2017年10期目次

  • Compatibility of packaging materials to live attenuated Japanese encephalitis vaccine and its diluent

    ZHUO Jin-rong;NAN Fang;LAN Wan-ling;ZHANG Luo-hong;HAN Lin;YANG Ye;YAN Dong-zhen;ZHOU Yang;HU Mei-ling;MENG Li;Chengdu Institute of Biological Products Co., Ltd.;

    Objective To investigate the compatibility of packaging materials to live attenuated Japanese encephalitis vaccine and its diluents so as to evaluate the suitability of packaging container. Methods The packaged vaccines and diluents under accelerated conditions and in long-time storage were observed for the appearance of containers and adsorptivity to rubber stoppers, determined for the migration of 2, 6-di-tert-butyl-4-methylphenol(BHT), an antioxidant in rubber stoppers, and harmful metal ions such as arsenic(As), antimony(Sb), lead(Pb) and cadmium(Cd) in glass containers, evaluated for the risk of abscission of inner surface of glass containers, and analyzed for the effect of containers on quality of vaccine or diluents as well as whether the containers were corroded by vaccine or diluents. Results The appearances of containers of three batches of packaged vaccines after storage at(25 ± 2)℃, RH(60 ± 5)% for 6 months,at(37 ± 2)℃, RH(75 ± 5)% for 4 weeks, and at(2 ~ 8)℃ for 24 months were consistent with those before storage,while the adsorption rates of stoppers were less than 0. 05%. The amounts of antioxidant BHT and harmful metal ions As,Sb, Pb and Cd migrated from packaging container to vaccine were far lower than the safety limit. The risk of abscission of inner surface of glass containers due to corrosion by the vaccine was low. However, the appearances of three batches of packaged diluents after storage at(40 ± 2) ℃, RH(75 ± 5) % for 12 months and at(2 ~ 30) ℃ for 42 months were consistent with those before storage. The amounts of antioxidant BHT and harmful metal ion As, Sb, Pb and Cd migrated from packaging container to diluent were far lower than the safety limit, while the risk of abscission of inner surface of glass containers due to corrosion by the diluent was low. Conclusion The risk of interaction between live attenuated JE vaccine or diluent and the containers was acceptable, indicating good compatibility. It confirmed that the current containers were suitable for the vaccine and diluent.

    2017 10 v.30 [Abstract][OnlineView][Download 387K]

  • Gene cloning, sequencing and enzymological property of aminopeptidase secreted by Photobacterium sp. QH

    WU Cui-ling;WAN Bing;LI Yu-na;ZHONG Niu-niu;ZHOU Jing;PEI Jin-hong;Department of Biochemistry, Changzhi Medical College;

    Objective To clone the aminopeptidase gene of Photobacterium sp. QH(Ps sp. QH)and analyze its sequence and enzymological property. Methods Water samples were taken from Qinghai Salt Lake, from which Ps sp. QH was screened and analyzed for the scquence of 16 S r RNA. Aminopeptidase was purified from the fermentation liquid of Ps sp. QH by ammonium sulfate precipitation, DEAE Sepharose FF and gel filtration chromatography for mass spectro-metry, gene cloning and analysis of enzymological property. Results The obtained Ps sp. QH belonged to the genus of Photobacterium halotolerans, by which the secreted aminopeptidase belonged to M28 family, at a full-length of 1 527 bp,and encoded 508 amino acids. It was predicted that the relative molecular mass of aminopeptidase was 55 900, while the homology of amino acid sequence was 98% to that of Photobacterium halotolerans(KKC99795. 1). The aminopeptidase showed the highest enzyme activity at 50 ℃ and high stability at 40 ~ 50 ℃ and p H 8. 5. The enzyme activity was promoted by cobalt ion while inhibited at different degrees by magnesium, manganese, zinc, calcium, iron and copper ions. Aminopeptidase showed high activity in 1 ~ 3 mol/L sodium chloride solution. Conclusion The aminopeptidase secreted by Photobacterium sp. QH showed high halo-tolerance, which was of potential value in application to industry.

    2017 10 v.30 [Abstract][OnlineView][Download 434K]

  • Authentication of working cell bank for production of vaccines for human use

    QIAN Xing-li;SONG Cai-hua;REN Fang-fang;ZHAO Yong;DUAN Nan;LI Ya-xian;YU Jian-kun;HONG Chao;YANG Xiao-lei;Institute of Medical Biology, Peking Union Medical College, Chinese Academy of Medical Science,Laboratory of Quality Control & Yunnan Provincial Key Laboratory For Development of Vaccines against Major Infections Disease;

    Objective To authenticate the working cell bank for production of vaccines for human use by two methods,and provide a guarantee for improving the accuracy of cell lines for production of vaccines for human use and the safety of vaccines. Methods Short tandem repeat(STR) markers were used to sequence the eight loci, i. e. D8S1106, D1S518,D6S1017, D17S1304, D4S2408, D5S1467, D19S245 and DYS389, in Vero cell lines, and the profiles were compared with those reported in documents. The prepared Vero cell lines for vaccine production were treated with stained with Giemsa staining, and the chromosomes of 100 cells were counted precisely under microscope, based on which the proportion of cells with 58 or 60 chromosomes was calculated. Results The profiles of the Vero cell lines are identical to those reported in documents. No three unit genes appeared, and the numbers of repeats were identical. The proportion of cells with 58 or 60 chromosomes was 79%. Conclusion The Vero cell lines from the working cell bank for vaccine production were correct cell lines without contamination or cross-contamination, which provided a guarantee for the safety and accuracy of produced vaccines for human use.

    2017 10 v.30 [Abstract][OnlineView][Download 284K]

  • Effect of silencing vascular endothelial cadherin gene on proliferation and migration of human high metastatic hepatocellular carcinoma cells

    CHEN Bei;JING Shen-rong;WANG Ling;LUO Li-lin;WANG Jun-feng;GENG Jia-wei;College of Medicine, Kunming University of Science and Technology;

    Objective To investigate the effect of silencing vascular endothelial cadherin(VE-cadherin) gene on the proliferation and migration of highly metastatic human hepatocellular carcinoma cell line HCCLM3. Methods Three sh RNA plasmids targeting VE-cadherin gene, GV248-shRNA-VE-cadherin(1),(2)and(3), were constructed, with which HCCLM3 cells were transfected, using those transfected with empty vector GV248-shRNA-NC as negative control and those untransfected as blank control. The mRNA transcription and protein expression levels of VE-cadherin gene were determined by qRT-PCR and Western blot respectively, while the most effective targets were screened. The cell proliferation in vitro was determined by MTT assay, while the migration ability by Transwell assay. Results The m RNA transcription and protein expression levels of VE-cadherin gene in HCCLM3 cells transfected with GV248-shRNA-VEcadherin(1),(2) and(3) decreased significantly as compared with those in blank and negative control groups(P <0. 05). GV248-shRNA-VE-cadherin(1) showed the highest inhibitory rate(P < 0. 05) to VE-cadherin gene, which contained the most effective target. Both the proliferation and migration abilities of HCCLM3 tranfected with the plasmid decreased significantly(P < 0. 05). Conclusion The silencing of VE-cadherin gene inhibited the proliferation and migration of HCCLM3 cells significantly.

    2017 10 v.30 [Abstract][OnlineView][Download 246K]

  • Effect of miR-25-3p on invasion and migration of human lung cancer A549 cells by regulating expression of cytoplasmic polyadenylation element binding protein 4

    ZHANG Hui-hui;JIA Fan-bai;WANG Tao;School of Medicine and Life Sciences, University of Jinan-Shandong Academy of Medical Sciences;

    Objective To investigate the expression of miR-25-3p and cytoplasmic polyadenylation element binding protein 4(CPEB4) in human lung cancer A549 cells, the targeting effect of miR-25-3p to CPEB4 gene as well as the effect of miR-25-3p on invasion and migration of A549 cells. Methods The expression of CPEB4 in A549 cells was determined by immunofluorescence cytochemical assay. The targeting effect of MiR-25-3p to CPEB4 gene was predicted by bioinformatics. The expressions of miR-25-3p and CPEB4 mRNAs in A549 cells transfected with miR-25-3p mimics and siRNA in mediation of liposome 2000 were determined by real-time PCR, while that of CPEB4 by Western blot. The invasion of A549 cells was determined by Transwell chamber test, while the migration by wound healing assay. Results CPEB4 was highly expressed in the cytoplasm of A549 cells, which showed green fluorescence. The miR-25-3p was well complementary with CPEB4 gene. The overexpression of miR-25-3p down-regulated the expressions of CPEB4 mRNA and protein, and inhibited the invasion and migration of A549 cells. Conclusion The miR-25-3p may down-regulate the CPEB4 expression in human lung cancer A549 cells and inhibit the cell invasion and migration.

    2017 10 v.30 [Abstract][OnlineView][Download 340K]

  • Expression of exosome TMEM88 protein in supernatant of A549 cells after X irradiation and its relationship to cell proliferation

    WANG Li-wei;TONG Zhi-min;HU Yuan-yuan;JIN Peng;LI Ning;CHEN Yu-bing;People's Hospital of Changchun City;

    Objective To investigate the effect of X irradiation on proliferation of lung adenocarcinoma A549 cells and observe the expression of exosome transmembrane protein 88(TMEM88) in the supernatant. Methods A549 cells in logarithmic growth phase were irradiated with X-ray at dosages of 0, 0. 5, 1. 0, 1. 5, 2. 0 Gy(dose rate: 1. 75 Gy/time,300 Mu/min), further cultured for 6 and 12 h, and determined for proliferation level by CCK-8 method. The supernatant was collected, from which the exosomes were extracted by ultracentrifugation. The expression of TMEM88 protein was determined by Western blot. Results The A450 value of cells 6 h after irradiation with 2 Gy X-ray was significantly lower than those with 0 and 0. 5 Gy(P < 0. 05). The A450 value decreased with the increasing irradiation dose, while showed no significant difference in various dosage groups(P > 0. 05). However, the A450 value 12 h after irradiation decreased in a dose-dependent pattern, and those in various dosage groups except 0. 5 Gy group showed significant difference with that in0 Gy group(P < 0. 05). The relative expression level of TMEM88 protein 6 h after irradiation showed a decreasing tendency with the increasing irradiation dosage, which showed significant difference in 2 and 0 Gy groups(P < 0. 05).However, the expression level 12 h after irradiation showed a gradually decreasing tendency with increasing irradiation dosage, which showed significant difference in various dose groups(P < 0. 05). Conclusion X irradiation inhibited the proliferation of lung adenocarcinoma cell line A549 and the expression of exosome TMEM88 protein in the supernatant.

    2017 10 v.30 [Abstract][OnlineView][Download 319K]

  • A comparative study of allergic components of human and BALB/c mice to portunid and separation/purification of main allergens

    MAO Lu-tian;YANG Hua-sheng;School of Life Science, Huizhou University;

    Objective To compare the allergic components of human and BALB/c mice to portunid, evaluate the reliability of construction of mouse model of allergy, and separate/purify the main allergens. Methods The mouse model of allergy was established by injecting s. c. with impregnating solution of portunid protein, of which the specific Ig E serum was obtained. Meanwhile, the serum samples of crab-allergic patients were collected and determined for allergic components by Western blot. The main allergic components were separated and purified by fractional salting out with saturated ammonia sulfate and DEAE ion-exchange chromatography. Results Mouse model of allergy was established by using the impregnating solution of portumid protein, and high titer s Ig E serum was obtained. A protein component with relative molecular mass of 89 000 in portunid was the common allergen of humans and mice, which was obtained by fractional salting out with saturated ammonia sulfate and DEAE ion-exchange chromatography. Conclusion The allergens of humans and mice in portunid were basically identical, indicating that mouse might be a reliable animal model for study on allergy, and the specificity of diagnosis and treatment might be improved by using purified single allergen.

    2017 10 v.30 [Abstract][OnlineView][Download 184K]

  • Regenerative repair effect of hepatocyte growth factor gene on gastric ulcer in rats

    HA Xiao-qin;GUO Wen-jia;ZHANG Ping;YANG Shu-juan;LU Run-lan;LI Bin;ZHAO Yong;YANG Zhi-hua;BAI Yan-qing;Department of Clinical Laboratory Medicine, Key Laboratory of Stem Cell and Gene Drug in Gansu Province, Lanzhou General Hospital of Lanzhou Military Region,People's Liberation Army;

    Objective To evaluate the promoting effect of hepatocyte growth factor(HGF) gene therapy on tissue regeneration in rats with gastric ulcer induced by acetic acid. Methods Gastric ulcer model was established by subserosal injection with acetic acid in Wistar rats. The model rats were randomly divided into three groups on day 5 after establishment, and treated with 10% sodium hydrogen carbonate solution containing 1 × 109 cfu TPH(attenuated salmonella carrying eukaryotic expression vector for HGF gene), 10% sodium hydrogen carbonate solution containing 1 ×109cfu TP(attenuated salmonella carrying empty eukaryotic expression vector) and 10% sodium hydrogen carbonate solution(control)at an equal volume(1 ml)by lavage respectively, once 3 d for 3 times. The stomach tissues of rats were collected on day 21 after the last lavage, observed for ulcer healing visually, and for regenerative repair by microscopy.The HGF expression levels in ulcer tissue on days 1, 3, 7, 14 and 21 after the last lavage were determined by ELISA.Results The wound surface of ulcer of rats in TPH group was small in area and shallow, while the basal cells were proliferated significantly, the gastric mucosa epithelial cells were proliferated and migrated significantly, and capillary vessels were observed in basal tissue. However, no obvious proliferation of basal cells or proliferation and migration of gastric mucosa epithelial cells were observed in TP and control groups. The expression levels of HGF at various time points after lavage in TPH group were significantly higher than those in TP and control groups(P < 0. 05 or P < 0. 01).Conclusion HGF gene therapy accelerated the healing of gastric ulcers via stimulating the proliferation of gastric epithelial cells and basal cells of gastric ulcer wound.

    2017 10 v.30 [Abstract][OnlineView][Download 265K]

  • Preparation and identification of monoclonal antibody against gD protein of bovine infectious rhinotracheitis virus

    BI Ying;WEN Xiao-bo;NI Hong-bo;College of Animal Science and Veterinary Medicine, Heilongjiang Bayi Agricultural University;

    Objective To prepare and identify the monoclonal antibody(Mc Ab) against gD protein of bovine infectious rhinotracheitis virus(IBRV). Methods The amplified IBRV gD gene was inserted into prokaryotic expression vector pET-28a(+). The constructed recombinant plasmid gD-pET-28 a was transformed to E. coli BL21(DE3)and induced with IPTG. The expressed recombinant gD protein was purified by Ni-NTA Agarous Kit, with which BALB/c mice were immunized. The splenocytes of immunized mice were fused with SP2/0 cells, from which positive hybridoma cells were screened and injected i. p. into mice. The ascites of mice were collected, purified by using Hi Trap Protein G HP kit,subclassed and identified by Western blot and indirect immunofluorescence assay. Results The recombinant gD protein with a relative molecular mass of 48 000 reached a concentration of 4. 19 mg/ml after purification. One hybridoma cell strain was screened, and the obtained McAb reached a protein content of 1. 81 mg/ml, and showed specific reaction with IBRV. Specific fluorescence was observed in MDBK cells inoculated with IBRV. Conclusion IBDV gD protein was expressed by prokaryotic expression system and purified, and the Mc Ab against IBDV gD protein was successfully prepared, which laid a foundation of identification of epitope and study on pathogenic mechanism of the virus.

    2017 10 v.30 [Abstract][OnlineView][Download 256K]

  • Characteristics and clinical significance of hepatitis C virus genotype distribution in Qingyang Region, Gansu Province, China

    HOU Juan-juan;ZHANG Zhi-feng;ZHANG Ji-ping;XI Wei-yue;ZHENG Hong-jun;ZHAO Hai-yan;LI Juan;Clinical Laboratory, The People's Hospital of Qingyang City;

    Objective To investigate the characteristics and clinical significance of hepatitis C virus genotype distribution in Qingyang Region, Gansu Province, China. Methods The clinical data and blood samples of 289 patients with hepatitis C in Qingyang Region were collected, and tested for the RNA and genotype of HCV by PCR-fluorescent probe, and the results were statistically analyzed. Results Of the 289 HCV RNA positive blood samples, 139 were of genotype for 1b(48. 1%), while 136 were of 2a(47. 1%), 8 were of 3a(2. 8%), 5 were of 3b(1. 7%), and one of undefined genotype(0. 3%). Genotype of hepatitis C virus was related to the gender and age(P < 0. 05). Genotype was related to transmission routes(P < 0. 05). HCV was mainly transmitted by blood transfusion, followed by hemodialysis. The sustained virological response(SVR)rate of HCV of genotype 2a was 77. 2%, which was insignificantly higher than that of genotype 1b(69. 8%)(P > 0. 05). The RNA load of HCV of various genotypes showed significant difference(P < 0. 05).The proportions of liver cirrhosis and liver cancer in patients infected with HCV of genotype 1b were significantly higher than those of other genotypes. Conclusion Genotypes 1b and 2a were prominent in the patients with HCV infection in Qingyang Region, of which the ratios were equal. Genotype of hepatitis C virus was related to the gender and age of patients, transmission routes, HCV RNA load and severity of liver diseases.

    2017 10 v.30 [Abstract][OnlineView][Download 146K]

  • Epidemic characteristics of tick-borne encephalitis in Jilin Province during 2010～2016

    DENG Li-quan;SHEN Bo;LI Ya-ming;Jilin Provincial Center for Disease Control and Prevention;

    Objective To investigate the epidemic of tick-borne encephalitis(TBE)in Jilin Province during 2010 ~ 2016 and analyze the temporal, spatial and demographic characteristics of case distribution. Methods The data of surveillance of TBE in Jilin Province were collected from January 1, 2010 to December 31, 2016 on the basis of address, the date of examination, the date of onset, age, gender and occupation, and made into a chart by using Excel software, based on which the atlas of morbidity in various regions was plotted by using Mapinfo software. Results The epidemic season of TBE appeared from May to mid-September, while the peak of morbidity appeared from the end of May to mid-June, and a small peak at the beginning of July. The cases were in obviously regional distribution, most of which occurred in the area of Changbai Mountain and the forest region in a branch of the mountain. The cases were mainly appeared in the population at ages of 50 ~ 60 years. The incidence was higher in male than in population, with a sex ratio of 1. 66 ∶ 1.However, 61. 47% of the total cases appeared in farmers. Conclusion Obvious temporal, spatial and demographic characteristics of TBE were observed in Jilin Province. Comprehensive measures should be taken to prevent and control the diseases.

    2017 10 v.30 [Abstract][OnlineView][Download 199K]

  • Analysis of significance of human epididymal protein 4 in diagnosis of ovarian cancer by ROC curve

    ZHANG Jia-jia;CUI Qian-qian;WANG Ping;The First Hospital Affiliated to Henan University of Science and Technology, The Second People's Hospital of Jiaozuo City;

    Objective To analyze the significance of human epididymal protein 4(HE4)in diagnosis of ovarian cancer by ROC curve. Methods A total of 107 patients with ovarian diseases were divided into normal control(21 cases), benign ovarian tumor(20 cases)as well as ovarian cancer of stagesⅠ~ Ⅱ(14 cases), Ⅲ(30 cases)and IV(22 cases)groups,then determined for serum HE4 level and subjected to imaging examination. The two methods were evaluated for significance in diagnosis of ovarian cancer in terms of sensitivity and specificity. However, the serum HE4 levels in various groups were analyzed by ROC curve to evaluate such significance of HE4. Results The sensitivity of serum HE4 level was significantly higher than that of imaging examination(P < 0. 05), while the specificity of the former showed no significant difference with that of the latter(P > 0. 05). The serum HE4 level increased with the increasing malignancy.The AUG of ROC curve was 0. 912, indicating an important significance in diagnosis of ovarian cancer. Conclusion Serum HE4 level showed higher sensitivity than imaging examination, as well as high specificity, which might be used for assistant diagnosis of ovarian cancer, and of an important significance in diagnosis of ovarian cancer.

    2017 10 v.30 [Abstract][OnlineView][Download 118K]

  • Development and verification of auantitative detection assay for glycoprotein(Gn)antigen of severe fever with thrombocytopenia syndrome bunyavirus

    CHEN Lei;DAI Xin-xian;HAO Chun-sheng;WEN Zhi-heng;LIU Yu;WU Yun-yi;MA Shu-hua;LU Wei-wei;ZHAO Zhong-xiang;LI Xiu-ling;National Vaccine & Serum Institute;

    Objective To develop and verify a double antibody sandwich ELISA for quantitative detection of glycoprotein(Gn)antigen of severe fever with thrombocytopenia syndrome bunyavirus(SFTSV)and use for monitoring of virus antigen content during SFTSV vaccine production. Methods Polyclonal and monoclonal antibodies against SFTSV were prepared from the serum of rabbits and the ascites of BALB/c mice immunized with SFTSV Gn antigen respectively. A double antibody sandwich ELISA method was developed using polyclonal antibody as coating antibody and HRP-labeled monoclonal antibody as enzyme-labeled antibody, and verified for specificity, precision, accuracy, stability and suitability.Results The linear range of the developed ELISA method was 0. 125 ~ 4. 000 μg/ml, with a R~2 value of 0. 994 1, while the quantitative detection limit was 0. 125 μg/ml. SFTSV strain was detected specifically by the developed method, while no cross reactions with other viruses belonging to Bunyavirus, culture supernatant of Vero cells or other subsidiary materials were observed. The recovery rates of samples at various concentrations were 85% ~ 115%, with CVs of less than15%. The CVs of test results by the ELISA reagent after storage at 37 ℃ for 3 d was less than 15%. The antigen content of unit protein in samples at various stages of preparation of bulk of SFTSV showed an increasing tendency, which reflected the purification process of antigen. Conclusion A quantitative detection assay for SFTSV Gn antigen was developed, which was broad spectrum, sensitive, specific and stable. It laid a foundation of quality control of production process of vaccine.

    2017 10 v.30 [Abstract][OnlineView][Download 313K]

  • Establishment and validation of multiplexed opsonophagocytic killing assay for 13-valent pneumococcus

    QIAO Rui-jie;LIU jia;WANG Hao;GE Yu-wen;WANG Xin-ru;FAN Feng-feng;SHI Xiao-ling;TAN Xiao-mei;XIE Gui-lin;Lanzhou Institute of Biological Products Co.Ltd.;

    Objective To establish and validate a standardized multiplexed opsonophagocytic killing assay(MOPA) for measuring functional antibody specific to 13-valent pneumococcal capsular polysaccharide. Methods A standardized MOPA was established by preparation of working bacterial seeds, identification of serotype, optimizing culture and differentiation of HL60 cells, complement screening and establishment of quality controls based on the protocol published by WHO and US NIH Bacterial Respiratory Pathogen Reference Laboratory, Department of Pathology, University of Alabama at Birmingham(UAB)(UAB-MOPA for short), and verified for specificity, linearity, precision, accuracy as well as robustness according to the guidance of International Conference Harmonization(ICH). Results All the indexes of prepared working bacterial seeds met the requirements in UAB protocol. The HL60 cells after differentiation for 3 ~ 6 d could be used as effector cells for MOPA. The complements with Lot No. of 27531 and 84321 could be used as working complements. The quality control ranges of five control serum samples, i. e. 09 CS, QC2, B, C and F, were confirmed. The specificity, linearity, precision, accuracy and robustness of MOPA met the specified criteria. Conclusion A standardized MOPA on 13-valent pneumococcus was successfully established and validated.

    2017 10 v.30 [Abstract][OnlineView][Download 331K]

  • Development, verification and application of ELISA for determination of antigen content of von Willebrand factor

    ZHOU Zhi-jun;LIN Lian-zhen;PENG Yan;Li Tao-jing;LI Ce-sheng;HU Yong;Department of Blood Products Laboratory, Sinopharm Wuhan Plasma-derived Biotherapies Co., Ltd.;

    Objective To develop, verify and preliminarily apply a double antibody sandwich ELISA for determination of antigen content in von Willebrand factor(VWF). Methods A sandwich ELISA for antigen content of VWF was developed through optimization of concentrations of capture antibody and detection antibody by chessboard titration of paired VWF antibody. The internal control standard(FⅧ-free VWF)was calibrated with international standard(NIBSC07/316)for VWF to determine the relationship between of international unit(Ag/ml)and quality unit(μg/ml)of VWF ∶ Ag.The linearity, accuracy and precision of the developed method as well as the stability of samples were verified. The method was applied to the monitoring of FⅧ purification process and the quality analysis of products. Results Both the dilutions of capture antibody and detection antibody were 1 ∶ 200. A portion of 1 Ag/ml VWF ∶ Ag corresponded to 16 μg/ml. The linear range of standard curve was 160 ~ 10 ng/ml. The recovery rates of samples at various concentrations were 95. 8% ~103. 2%, with a CV of less than 5% and a R2 value of not less than 0. 99. The recovery rates of samples at various concentrations in intra-and inter-assays were 93. 0% ~ 113. 9% and 95. 1% ~ 105. 5%, while the CVs of determination results were 0. 7% ~ 8. 2% and 3. 8% ~ 9. 6%, respectively. The recovery rate in sample adding experiment was 96. 9% ~114. 7%. The samples with biological activity should be preserved at-80 ℃ and immediately tested after reconstitution.PEG precipitation and ion exchange chromatography in the purification process of Sinopharm Wuhan Plasma-derived Biotherapies Co., Ltd. removed more than 95% of VWF. The purification process was stable, while the F Ⅷ ∶ Ag/VWF ∶ Ag ratio of final product was closed to that of plasma. Conclusion The developed ELISA for antigen content of VWF showed good linearity, accuracy and precision, which might be used for the internal quality control of purification process of F Ⅷ.

    2017 10 v.30 [Abstract][OnlineView][Download 211K]

  • Comparison of effectiveness of two kinds of gel filtration medium in purification of polio virus Sabin strain

    WANG Xiao-yu;ZHU Yun;SHEN Wu-ling;PING Ling;CAI Wei;SUN Ming-bo;Institute of Medical Biology, Chinese Academy of Medicine Science & Peking Union Medical College, Yunnan Key Laboratory of Vaccine Research & Development on Severe Infectious Diseases;

    Objective To compare the effectiveness of two kinds of gel filtration medium in purification of polio virus Sabin strain. Methods Three batches of harvested liquids of polio virus Sabin strain of types Ⅰ, Ⅱ and Ⅲ were concentrated by ultrafilitration, and purified by Sepharose CL-6B sepharose 6FF gel filtration chromatography separately.The purification peaks were collected and determined for D antigen and protein contents to calculate the specific activity,protein removal rate and antigen recovery rate, based on which the collection range of virus peaks was confirmed. The harvested virus liquid with the highest antigen content was purified by DEAE Sepharose FF ion exchange chromatography,and determined for residual Vero cell DNA, residual Vero cell protein, residual bovine serum albumin and residual antibiotics contents. Results In Sepharose 6FF gel filtration chromatography, samples were loaded at 6% of column volume, while the mean recovery rates of virus of types Ⅰ, Ⅱand Ⅲ were 78. 69%, 78. 03% and 78. 37% respectively.However, in Sepharose CL-6B chromatography, the samples were loaded at 3% of the column volume, and the mean recovery rates were 77. 29%, 78. 60% and 77. 36% respectively, which showed no significant difference with those by Sepharose 6FF chromatography(P > 0. 05). After further purification by ion exchange chromatography, the protein removal rate, specific activity, residual Vero cell DNA content, residual Vero cell protein content, residual bovine serum albumin content and residual antibiotics content of harvest liquids of various types met the requirements in Chinese Pharmacopoeia(Volume Ⅲ, 2015 edition), which showed no significant difference in those previously purified by Sepharose 6FF and Sepharo se CL-6B chromatography. Conclusion The recovery rate and specific activity of virus antigen of various types purified by Sepharose 6FF chromatography showed no significant difference with those by Sepharose CL-6B chromatography. However, the times of sample loading decreased, and the purification efficacy increased remarkably, indicating that Sepharose CL-6B might be substituted with Sepharose 6FF.

    2017 10 v.30 [Abstract][OnlineView][Download 401K]

  • Development and verification of ultraviolet spectrophotometric method for determination of chitosan content

    DENG Xia;ZHANG Yu;SHAN Pu;LI Shu-xiang;WANG Xin-yi;WANG Zhi-biao;XU Jing;National Vaccine & Serum Institute;

    Objective To develop and verify a ultraviolet(UV) spectrophotometric method for determination of chitosan(CTS) content. Methods A UV spectrophotometric method for determination of CTS content was developed by optimization of reaction system solution and dilution factor on standard curve, and verified for specificity, linear range,accuracy, precision(reproducibility and intermediate precision) and robustness. Results The optimal reaction system solution was 0. 2% acetic acid solution, while the optimal dilution factor was 2-fold serial dilution. The recovery rate of CTS in 0. 2% acetic acid solution was 8. 07%, while that of CTS solution was 101. 38%. The linear range of the deve-loped method was 0 ~ 62. 5 μg/ml. The method showed high accuracy at a sample concentration of 25. 0 ~ 75. 0 μg/ml.The CVs of test results of reproducibility, intermediate precision and robustness were less than 20%. Conclusion The UV spectrophotometric method for determination of CTS content was developed, which showed high specificity, repro-ducibility and intermediate precision, as well as high accuracy at sample concentration of 25. 0 ~ 62. 5 μg/ml and good linearity at 0 ~ 62. 5 μg/ml.

    2017 10 v.30 [Abstract][OnlineView][Download 145K]

  • Establishment of a procedure for serum-free suspension culture of MDCK-siat7e cells in bioreactor

    CHEN Jun;SUN Yan;ZHANG Sheng-yan;SUN Zhen-peng;FAN Xiu-juan;MA Chao;LIU Peng;LI Wei;Lanzhou Institute of Biological Products Co., Ltd.;

    Objective To establish a procedure for suspension culture of MDCK-siat7 e cells in serum-and protein-free medium BD001 from non-animal origin, and provide a basis for large-scale culture of MDCK-siat7 e cells and preparation of viral vaccines. Methods A master cell bank for MDCK-siat7 e cells derived from BD001 and serum-free cryopreservation solution was established and subjected to routine control tests, while the cells were observed for growth status. The time for formation of platform period as well as the cell density and cell viability in the period of MDCK-siat7 e cells at various initial inoculum densities(4. 0 × 10~5, 8. 0 × 10~5 and 1. 6 × 10~6 cells/ml) were compared. The specific growth rate(μ), times of division(Cd), specific consumption rate of glucose(q_(gluc))and transformation rate of lactic acid to glucose(Y_(lac/gluc))of MDCK-siat7 e cells in various volumes of bioreactor(3, 15 and 60 L)were compared, and the growth and metabolism of the cells were observed. Results The mean viability of MDCK-siat7 e cells cultured in BD001 medium was(96. 20 ± 2. 95) % after resuscitation. The cells after resuscitation grew well in BD001 medium and reached the logarithmic phase after an incubation period of 24 h, which reached the platform period on day 6, reached the maximum cell density of 6. 20 × 10~6cells/ml on day 8 and was maintained to day 12. The cell density and viability began to decrease from day 12 and entered to decay period. MDCK-siat7 e cells grew well in serum-free medium. The time for formation of platform period of cells at various initial inoculum densities showed significant difference(P < 0. 05), while the density and viability of cells in platform period showed no significant difference(P > 0. 05). There were no significant differences in the μ, Cd, q_(gluc)and Y_(lac/gluc)of MDCK-siat7 e cells in various volumes of bioreactor(P > 0. 05), and the cells grew well. Conclusion A procedure for serum-free suspension culture of MDCK-siat7 e cells in bioreactor was developed,which provided an experimental basis for culture of MDCK-siat7 e cells in serum-and protein-free medium BD001 from non-animal origin in bioreactor.

    2017 10 v.30 [Abstract][OnlineView][Download 226K]

  • Progress in research on novel tuberculosis vaccine

    MU Shan-shan;SUN Shu-xue;Department of Purification, Beijing Chengda Tehe Bio-technology Co., Ltd.;

    Tuberculosis(TB)is a chronic infectious disease caused by Mycobacterium tuberculosis complex. At present,Bacillus Calmette-Guérin(BCG) is the most widely used vaccine against TB, though its immune protection is less ideal.Along with the increases of multi-drug and extensive-drug resistant strains and increasing seriousness of problems such as co-infection of HIV, it is imperative to develop novel TB vaccines. Considerable progress in some novel TB vaccines which mainly include protein adjuvant booster vaccines, viral-vectored booster vaccines, priming vaccines and therapeutic vaccines have been achieved in decades. Therein, booster vaccines targeting adolescents and adults have attracted significant attention because of their significant effects on epidemic of tuberculosis worldwide. This review outlines the progress in research on novel TB vaccines in recent years.

    2017 10 v.30 [Abstract][OnlineView][Download 218K]

  • Advances in research on candidate antigens of Mycoplasma pneumoniae vaccine

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

    Mycoplasma pneumonia(MP) is the main pathogen of community-acquired pneumonia, which may cause respiratory infection in humans, resulting in anaphylactic asthma in partial population. MP may also aggravate lung diseases and cause some complications. At present, MP infection is mainly treated with macrolide antibiotics. However,with the wide application of antibiotics, the epidemic of macrolide-resistant MP(MRMP) appeared, with a resistance rate of 90%, to which no protocol of therapy has been developed. For this reason, MP vaccine is very important to prevent and control MP infection. In this paper, the research on whole cell antigen, protein antigen and glycolipid antigen in candidate MP vaccine antigen are summarized.

    2017 10 v.30 [Abstract][OnlineView][Download 142K]

  • Advances in research on role of UbcH7 in cancer therapy

    HE Rong;LV Lin-yue;CHENG An-yi;ZHANG Lei;Institute of Biomedical and Pharmaceutical Sciences, Hubei Provincial Cooperative Innovation Center, College of Bioengineering, Hubei University of Technology;

    As a member of ubiquitin-conjugating enzyme E2 family, ubiquitin conjugating enzyme L3(UbcH7)contributes the ubiquitination modification of substrate proteins in combination with E3 ubiquitin ligase. Recently,emerging results showed that UbcH7-dependent ubiquitination and proteasome-dependent degradation are involved in the regulation of p53 binding protein(53BP1). Normal degradation of 53BP1 protein drives cancer cells to repair doublestrand breaks(DSBs) with homologous recombination(HR). When UbcH7 is suppressed, nonhomologous end-jointing(NHEJ)is used because of the enhanced 53BP1 retention. Decreased UbcH7 level leads to an increased sensitivity to anticancer therapies and cells death caused by impropriate genes arrangement. Functional significance of UbcH7 in the process of DNA damage response suggests it may be used as a potential target for cancer therapy.

    2017 10 v.30 [Abstract][OnlineView][Download 177K]


@2012 Editorial Department of "Chinese Journal of Biologicals"