. The rapid development of antibody engineering technology promotes the exploration and new attempts in anti-infectious nanobodies. Two nanobody drugs have been in the late phase of clinical trials,of which the relevant results need to be further observed. Nanobodies may play a role in the treatment of infectious diseases with the promoting of research progress in the future.

    2018 05 v.31 [Abstract][OnlineView][Download 241K]
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@2012 Editorial Department of "Chinese Journal of Biologicals"