2017年02期目次

  • Effect of alendronate as an oral adjuvant on immunogenicity of M. RCAg-1, a multi-epitope protein vaccine against Plasmodium falciparum

    ZHANG Jia-jia;DENG Wei-wei;GAO Yu-hui;WANG Heng;Department of Microbiology and Parasitology, Institute of Basic Medical Sciences,Chinese Academy of Medical Sciences;

    Objective To evaluate the effect of alendronate(ALD)as an oral adjuvant on immunogenicity of M. RCAg-1,a multi-epitope protein vaccine against Plasmodium falciparum. Methods BALB / c mice were immunized with ALD at various dosages(0. 05, 0. 1 and 1 mg) plus M. RCAg-1. M. RCAg-1 was injected subcutaneously at a dosage of 20 μl,while ALD was administered by oral route. The mice were immunized for 3 times, each at an interval of 14 d. Serum samples were collected 10 d after the last immunization, and determined for antibody titer, Ig G antibody subtype and recognition of single epitopes by serum antibody by ELISA, for recognition of natural P. falciparum protein with serum antibody by IFA, and for cytokine level by ELISPOT. Results The GMTs of serum antibody of mice immunized with0. 05 mg ALD + M. RCAg-1 and 0. 1 mg ALD + M. RCAg-1 reached 1 ∶ 10~8. The Ig G antibodies were mainly Ig G1. All the 11 single epitopes(E1 ~ E11)of M. RCAg-1 were recognized. The numbers of specific T lymphocyte clones secreting IL-4, induced by various epitope antigens, except E2,were significantly larger than those secreting IFNγ(P < 0. 000 1).The recognition level of natural P. falciparum protein by serum antibody of mice immunized with 0. 1 mg ALD + M. RCAg-1was significantly higher than that with 0. 05 mg ALD + M. RCAg-1(P < 0. 000 1). Conclusion As an adjuvant of M. RCAg-1, ALD enhanced the humoral and cellular immunities significantly.

    2017 02 v.30 [Abstract][OnlineView][Download 287K]

  • Prokaryotic expression of recombinant Staphylococcus aureus enterotoxin B and heat shock protein 65 fusion protein and its immune effect in mice

    HOU Tian-quan;ZHANG Guo-li;TENG Li-rong;YUE Yu-huan;WU Guan-mou;TIAN Yuan;FU Yu-he;ZHAO Xin;ZHANG Pei-pei;College of Life Sciences, Jilin University;

    Objective To express recombinant Staphylococcus aureus enteroxtoxin B(r SEB) and heat shock protein 65(HSP65)fusion protein in prokaryotic cells and investigate its humoral immune effect in mice. Methods Recombinant plasmid p ET-r SEB-HSP65 was constructed by fusion of a SEB gene in which TCR Vβ binding region was modified with HSP65 gene by molecular biological technique, and transformed to E. coli BL21(DE3) for expression under induction of IPTG. The expressed product was purified by affinity chromatography, with which mice were injected s.c. on days 0,14 and 28. Serum samples were collected on days 14, 28 and 42, and determined for anti-SEB Ig G by indirect ELISA.Results Restriction analysis and PCR proved that recombinant plasmid p ET-28a-r SEB-HSP65 was constructed correctly.The expressed r SEB-HSP65, with a relative molecular mass of about 85 000, mainly existed in a form of inclusion body,contained about 40. 81% of total somatic protein, and reached a purity of about 90% after purification. High antibody titers were induced in mice in various groups. Most of the GMTs of mice on days 14, 28 and 42 after immunization with aluminum adjuvant-containing vaccine were significantly higher than those with aluminum adjuvant-free vaccine at the same dosages(each P < 0. 05). However, the GMTs of mice 42 d after immunization with aluminum adjuvant-containing vaccine at a low dosage showed no significant difference with those with aluminum adjuvant-free vaccine at the samedosage(P > 0. 01). Conclusion Recombinant vaccine r SEB-HSP65 was successfully expressed in E. coli BL21(DE3),which induced high antibody level in mice.

    2017 02 v.30 [Abstract][OnlineView][Download 344K]

  • Secretory expression of recombinant human pepsinogen Ⅰ in Hansenula polymorpha as well as purification and application of expressed product

    CHEN Dan;LI Lu;BAN Jing-yang;GUO Yu-zheng;WANG Wei-long;LIU Juan;LI Ding-feng;LIU Yong;Beijing ABZYMO Biosciences Co., Ltd.;

    Objective To achieve high-level secretory expression of recombinant human pepsinogenⅠ(PGⅠ)in Hansenula polymorpha and prepare the purified recombinant PG Ⅰ into a calibrator for in vitro diagnostic reagents. Methods According to the H. polymorpha-prefered codon, PG Ⅰ gene was designed, synthesized and cloned into expression vector p RMHP2. 1. The constructed recombinant plasmid was transformed to H. polymorpha 26012 by electroporation for several rounds of passage and stabilization, and the strain in which PG Ⅰwas highly expressed in a secretory form was screened and identified for the integrated site and copy number of heterologous gene. Fermentation liquid was prepared in a 200 L fermenter, from which recombinant PG Ⅰprotein was purified by nickel ion affinity chromatography and determined by a latex-enhanced immunoturbidimetric kit. The purified PG Ⅰprotein was prepared into calibrators at concentrations of 100 and 50 μg / L respectively and tested for stability. Results Restriction analysis and DNA sequencing proved that recombinant plasmid p RMHP2. 1-PGⅠwas constructed correctly. After rounds of screening, a recombinant H. polymorpha strain was obtained, in which PG Ⅰ protein with a relative molecular mass of about 45 000 was expressed. The yield of expressed product was more than 100 mg / L, while PG Ⅰ gene of was integrated into the host r DNA site, and the gene copy number was not less than 20. The purity of purified recombinant PG Ⅰ protein was 94. 5%. The mean degradation limit of active ingredient contents of PGⅠ as a calibrator in accelerated stability, real time stability and on-board stability tests were not more than 10%. Conclusion Recombinant PG Ⅰ protein was highly expressed in a secretory from in H. polymorpha, which showed high stability and might be used as a calibrator for in vitro diagnostic reagents.

    2017 02 v.30 [Abstract][OnlineView][Download 387K]

  • Effect of LTD protein on cytotoxicity of He La cells in vitro

    WANG Wei-fang;LIU Ming-yuan;WU Guang-mou;WU Xiu-ping;Changchun University of Chinese Medicine;

    Objective To investigate the effect of LTD protein on cytotoxicity of He La cells in vitro. Methods He La cells were treated with LTD protein at various concentrations, observed for morphological change by optical microscopy and for location of protein by laser confocal microscopy. The cell prolferaton was determined by MTT assay, while the apoptosis by HE staining and TUNNEL method. Results LTD protein showd toxicity to He La cells, which inhibited the cell proliferation and induced the cell apoposis. With the increasing protein concentration and time duration for treatment,the apoptosis and growth inhibition phenomenons of He La cells were more and more obvious, and the protein con-centration was significantly related to the A_(490) value of cells. However, the location of LTD protein changed from the cell membrane to the cytoplasm and nucleus. Conclusion LTD protein promoted the apoptosis and inhibited the proliferation of He La cells.

    2017 02 v.30 [Abstract][OnlineView][Download 276K]

  • Construction of attenuated Salmonella TPHK carrying hypoxia inducible factor-1α and keratinocyte growth factor genes and expression of the two genes in gastrointestinal tissue of rats with hypoxia stress

    BAI Yan-qing;XU Qian;ZENG Tong-xu;CAO Hui-zhe;HA Xiao-qin;Provincial-Level Key Laboratory for Molecular Medicine of Major Diseases and the Prevention and Treatment with Traditional Chinese Medicine Research in Gansu Colleges and Universities, Gansu University of Traditional Chinese Medicine;

    Objective To construct an attenuated Salmonella TPHK carrying hypoxia inducible factor(HIF)-α and keratinocyte growth factor(KGF) genes, and determine the expression of the two genes in gastrointestinal tissue of rats with hypoxia stress. Methods KGF gene was amplified from plasmid p IRES-KGF by PCR, digested with XbaⅠ and NotⅠ,and inserted into vector p IRES-HIF. The obtained plasmid p IRES-HIF-KGF was transformed to attenuated Salmonella Ty21 a by calcium chloride method. Rats were treated with the constructed recombinant Salmonella strain TPHK by gavage,109 cfu for each daily, for 7 d, and transferred to the hypoxia cabin with mimic environment of plateau at a height of 6 000 m above sea level and further raised for 7 d. The gastrointestinal tissues of rats were collected and determined for the expression levels of HIF-1α and KGF by ELISA. Results Restriction analysis and sequencing proved that recombinant plasmid p IRES-HIF-KGF and recombinant Salmonella TPHK were constructed correctly. The expression levels of HIF-1αand KGF in gastrointestinal tissues of rats increased significantly via gavage with TPHK strain(P < 0. 001). Conclusion An attenuated Salmonella TPHK strain carrying HIF-1α and KGF genes was successfully constructed, which increased the expression levels of the two genes in gastrointestinal tissue of rats with hypoxia stress.

    2017 02 v.30 [Abstract][OnlineView][Download 191K]

  • Effect of various signal peptides on expression of recombinant serogroup B Neisseria meningitidis factor H-binding protein

    GONG Yue;CHEN Chen;LI Ya-dong;ZHANG Hui;LI Zhi-hua;XIAO Hong-jian;BI Yan-wei;CUN Wei;Institute of Medical Biology, Chinese Academy of Medical Sciences & Peking Union Medical College,Yunnan Key Laboratory of Vaccine Research and Development on Severe Infectious Disease;

    Objective To investigate the effect of various signal peptides on expression of factor H-binding protein(f HBP)of serogroup B Neisseria meningitides. Methods The original signal peptide of f HBP of serogroup B N. meningitides was substituted with the major outer membrane lipoprotein(MLP), lipoprotein-28(LP) and P4 outer membrane protein of Haemophilus influenzae by PCR respectively, and inserted into vector p ET-30 a. The constructed recombinant plasmids were transformed to competent E. coli BL21(DE3)for expression under induction of IPTG. The expression levels of target proteins were determined by SDS-PAGE. Results The expression levels of f HBP of serogroup B N. meningitides in which original signal peptide were substituted with P4, LP and MLP increased by 2. 68, 1. 94 and 2. 91 folds compared with that before substitution, respectively. Conclusion MLP showed significant effect on the expression of f HBP of recombinant N. meningitides, which provided a novel route for study on increasing the expression level of f HBP.

    2017 02 v.30 [Abstract][OnlineView][Download 336K]

  • Effect of Flc A in Azospirillum brasilense Sp7 on thickness of cell wall

    ZHANG Yuan-yuan;LIU Shuai;CUI Jia-bin;WANG Wei;HOU Xing-sheng;WANG Gui-qin;Department of Microbiology and Immunology.Shanxi Medical University;

    Objective To evaluate the effect of Flc A in Azospirillum brasilense Sp7 on the thickness of cell wall.Methods Wild type A. brasilense strain Sp7, Sp7-flc AΔ strain with Flc A gene deleted and Sp72001 strain with Tn5 gene inserted were cultured in nutrient broth(NB)medium and flocculation medium separately, observed for cell morphology by transmission electron microscopy, and measured for thickness of cell wall, based on which the effects of Flc A on bacterial appearance and thickness of cell wall were evaluated. Results The thicknesses of cell walls in Sp7, Sp7-flc AΔ and Sp72001 strains in NB medium were(34. 13 ± 12. 47),(29. 94 ± 9. 25) and(34. 90 ± 14. 88) nm respectively, which showed no significant difference(P > 0. 05). However, in flocculation medium, the thicknesses were(75. 03 ± 17. 67),(27. 00 ± 12. 17)and(42. 22 ± 15. 54)nm respectively, which showed significant difference(P < 0. 001). Meanwhile,the thicknesses of cell walls of the same strain in two media showed significant difference(P < 0. 05). Conclusion The cell wall in Sp7-flc AΔ strain in flocculation medium was significantly thinner than that in Sp7 strain, indicating that Flc A was involved in cell wall development.

    2017 02 v.30 [Abstract][OnlineView][Download 304K]

  • Effect of Musashi2 on invasion potential of leukemia K562 cells in vitro

    SHAO Hui-yuan;MIAO Zong-yu;ZHANG Zheng-fang;ZHANG Gui-li;SUN Cheng-ming;Medical Laboratory Center, Yantai Yu Huang Ding Hospital of Qingdao University;

    Objective To investigate the effect of Musashi2(Msi2)on invasion potential of leukemia K562 cells in vitro.Methods K562 cells were transfected with the si-RNA1, si-RNA2, targeting Msi2 gene, and the negative control sequence respectively, using those untransfected as blank control. The m RNA transcription and protein expression levels of Msi2 in K562 cells were determined by real-time fluorescent quantitative PCR and Western blot respectively. The proliferation potential in vitro of K562 cells was observed by using cell growth curve, while the adhesion ability in vitro by cell adhesion assay, and the migration and invasion potential in vitro by Transwell assay. Results Compared with those in negative and blank control groups, scramble and K562 groups, both the m RNA transcription and protein expression levels of Msi2, as well as the adhesion, migration and invasion abilities in vitro of K562 cells in Msi2-1 and Msi2-2 groups decreased significantly(each P < 0. 05). Conclusion The inhibition of Msi2 expression inhibited the invasion in vitro of K562 cells signifiantly, which laid a foundation of further study on role of Msi2 in regulation of pathogenesis and progress of leukemia.

    2017 02 v.30 [Abstract][OnlineView][Download 220K]

  • Effect of proteasome inhibitor MG132 on proliferation of He La cells

    SHI Min;WEI Wei;SUN Yue;SONG Ling;KONG Fan-dou;ZHAO Chun-yan;College of Laboratory Medicine, Dalian Medical University;

    Objective To investigate the effect of proteasome inhibitor MG132 on proliferation of He La cells. Methods He La cells were treated with MG132 at various concentrations(1, 3, 5, 8 and 10 μmol / L), and determined for survival by MTT assay, based on which a cell growth curve was plotted, and the effect of MG132 on cell proliferation was observed.Results The MG132 at a concentration of 1 μmol / L showed no inhibitory effect on He La cells(P > 0. 05). However,when the MG132 concentration was not less than 3 μmol / L, the inhibitory effect was enhanced with the increasing MG132 concentration(P < 0. 05). The inhibitory effect was not enhanced when the MG132 concentration was not less than 8 μmol / L. Conclusion MG132 showed inhibitory effect on the proliferation of He La cells. This study provided an experimental basis for development of novel drugs based on ubiquitin proteasome pathway.

    2017 02 v.30 [Abstract][OnlineView][Download 141K]

  • Effect of astragalus polysaccharide on expression of proprotein convertase subtilisin/kexin type 9 in THP-1 cells

    ZOU Hong-yu;LIU Zeng-zhang;Department of Cardiology,The Second Affiliated Hospital of Chongqing Medical University;

    Objective To investigate the effect of astragalus polysaccharide(APS)on the expression of proprotein convertase subtilisin / kexin type 9(PCSK9)in THP-1 cells as well as the relevant mechanism. Methods THP-1 was differentiated into THP-1-derived macrophages by induction with TPA. The effect of APS at various concentrations on proliferative activity of THP-1-derived macrophages was determined by CCK-8 assay,based on which the APS concentration was optimized. THP-1-derived macrophages were divided into five groups. The macrophages in four test groups were treated with 30 μg low density lipoprotein(LDL),LDL(30 μg / ml) + APS(400 μg / ml),LDL(30 μg / ml) + GW0742(1 μmol)[an activator of peroxisome proliferator-activated receptor β / δ(PPAR-β / δ)],LDL(30 μg / ml)+ GSK0660(1 μmol,an inhibitor of PPAR-β / δ)+ APS(400 μg / ml)respectively,while those in control group were untreated. The expressions of PCSK9,LDL receptor(LDLR) and PPAR-β / δ at gene and protein levels in macrophages of various groups were determined by RT-PCR and Western blot respectively 24 h after treatment. Results The optimal APS concentration for treatment of THP-1-derived macrophages was 400 μg / ml. Both the expression levels of PCSK9 gene and protein were significantly higher in LDL group than in control group(P < 0. 01),while was significantly lower in LDL +APS and LDL + GW0742 groups than in LDL group(P < 0. 01),and significantly higher in LDL + GSK0660 + APS group than in LDL + APS and LDL + GW0742 groups(P < 0. 01). However,the expression levels of LDLR gene in LDL,LDL + APS,LDL + GW0742 and LDL + GSK0660 + APS groups were significantly lower than that in control group(P < 0. 01). The expression level of LDLR protein was lower in LDL group than in control group(P < 0. 01),higher in LDL + APS and LDL + GW0742 groups than in LDL group(P < 0. 01),and significantly lower in LDL + GSK0660 +APS group than in LDL + APS and LDL + GW0742 groups(P < 0. 01). The expression levels of PPAR-β / δ gene and protein were significantly higher in LDL + APS and LDL + GW0742 groups than in control group(P < 0. 01),while were significantly lower in LDL + GSK0660 + APS group than in LDL + APS 和 LDL + GW0742 groups(P < 0. 01).Conclusion APS down-regulated the expression of PCSK9 in THP-1-derived macrophages by a possible PPAR-β / δroute.

    2017 02 v.30 [Abstract][OnlineView][Download 291K]

  • Preparation and identification of monoclonal antibody against 1-amino-hydantion

    WANG Wei-ping;ZHAO Yun;LIU Ai-chun;CHEN Fei-dong;HU Ye-jun;YE Cong-ying;Hangzhou Nankai Biotech Co.Ltd.;

    Objective To prepare and identify the monoclonal antibody(Mc Ab) against 1-amino-hydantion(AHD).Methods The 4-CPAHD, a derivative of AHD, was synthesized by using 4-CBA, and covalently attached to bovine serum albumin(BSA)and ovalbumin(OVA)by modified active ester method to obtain antigens 4-CPAHD-BSA and 4-CPAHDOVA for immunization and coating, respectively. The coupled product was confirmed by ultraviolet spectrophotometry.BALB / c mice were immunized with the synthetic antigen 4-CPAHD-BSA, of which the splenocytes were fused with Sp2 / 0cells by hybridoma technique, and the cell strains stably secreting Mc Ab were screened. The titer of secreted Mc Ab and the median inhibitory concentration(IC50) of 2-NPAHD to the Mc Ab were determined by indirect ELISA, and the specificity of Mc Ab was analyzed. Results The maximum absorption peak of 4-CPAHD-BSAs showed significant shift after coupling, indicating that 4-CPAHD was conjugated with BSA successfully. The mean coupling ratio was 17. 4 ∶ 1,while the titer of prepared Mc Ab was 1 ∶ 64 000, and the IC50 of 2-NPAHD to the Mc Ab was 1. 45 μg / L. The Mc Ab showed no cross reaction to the antibiotics of the same kind or other metabolites. Conclusion All the indexes of prepared Mc Ab against AHD met the relevant requirements, which laid a foundation of development of immunoassay kit for nitrofurantoin metabolites.

    2017 02 v.30 [Abstract][OnlineView][Download 213K]

  • Enterovirus DNA test and etiological analysis of patients with hand-foot-mouth disease in Yantai City, Shandong Province,China during 2010 2015

    SUN Zhen-lu;GONG Lian-feng;HAN Wen-qing;LIU Juan;XU Xiao-wen;LIU Jun;WANG Zhi-yu;GAO Qiao;ZHANG Cai-ji;Yantai Municipal Center for Disease Control and Prevention;

    Objective To perform enterovirus(EV) DNA test and etiological analysis on the patients with hand-footmouth disease(HFMD) in Yantai City, Shandong Province, China during 2010 ~ 2015. Methods Throat swabs were collected from five common patients with HFMD who visited the medical institutions for the first time in each month, from all the patients with severe HFMD and from the patients who died from the disease in Yantai City during 2010 ~ 2015,and tested for EV DNA by RT-PCR. Results The annual average incidence rate of HFMD in Yantai City during 2010 ~2015 was 68. 68 / 100 000, of which the severe cases accounted for 0. 36%. The ratio of male to female patients was1. 81 ∶ 1. A portion of 91. 22% of the cases appeared in the children at ages of less than 5 years. The peak incidence of the disease appeared from May to August every year. There were significant differences in the composition of pathogen spectrum(P < 0. 01). Of the cases, 769(30. 95%)were caused by EV71, while 814(32. 76%)by coxsa-ckievirus A16(Cox A16), and 902(36. 30%)by other EV types. The main pathogens of HFMD in Yantai were EV71 and Cox A16.However, 52. 54% of severe cases were caused by EV71. Conclusion EV71, Cox A16 and EV of other types were the dominant pathogens of HFMD in Yantai during 2010 ~ 2015. The incidences of HFMD were significantly different in various seasons and in the population of both genders and at various ages. However, the main pathogen of severe cases was EV71 on which the monitoring should be strengthened. HFMD prevention and control work should focus on the children in kindergarten and lower grade pupils.

    2017 02 v.30 [Abstract][OnlineView][Download 196K]

  • Analysis on surveillance result of adverse events following immunization in Ninghe District, Tianjin City during 2005 2014

    GAO Hong-min;CHANG Li-min;Xingang Community Health Service Center of Tanggu District;

    Objective To analyze the characteristics of adverse events following immunization(AEFI)in Ninghe District,Tianjin City, evaluate the operation of AEFI monitoring system and investigate the measure to improve the monitoring quality. Methods The data on 434 cases of AEFI reported from 2005 to 2014 through the China AEFI Information Management System were collected and subjected to epidemiological analysis by descriptive method. Results The 434 cases were distributed in 19 vaccination sites, while the reported coverage was 100%. Of the 434 cases, 335 were common reactions, 93 were abnormal reactions, 5 were coincidental events, and 1 case was psychogenic reaction. Most(94. 9%)of the reported cases occurred 0 ~ 3 d after vaccination. The sex ratio in the cases was 1. 3 ∶ 1. A portion of 70. 7% of cases occurred in the children at ages of 0 ~ 1 year. The reported cases were involved in 22 kinds of vaccines, in which those involved in acellular DTP, measles and hepatitis B(yeast) vaccines accounted for 35. 5%, 12. 7% and 10. 4% respectively. Fever / redness and swelling accounted for 71. 2% of the AEFI, seconded by allergic skin rash. All the 434 cases were recovered. Conclusion The AEFI surveillance system in Ninghe District worked well, while the vaccination was safe and reliable.

    2017 02 v.30 [Abstract][OnlineView][Download 151K]

  • Epidemiological characteristics of rabies-exposed population in Xingyang City, Hubei Province, China during 2013 2015

    BAO Chun;SI Qian;Department of Prevention and Health Care, The Traditional Chinese Medical Hospital of Xiangyang City;

    Objective To analyze the epidemiological characteristics of rabies-exposed population in Department of Prevention and Health Care, The Traditional Chinese Medical Hospital of Xiangyang City, Hubei Province, China during2013 ~ 2015. Methods The data of 5 855 rabies-exposed cases were collected from 2013 to 2015 and analyzed by descriptive epidemiological methods. Based on the Protocol for Prevention and Treatment of Rabies Exposure issued by the Ministry of Health, the wounds were classified and treated with rabies vaccine and human rabies immunoglobulin accordingly. Results Among the 5 855 rabies-exposed cases, the ratio of male to female victims was 0. 98 ∶ 1. The numbers of cases from rural areas in 2013, 2014 and 2015 were 1 018, 1 085 and 1 287, while those from urban were624, 787 and 1 054, respectively, both of which showed increasing tendencies year by year. A total of 3 239 cases appeared in spring and summer, while 2 616 cases in autumn and winter. A portion of 20. 03%, 28. 9% and 21. 6% of cases in 2013, 2014 and 2015 occurred in the population at ages of 0 ~ 10 years, while 22. 3%, 18.1% and 19. 3% in those at ages of 41 ~ 50 years. All the cases were followed-up for 1 year, and no rabies occurred. Conclusion Spring and summer are the risk seasons for rabies exposure, in which the cats, dogs and other animals should be carefully under control, and children should be cared well especially. If exposure happened, the wounds should be treated thoroughly,regularly and timely, and adequate human rabies immunoglobulin and rabies vaccine should be used according to the class of wounds to effectively prevent the occurrence of rabies.

    2017 02 v.30 [Abstract][OnlineView][Download 142K]

  • Development of pilot purification procedure of monoclonal antibody against vascular endothelial growth factor

    YANG Hui;ZHOU Zhang-zhang;MA Xu-tong;LIN Xiao-que;SUN Wen-zheng;YANG Bin;Sunshine Lake Pharma Co.,Ltd.;

    Objective To optimize the condition for small scale purification of monoclonal antibody(m Ab) against vascular endothelial growth factor(VEGF) so as to develop a pilot purification procedure. Methods Harvested cell culture fluid(HCCP) containing crude m Abs were purified by protein A affinity chromatography followed by a cationexchange chromatography step. The effects of p H value and salt concentration for elution as well as sample loading on antibody quality in harvested liquid were investigated by single factor test. The purified m Ab was determined for concen-tration by protein A-HPLC, for purity by high performance capillary electrophoresis, and for polymer content by SECHPLC. Results The optimal p H value for elution in protein A affinity chromatography in small scale was 3. 8. For cation-exchange chromatography, the optimal sample loading was 40 mg / ml, while the optimal p H value for elution was 6. 5, and the optimal salt solution for elution was 25 mmol / L PBS containing 0. 10 mol / L sodium chloride. These optimal conditions were successfully scaled up to the pilot procedure. The purity of purified m Ab was(98. 3 ± 0. 2)%, while the polymer content was(1. 6 ± 0. 3)%, which were superior to those of reference preparation. Conclusion The key quality indexes of m Ab against VEGF purified by affinity chromatography + cation-exchange chromatography were consistent with those of reference preparation, which provided an experimental basis for scale-up of production procedure of the m Ab.

    2017 02 v.30 [Abstract][OnlineView][Download 249K]

  • Determination and analysis of titer of coagulation factor Ⅷ in manufacturing process

    LI Qing;LIU Xiao;WEN Yuan;DOU Zi;ZHANG An-shan;ZHANG Jin;HE Yan-lin;Lanzhou Institute of Biological Products Co., Ltd., Center for Gansu Provincial Vaccine Engineering Research;

    Objective To determinate the titer coagulation factor Ⅷ(FⅧ)in each critical stage of manufacturing process and calculate t he recovery rate. Methods One stage-assay was used to determine the titer of F Ⅷ in material plasma,solution of cryoprecipitate, solution of chemical precipitate, solution after virus inactivation by S / D method, eluate of chromatography, bulk obtained after ultrafiltration, as well as samples taken after lyophilization and virus inactivation by dry heating. The protein content of each sample was determined by Lowry method, based on which the specific activity was calculated. The recovery rate of F Ⅷ activity in each process was calculated according to the titer and weight of product. Results The mean FⅧ titer in 10 batches of plasma pool was(0. 62 ± 0. 17)IU / ml, while the mean specific activity was(0. 011 ± 0. 003) IU / mg. The recovery rates of F Ⅷ activity in the process of centrifugation, chemical precipitation, virus inactivation by S / D method, ion exchange chromatography, ultrafiltration, lyophilization and virus inactivation by dry heating were 73. 78%, 79. 64%, 93. 10%, 64. 96%, 94. 20%, 90. 33% and 79. 25%, respectively,while the total recovery rate in the whole manufacturing process was 23. 96%. Calculated with a mean F Ⅷ content of0. 62 IU / ml, 149 IU FⅧ could be obtained from each liter of plasma. The specific activity of F Ⅷ in the bulk increased by 170 fold in average compared with that in cryprecipitate during production of seven batches of F Ⅷ. Conclusion The recovery rate of FⅧ activity in each process was obtained, which provided a support for the production of F Ⅷ with high purity and high quality.

    2017 02 v.30 [Abstract][OnlineView][Download 317K]

  • Optimization of two-wavelength microplate Reitman-Frankel assay for ALT testing

    HAO Chun-yu;YE Xiao-shu;YUAN Pan;ZENG Lang-xia;MU Jia-wei;HE Xue-xin;DUAN Yan;ZHANG Yan;Chengdu Rongsheng Pharmaceutical Co., Ltd.;

    Objective To optimize the two-wavelength microplate Reitman-Frankel assay for ALT testing. Methods The blank reagent(PBS or purified water) and concentration of sodium hydroxide for two-wavelength microplate ReitmanFrankel assay were optimized by orthogonal test, and the optimized method was evaluated for stability, linearity, precision and accuracy. Results The optimized method showed good stability and linearity when purified water was used as bland reagent and 1. 0 mol / L sodium hydroxide as stop solution(R2 = 0. 998 3 > 0. 990 0). Both the CVs of A492 / 630 values in intra-assay on 38 and 57 Karmen units of standard pyroracemic acid were less than 10%, and all the determination results of the samples were within the acceptable range. Conclusion The optimized two-wavelength microplate Reitman-Frankel assay showed good stability, linearity, precision and accuracy and high flux, which was suitable for large-scale determination of ALT.

    2017 02 v.30 [Abstract][OnlineView][Download 170K]

  • Selection of nanomembrane in manufacturing process of recombinant human coagulation factor Ⅷ

    KOU Peng;YANG Jin-liang;LIANG Hong;LI De-kuan;LI Cheng;WU Li-heng;JIANG Xiao;LEI Tao;State Key Laboratory of Biotherapy, West China Hospital, Sichuan University;

    Objective To select the nanomembrane applicable for the manufacturing process of recombinant human coagulation factor Ⅷ(rhFⅧ). Methods The harvested liquids Ⅰ(containing Tween 80)and Ⅱ(containing no Tween80) were pre-filtrated by using the filters of four types(Duropore~, Kleenpak~, Minisart~ and Virosart~ max)respectively, and determined for activity recovery rate and protein recovery rate, then further filtrated with nanomembranes(Nano1 ~ Nano7) from various manufacturers, each at a pore diameter of 20 nm, and determined for activity recovery rate, protein recovery rate and filtration flux. The screened nanomembranes were verified for virus removal using minute virus of mice(MVM) as an indicator. Results The optimal nanomembrane for pre-filtration was Minisart~, with which the activity recovery rates of harvested liquids Ⅰ and Ⅱ were 87. 9% and 99. 1%, while the protein recovery rates were97. 3% and 98. 2%, respectively. The optimal nanomembrane for filtration of harvested liquid Ⅰ was Nano6, with which the activity recovery rate, protein recovery rate and filtration flux were 100%, 92. 6% and 16. 44 L /(m2 · h)respectively. However, the optimal nanomembrane for filtration of harvested liquid Ⅱ was Nano7, with which the activity recovery rate, protein recovery rate and filtration flux were 98%, 86% and 26. 54 L /(m2 · h) respectively. Both Nano6 and Nano7 were qualified in verification test for virus removal. Conclusion The screen nanomembranes may be used for the industrial manufacturing process of rh FⅧ.

    2017 02 v.30 [Abstract][OnlineView][Download 178K]

  • Development and verification of determination method for residual CDAP in polysaccharide conjugate vaccine

    WANG Zhen;CHU Zi-qing;LIU Xiao-lei;ZHANG Jing-chun;MA Hong-chu;SUN Shu-xue;Beijing Chengda Tianhe Biotechnology Co., Ltd.;

    Objective To develop a high performance liquid chromatography(HPLC)method for determination of residual1-cyano-4-dimethylamino pyridinium tetrafluoroborate(CDAP) and provide a basis for quality control of polysaccharide conjugate vaccine. Methods The condition for HPLC was as follows: TSKgel ODS-100 V C18 column(4. 6 mm × 250 mm,5 μm)was used, serving acetonitrile-water-triethylamine(60 ∶ 40 ∶ 0. 1)solution as mobile phase at a flow rate of 1 ml / min.The detection wavelength was 300 nm, while the sample loading was 15 μl. The number of theoretical plate calculated according to CDAP peak was not less than 2 000. The developed method was determined for linear range by linear regression of peak area against concentration, and verified for specificity, limit of detection, limit of quantitation, precision,stability and accuracy. Results The method showed good specificity in determination of residual CDAP, of which the detection and quantitation limits were 35 and 40 ng / ml respectively. The standard curve showed good linearity within a CDAP concentration range of 160 ~ 4 000 ng / ml(R2 = 0. 999 9). The RSDs of test samples and standard CDAP in 6consecutive loading were 0. 26% and 0. 57% respectively. The test samples were stable within 8 h, of which the mean recovery rate was 94. 5%, with a RSD of 2. 5%. Conclusion A HPLC method for determination of residual CDAP in polysaccharide conjugate vaccine was developed, which was accurate, simple and reliable for quality control of the vaccine.

    2017 02 v.30 [Abstract][OnlineView][Download 158K]

  • Progress in research on plasminogen / plasmin

    YUE Sheng-lan;LI Ce-sheng;Department of Blood Products Laboratory, Wuhan Institute of Biological Products Co., Ltd.;

    As the active form of human plasminogen(Plg), plasmin(Plm) is an essential component of fibrinolytic system. The physiological function refers Plg / Plm an extensive prospects of application in clinical therapy. Plg / Plm has been studied in clinical trials. This paper reviews the conversion mechanism, related diseases, main function, pre-clinical /clinical studies and manufacturing process of Plg / Plm.

    2017 02 v.30 [Abstract][OnlineView][Download 198K]

  • Progress in research on avian influenza H7N9 vaccine for human use

    GUO Qi;LIAO Guo-yang;Institute of Medical Biology, Chinese Academy of Medical Sciences and Peking Union Medical University;

    In early 2013, a novel avian influenza virus(H7N9)strain was discovered in some parts of East China, which caused more than 500 cases of infection and a mortality of about 30% in humans. The experts of WHO predicted that the novel avian influenza virus(H7N9) strain may cause a pandemic, therefore the development of H7N9 vaccine is eagerly needed. At present, couples of candidate H7N9 vaccine strains have passed the safety evaluation by the WHO and may be used for the vaccine development. On the basis of this, satisfactory results have been obtained in the development of inactivated, live attenuated and recombinant avian influenza H7N9 vaccines. This paper reviews the progress in research on avian influenza H7N9 vaccine.

    2017 02 v.30 [Abstract][OnlineView][Download 165K]

  • Research progress and application prospect of bifidobacterium

    XU Zhen-guo;CAI Yu-hua;LIU Xiu-shu;FAN Gao-fu;DAI Yin;Hefei College of Vocational Technology;

    Bifidobacterium is a probiotics living in human body, and has become one of important indexes for human health. As a probiotics, bifidobacterium has several beneficial functions including nourishing, antagonism, purging,increasing immunity, anti-tumor and anti-aging, which has become a hotspot in microecological field. This paper summarizes the progress in research on biological properties, physical functions and molecular biological assay as well as the prospect of bifidobacterium.

    2017 02 v.30 [Abstract][OnlineView][Download 193K]

  • Progress of application of lyophilization stabilizer in live vaccine production

    LI Jian;School of Life Sciences and Biotechnology, Shanghai Jiao Tong University;

    Lyophilization is a key technique for the development and production of vaccines. During lyophization and storage, the activity of vaccine is influenced by various factors, including the chemical composition, temperature and rate for freezing, temperature for drying, residual moisture in dried solid as well as temperature and humidity of environment for storage. In order to prevent the active composition of vaccine from degeneration during lyophilization, it is necessary to add an appropriate stabilizer to minimize the loss of vaccine activity due to lyophization and storage. This paper reviews the action mechanism, methods for screening, challenge and development tendency of various lyophilization stabilizers.

    2017 02 v.30 [Abstract][OnlineView][Download 135K]
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@2012 Editorial Department of "Chinese Journal of Biologicals"