2019年09期目次

  • Analysis of interfering factors of determination of D-ribose in quality control of Haemophilus influenzae type b vaccine

    QIU Yuan-tao;LEI Yong-hong;YANG Fan;WANG Zhi-wei;DANG Xiao-yan;GAO Qiang;LIN Ji-sheng;Sinovac Research & Development,Co.,LTD;

    Objective To investigate the interference of cetyltrimethyl ammonium bromide(CTAB),ethyl alcohol and sodium chloride to determination of D-ribose in quality control of Haemophilus influenzae type b(Hib)vaccine. Methods Simulating the concentrations of CTAB,ethyl alcohol and sodium chloride during production of Hib vaccine,the D-ribose samples containing CTAB at final concentrations of 0. 03%,0. 06%,0. 13%,0. 25%,0. 50% and 1. 00%,ethyl alconhol at final concentrations of 10%,20%,30%,40%,45% and 50% and sodium chloride at final concentrations of0. 06,0. 13,0. 25,0. 50 and 1. 00 mol/L were prepared and determined for D-ribose content,based on which the recovery rates were calculated. Results When the final CTAB concentrations were 0. 25% and 1. 00%,the recovery rates of D-ribose were 92. 0% and 53. 6% respectively. However,when the final concentrations of ethyl alcohol were at40%(V/V)and 50%(V/V),the recovery rates of D-ribose were 93. 6% and 88. 0% respectively. The recovery rate of D-ribose decreased with the increasing concentrations of CTAB and ethyl alcohol. When the sodium chloride concentration was 0 ～ 1 mol/L,the recovery rate of D-ribose was 97. 6% ～ 100. 8%. Along with the increasing sodium chloride con-centration,the recovery rate of D-ribose showed a static tendency. Conclusion High CTAB and ethyl alcohol concen-trations interfered the determination of D-ribose content,while sodium chloride concentration showed no influence on the determination.

    2019 09 v.32 [Abstract][OnlineView][Download 192K]

  • Immunomodulatory effect of mung bean protein peptides on RAW264. 7 macrophages

    DIAO Jing-jing;CHI Zhi-ping;SUN Di;CHEN Hong-sheng;ZHANG Li-ping;ZUO Feng;National Coarse Cereals Engineering Research Center,Heilongjiang Bayi Agricultural University;

    Objective To investigate the immunomodulatary effect of mung bean protein peptides(MBPs)on RAW264. 7 macrophages. Methods RAW264. 7 macrophages were cultured in vitro and added with MBPs at concentrations of 10,100,200 and 400 μg/mL respectively,then determined for proliferation ability,phagocytosis ability,SOD activity,nitric oxide content and secretion level of cytokines. Meanwhile,the effects of MBPs on secretion of cytokines and expression levels of LC3 and p62 in RAW264. 7 macrophages induced by LPS were evaluated. Results Compared with those in blank control group,the proliferation and phagocytosis rates of RAW264. 7 macrophages treated with MBPs at various concentrations increased significantly(P < 0. 05),while the SOD activity,nitric oxide content and secretion levels of TNF-α,IL-1β and IL-6 of those treated with 400 and 200 μg/mL MBPs increased significantly(P < 0. 05).The MBPs at various concentrations inhibited the secretions of TNF-α,IL-1β and IL-6 in RAW264. 7 macrophages induced by LPS significantly(P < 0. 05). However,the secretion levels of the cytokines after treatment with 400 μg/mL MBPs showed no significant difference with those in blank control group(P > 0. 05). The MBPs at various concentrations decreased the LC3 content while up-regulated the p62 expression in RAW264. 7 macrophages induced by LPS.Conclusion MBPs activated macrophages,increased the activity of autometabolic enzymes and released the cytokines,which improved the immune function of host cells by anti-inflammatory effect and inhibition of autophagy.

    2019 09 v.32 [Abstract][OnlineView][Download 379K]

  • Epidemiological characteristics and molecular evolution of structural protein of Zika virus in Southeast Asia

    LIN Yao;HONG Shan;LAN Qing-ping;SUN Qiang-ming;Institute of Medical Biology,Chinese Academy of Medical Science & Peking Union Medical College,Yunnan Key Laboratory of Vaccine Research and Development on Severe Infectious Disease;

    Objective To evaluate the transmission of Zika virus(ZIKV)in Southeast Asia and to explore the evolution of molecular characteristics. Methods The outbreak of ZIKV in Southeast Asia was retrieved by keywords. The whole gene sequence of ZIKV Southeast Asia strain was obtained from GenBank. Sequence alignment was conducted by using BIOEDIT software,and the phylogenetic tree was constructed by using MEGA 7. 0 software. Nucleic acids and amino acids of structural protein were used to analyze mutation site and mutation rate among different strains. The secondary structure of Envelop domainⅢ(ED-Ⅲ) was predicted by using PredictProtein. Results ZIKV has been reported to be serologically infected or retrospectively positive in Southeast Asian countries. Of these countries,Vietnam,Singapore and Thailand had more cases of ZIKV infection and higher burdens of disease. Phylogenetic tree showed that all the strains belonged to Asian type. The Singapore strain in 2016(KY241788)had the closest relationship with the Brazilian strain in2015. However,the Malaysian strain isolated in 1966 had a distant relationship with other Southeast Asian strains. A total of 22 base mutation sites were observed in the capsid(C)protein of various types of ZIKV strains in Southeast Asia,while the base mutation rate was 6. 36%. However,19 non-synonymous amino acid mutation sites were observed,while the mutation rate was 16. 52%. A total of 51 base mutation sites of pre-membrane protein(prM) were observed,while the mutation rate was 10. 12%. Correspondingly,37 non-synonymous amino acid mutation sites were observed,while the mutation rate was 22. 02%. A total of 141 base mutation sites of envelope(E)protein were observed,while the mutation rate was 9. 33%. Correspondingly,101 non-synonymous amino acid mutation sites were observed,while the mutation rate was 20. 04%. Prediction of secondary structure of ED-Ⅲ showed the same number of protein binding sites in this region of two representative strains,and an enrichment region of protein binding sites of ED-Ⅲ:55-63 in representative strain 2(1966 Malaysia KX377336),while the protein binding sites of representative strain 1(2014 Thailand KU681081)were well distributed. There is a disulfide bond in the ED-Ⅲ of the representative strain 2,while no disulfide bond was observed in the representative strain 1. However,a helix was observed in the representative strain 1,while no helix in the representative strain 2. Conclusion By means of statistical analysis of reported cases,phylogenetic analysis,protein nucleotide sequence and amino acid comparative analysis as well as prediction of secondary structure of ED-Ⅲ in Southeast Asia,this study provided references for research on the biology and differences in epidemiological pathogenicity of various ZIKV strains in Southeast Asia.

    2019 09 v.32 [Abstract][OnlineView][Download 775K]

  • Effect of lactobacillus on proliferation of endometrial epithelial cells

    HE Jin-ying;ZHU Su-fang;SARULA;SUN Wen-fang;MA Yu-zhen;Reproductive Center,People′s Hospital of Inner Mongolia Autonomous Region;

    Objective To co-culture human endometrial epithelial cells with lactobacillus so as to investigate the effect of latter on proliferation of the former. Methods Vaginal secretions were collected from 68 healthy women at reproductive age in Inner Mongolia,from which lactobacillus was isolated and purified by using conditioned medium,analyzed for biological characteristics by ammonium oxalate crystal violet staining,enzymatic reaction and hydrogen peroxide production test,and was classified by 16 sRNA gene amplification and seq-uencing. The dominant lactobacillus at various concentrations(10、102、103 and 104 bacteria/mL)were co-cultured with endometrial epithelial cells for 12,24,36 and48 h,and determined for the proliferation level of endometrial epithelial cells by CCK-8 assay to optimize the concentration and time of lactobacillus for treatment of endometrial epithelial cells. Results A total of 141 lactobacillus strains were obtained,which were stained purple with ammonium oxalate crystal violet and were Gram-positive. All the colonies produced hydrogen peroxide,of which the abilities in production were different. Six species of lactobacillus were identified by 16 sRNA gene amplification and sequencing,of which L. crispatus,L. asseri and L. vaginalis were the dominant lactobacillus species in the female population in Inner Mongolia,accounting for 54. 61%,24. 82%,and 12. 06%respectively. Lactobacillus at various concentrations promoted the proliferation of endometrial epithelial cells. However,the proliferation of endometrial epithelial cells was dominant after treatment with lactobacillus at a concentration of103 bacteria/mL for 36 h,of which the rate reached 93. 10%. Conclusion Lactobacillus promoted the proliferation of endometrial epithelial cells,which provided a theoretical basis for promoting the repair of endometrial local damage by lactobacillus.

    2019 09 v.32 [Abstract][OnlineView][Download 363K]

  • Analysis of crest trait and prediction of biological function of candidate gene SOX5 in Zi-goose

    ZHANG Yu-chen;ZHOU Rui-jin;YUAN Jian-bin;LI Wen-jie;ZANG Shu-cheng;WANG Li-peng;LANG Li-min;CHEN Yan;YANG Huan-min;Laboratory of Animal Stress,College of Animal Science and Veterinary Medicine,Heilongjiang Bayi Agriculture University;

    Objective To analyze the phenotypic trait of top head of Zi-geese(crest trait)and predict the biological function of candidate gene SOX5 so as to further reveal the phenotypic trait of poultry as well as the action mechanism of regulatory gene. Methods Six male geese without crest,15 female geese with and 15 female geese without crest were selected to establish two resource populations,i. e. crest(C)population consisting of 3 male geese without crest and 15 female geese with crest and non-crest(NC)population consisting of 3 male geese without crest and 15 female geese without crest. The two populations were subjected to group feeding in the same birdhouse,and the eggs were collected and hatched to observe the phenotypic separation ratio of offspring,based on which the genetic regularity of crest trait was analyzed. The expression levels of SOX5 gene in liver and head skin tissues of offspring at ages of 1,14 and 21 days were determined by qRT-PCR for analysis of association by Bioinformatics. Results The phenotypic separation ratio showed that the non-crest trait were in line with the regularity of recessive inheritance,while the crest trait were regulated by autosomal dominant genes. The relative expression level of SOX5 gene in liver tissue of offspring at age of one day was significantly higher in C population than in NC population(P < 0. 01),while those at the other ages were insignificantly higher(P > 0. 05). The body weight of male geese showed an increasing tendency with the increasing age,which was consistent with the change tendency of relative expression level of SOX5 gene in skin. The prediction by the SMART and David software showed a conservative domain of SOX5 gene,with a capable of forming biologically functional protein.Conclusion The crest trait of Zi-geese is similar to the phenotype inheritance of comb,which is regulated by autosomal genes. SOX5 is the regulatory gene of crest trait,which participates in the development process of the relevant poultry tissues.

    2019 09 v.32 [Abstract][OnlineView][Download 441K]

  • Prokaryotic expression and activity of Expansin of Bacillus subtilis

    YE Kai-xia;ZHU Hua-chen;ZHAO Qing-xin;CHEN Hong-sheng;College of Biotechnology and Pharmaceutical Engineering,Nanjing Tech University;

    Objective To express the Expansin of Bacillus subtilis in prokaryotic cells and analyze its activity in promoting the saccharification of polysaccharide. Methods Using Bacillus subtilis subsp. subtilis str. 168 genome as template,expansin gene was synthesized. The 6-his tag was added to the N-terminus of expected protein by primer design,based on which a fusion gene 6-his-expansin was synthesized by PCR,based on which recombinant plasmid 6 his-expansin-6 his-pET28 a(+)[heh-p ET28 a(+)]was constructed,transformed to E. coli BL21(DE3)and induced with IPTG for expression of fusion protein 6 His-Expansin-6 His(HEH). The final IPTG concentration,temperature and time for induction were optimized. The expressed product was purified by Ni-NTA chromatography and determined for synergistic activities with various polysaccharide degrading enzyme(PDE)preparations(cellulose,xylanase and pectinase)and poly-saccharide complex enzyme(PCE). Results Restriction analysis proved that recombinant plasmid heh-pET28 a(+)was con-structed correctly. After induction with 0. 05 mmol/L IPTG at 30 ℃ for 30 h,the expression level of HEH reached about 1. 2 mg/m L,while the relative molecular mass was 26 400,and the purity reached 95% after purification. The synergistic activities of the expressed protein with cellulose,xylanase and pectinase were 171. 53%,61. 95% and 230. 00%,while those with PCE in degrading filter paper and rice straw were 312. 83% and 356. 59%,respectively. Conclusion Recombinant fusion protein HEH showed high synergistic activities with various PDEs,which laid a foundation of application of Expansin in the saccharification of biomass lignocellulose.

    2019 09 v.32 [Abstract][OnlineView][Download 295K]

  • Antimicrobial activity of baicalin to E. coli and relevant mechanism

    LIU Hao;ZHAO Zi-bing;WANG Xin;College of Animal Science and Veterinary Medicine,Heilongjiang Bayi Agricultural University;

    Objective To investigate the antimicrobial activity and possible antimicrobial mechanism of baicalin to E. coli. Methods On the basis of determination of the minimum inhibitory concentration(MIC) and bacteriostatic activity of baicalin to E. coli,the effect of baicalin at a single MIC on activity of extracellular lactate dehydrogenase,forward scattering and DNA content were evaluated. Results The MIC of baicalin to E. coli was 7 mg/mL,while the bacteriostatic activity reached the peak value after intervention for 2 h. The lactate dehydrogenase activity of E. coli after treatment with baicalin at a single MIC increased gradually and reached(201. 48 ± 19. 06) U/L 8 h later. However,after treatment with baicalin at a single MIC for 2 h,the E. coli cells decreased in volume and DNA content,which showed significant difference with those in control group(P < 0. 05). Conclusion Baicalin increased the permeability of cell membrane of E. coli by causing a certain damage,resulting in the infiltration of bacterial substance in a large quantity so as to realize the bacteriostatic effect.

    2019 09 v.32 [Abstract][OnlineView][Download 192K]

  • Antitumor activity of a novel antibacterial peptide RGD-chimera targeting melanoma

    ZHANG Qiu-li;LIU Meng-yue;JIANG Xuan;JIN Tian-ming;ZHAO Rui-li;MA Ji-fei;FU Chao;SHANG Zi-yu;College of Animal Science and Animal Medicine,Tianjin Agricultural University;

    Objective To investigate the anti-tumor targeting activity of a novel antimicrobial peptide RGD(arginineglycine-aspartic acid). Methods The antitumor peptide with high antitumor activity was screened by MTT assay,while the tumor cells for high expression of cell surface integrin αvβ3 by flow cytometry,based on which a model of B16 subcutaneous tumor in nude BALB/c mice was established. The nude mice were divided randomly into T-La(FS),RGDTLa(FS),T-LA and RGD-T-La groups,and injected i.v. with the corresponding drugs at a dosage of 600 μg/m L,100 μL for each,serving 1 mg/mL cisplatin solution as positive control and PBS as blank control. The effects of antitumor peptides on expression of vascular endothelial growth factor(VEGF)and apoptosis related protein Caspase-3 in melanoma cells were evaluated. Results RGD-T-La(FS)was the most sensitive to B16 cells. The survival rate of B16 cells was 25. 12%at RGD-T-La(FS) concentration of 10 μg/mL. However,the survival rates of B16 cells treated with T-La(FS) and RGD-T-La(FS) each at a concentration of 50 μg/mL were 6. 61% and 24. 65% respectively. The expression level ofαvβ3 in B16 cells was the highest,with a PE-A fluorescence intensity of 868,followed by those in H460,HepG2 and3 T3 cells,with PE-A fluorescence intensities of 840,446 and 265 respectively. T-La(FS)and RGD-T-La(FS)inhibited the expression of VEGF while induced the expression of Caspase-3, which induced the apoptosis of melanoma cells collaboratively. Conclusion This study provides a scientific basis for the application of small molecular antibacterial peptides as targeted antitumor peptides,and lays a theoretical foundation of the development and application of antitumor drugs in clinic.

    2019 09 v.32 [Abstract][OnlineView][Download 448K]

  • Preparation of national reference for anti-D(IgM)blood grouping reagent(monoclonal antibody)

    SUN Bin-yu;SUN Nan;HU Ze-bin;YU Ting;HUANG Jie;QU Shou-fang;Division of In Vitro Diagnostic Control,National Institutes for Food and Drug Control;

    Objective To prepare the national reference for anti-D(IgM)blood grouping reagent(monoclonal antibody).Methods The culture supernatant of anti-D(IgM)hybridoma was filtered by using a 0. 22 μm filter membrane,filled and lyophilized to prepare the national reference for anti-D(IgM)blood grouping reagent(monoclonal antibody). According to the relevant requirements in Industry Standard for RhD(IgM) Blood Grouping Reagent(Monoclonal Antibody),the prepared national reference was tested for appearance, pH, specificity, affinity and titer, and was calibrated in collaboration by four laboratories to evaluate the precision between containers and thermal stability. Results The national reference was a white freeze-dried powder,which was colorless or light yellow transparent liquid without precipitate not dispersed on shaking or foreign matter after reconstitution. The pH value of the reference was 7. 2,while the titer was1 ∶ 64. The reference showed agglutination with RhD positive cells while no agglutination with RhD negative cells. All the agglutinations appeared within 15 s,while the agglutination blocks formed in 3 min were more than 1 mm2 in size. Both the titers of two containers of reference in collaborative calibration were 1 ∶ 64. However,all the titers of references in stability test were 1 ∶ 64 after storage at 37 ℃ for 3,7 and 14 d. Conclusion The national reference for anti-D(IgM)blood grouping reagent(monoclonal antibody)was successfully prepared,which showed high precision between containers and stability,and might be used for the determination of titer of anti-D(IgM) blood grouping reagent(monoclonal antibody).

    2019 09 v.32 [Abstract][OnlineView][Download 163K]

  • Prokaryotic expression of Tachypleus tridentatus factor G

    WU Shang;HUANG Hui;YU Hua-jun;PANG Qi-ming;LU Xin-jian;ZHANG Hai-tao;Guangdong Medical University;

    Objective To express recombinant Tachypleus tridentatus factor G in E. coli and determine its enzyme activity. Methods According to codon preference of E. coli,the alpha and beta subunit genes of factor G were optimized and cloned into the prokaryotic expression vector p ET32 a-sumo. The constructed recombinant plasmids pET32 a-sumo/factorG-α and p ET32 a-sumo/factorG-β were transformed to E. coli BL21(DE3)and induced with IPTG. The expressed protein was purified by Ni-NTA affinity chromatography,while the tag protein was digested with SUMO protein to obtain recombinant target proteins factorG-α and factorG-β. Recombinant factor G was obtained by isomolar mass recombination,and determined for enzyme activity by using a fluorogenic peptide substrate Boc-Glu-(OBzl)-Gly-Arg-MCA. Results PCR and sequencing showed that recombinant plasmids pET32 a-sumo/factorG-α and pET32 a-sumo/factorG-β were constructed correctly. The expressed protein was in a form of inclusion body,which contained 30% and 45% of total somatic protein. The relative molecular masses of purified factorG-α and factorG-β were about 74 000 and 31 000,of which the recoveries in 1 L of LB medium were 0. 7 and 1. 7 mg,respectively. FactorG-α and factorG-β showed little reaction with fluorescent substrate. However,recombinant factor G showed strong reaction with the substrate.Conclusion Recombinant factor G with enzyme activity was expressed successfully,which laid a foundation of further study on the biological function of Tachypleus tridentatus factor G and the development of a new detection kit for fungal infection.

    2019 09 v.32 [Abstract][OnlineView][Download 296K]

  • Epidemiological characteristics and homology of foodborne Salmonella enteritis in Jilin Region,Jilin Province,China

    ZHOU Feng-yan;FENG Chun-ya;GUO Shi-jie;TAO Na;Center for Disease Prevention and Control of Jilin City;

    Objective To analyze the homology and molecular epidemiological characteristics of Salmonella enteritidis isolated from the fecal samples of patients with foodborne diseases in Jilin Region during 2014 ～ 2018 years by pulsed field gel electrophoresis(PFGE)and provide a data support for tracing to the source and early warning of prevention and control of the diseases. Methods The S. enteritidis strains isolated from 1 000 fecal samples of patients with foodborne diseases in Jilin Region during 2014 ～ 2018 were identified,determined for serotype and subjected to PFGE molecular typing. The data on PFGE profile were analyzed by using Bio Numerics version 6. 6 software,based on which a dendrogram was constructed. Results A total of 51 strains of S. enteritidis of serotype O(1,9,12):H(g,m) were obtained,indicating a postivie rate of 5. 1%. Clustering analysis showed a fingerprint similarity of 51. 39% ～ 100%.Twelve kinds of fingerprints were observed. The similarity of fingerprints PS3 and PS4 was 93. 7%,indicating that the strains with the two fingerprints were highly homologous and were prominent in recent years. Conclusion PFGE classification results reflect the diversity,complexity and prominent clones of PFGE type of S. enteritidis in Jilin Region.

    2019 09 v.32 [Abstract][OnlineView][Download 429K]

  • Immune effect of live attenuated Japanese encephalitis vaccine on virus strains of major circulating genotypes in China

    LIU Xin-yu;LU Xu;ZHAO Dan-hua;XU Hong-shan;LI Yu-hua;National Institutes for Food and Drug Control,Key Laboratory of the Ministry of Health for Research on Quality and Standardization of Biotech Products;

    Objective To evaluate the immune effect of live attenuated Japanese encephalitis(JE)vaccine prepared with SA14-14-2 strain on JE virus(JEV)strains of major circulating genotypes(Ⅰand Ⅲ)in China. Methods Serum samples were collected from healthy children before and 28 d after immunization with live attenuated JE vaccine,and determined for the neutralizing antibody against JEV of genotypesⅠand Ⅲ by plaque reduction neutralization test(PRNT)to calculate the neutralizing antibody positive conversion rate. Results Immunization with live attenuated JE vaccine induced high neutralizing antibody levels against JEV genotypesⅠand Ⅲ,with seroconversion rates of 90. 0% and 92. 0% and GMTs of 1 ∶ 26. 2 and 1 ∶ 27. 5,respectively,which showed no significant difference(P > 0. 05). Conclusion Live attenuated JE vaccine showed good immune effect on JEV of genotypesⅠand Ⅲepidemic in China.

    2019 09 v.32 [Abstract][OnlineView][Download 161K]

  • Development and verification of quantitative 1H-NMR method for determination of capsular polysaccharide of Haemophilus influenzae type b

    XU Mei-feng;MAO Qi-qi;ZHAO Dan;CHEN Su-jing;LI Ya-nan;LI Mao-guang;YE Qiang;National Institutes for Food and Drug Control,Key Laboratory of the Ministry of Health for Research on Quality and Standardization of Biotech Products;

    Objective To develop and verify a method for determination of the absolute content of capsular polysaccharide of Haemophilus influenzae type b(Hib)by quantitative proton nuclear magnetic resonance(~1H-NMR). Methods Hib capsular polysaccharide powder was weighed precisely and dissolved with heavy water to prepare the test sample solution at a certain concentration. The quantitative~1H-NMR was developed by using Bruker Avance-600 analyzer,serving heavy water as solvent and dimethyl sulfone as internal standard,of which the parameters were set. The developed method was verified for specificity,accuracy,linearity,precision and durability,and used for determination of capsular polysaccharide content in five batches of Hib. Results The~1H-NMR,~(13)C-NMR,~1H-~1H COSY and ~1H-~(13)C HSQC spectra showed that that the quantitative proton signal of sample(δ_(H)5. 06)and internal standard(δ_(H)3. 14)were separated well without any mutual interference or the interference of other peaks in the spectra,indicating high specificity of the developed method.The spike recoveries of samples added with 100,1 50 and 200 μL international standard were 101% ～ 102%. The linearity range of the method was 1 ～ 20 mg/mL,with an equation of y = 0. 234 3 x-0. 002 6(R~2 = 1. 000 0). The RSDs of quantitative peak integral area ratios of test samples determined in six reproducibility tests and in intermediate precision test were 0. 78% and 0. 60% respectively,indicating a high precision. The RSDs of quantitative peak integral area ratios of test sample solution at a concentration of 5 mg/mL at various pulse angles and various relaxation delays were 0. 07%and 0. 13% respectively,indicating a good robustness. The capsular polysaccharide content in five batches of Hib determined by~1H-NMR and chemical method met the requirements(320 ～ 410 mg/g)in Chinese Pharmacopoeia(Volume Ⅲ,2015 edition). Conclusion The quantitative ~1H-NMR method for determination of capsular polysaccharide content in Hib was developed successfully,which showed good specificity,accuracy,linearity,precision and robustness. It laid a foundation of study on quality control of Hib vaccine.

    2019 09 v.32 [Abstract][OnlineView][Download 294K]

  • Development,validation and primary application of a single dilution enzyme-linked immunosorbent assay for human papillomavirus specific antibody

    ZHAO Hui;PAN Hui-rong;LI Juan;LIN Zhi-jie;LIN Bi-zhen;LI Chang-gui;National Institutes for Food and Drug Control,Key laboratory of the Ministry of Health for Research on Quality and Standardization of Biotech Products,Division of Respiratory Virus Vaccines;

    Objective To develop,validate and apply a single dilution enzyme linked immunosorbent assay(ELISA)for human papillomavirus(HPV)specific antibody. Methods A single dilution ELISA was developed using HPV16 and the virus-like particles(VLPs) of HPV18 L1 as coating antigens,in which the serum samples before immunization were diluted by 100 folds,while those after immunization by 500 folds. The developed method was validated for specificity,accuracy,linear range,detection limit and precision,by which the serum samples of 28 patients naturally infected with HPV and 28 subjects vaccinated were determined,and the results were compared with those by pseudovirus-based neutralization assay(PBNA). Results All the 100 negative serum samples were negative for HPV16 and HPV18 IgGs.The mean recovery rates of HPV16 and HPV18 antibody titers were 74% and 73%,while the quantitative ranges were 0. 049 ～ 0. 370 and 0. 030 ～ 0. 231 IU/mL,respectively. The CVs in reproducibility test was 2. 9% ～ 6. 6%,while those in inter-precision test was 7. 7% ～ 11. 9%. The measured values of serum samples of subjects vaccinated were significantly higher than those of patients naturally infected(P < 0. 05),which was significantly related to the determination result by PBNA(the r values of determination results of HPV16 and HPV18 antibodies were 0. 727 and0. 800 respectively). Conclusion The single dilution ELISA for HPV specific antibody was developed,which showed high specificity,accuracy and precision,and might be used for the evaluation of immune effect of HPV vaccine and the monitoring of antibody level of the vaccine in market.

    2019 09 v.32 [Abstract][OnlineView][Download 244K]

  • Determination of antibody coupling efficiency on nanoscale polymer micelle surface by flow cytometry

    TIAN Jiu-bo;ZHANG Li-na;WANG Xuan;MA Pei-yuan;MU Xiu-li;MENG Tao-tao;MENG Fan-xiu;GONG Tao;YU Bao-feng;Department of Biochemistry and Molecular Biology,Shanxi Medical University;

    Objective To develop a method for determination of antibody coupling efficiency on nanoscale polymer micelle(PM)surface by flow cytometry(FCM). Methods DMPE-PEG-Maleimide,DPPE-PEG-Maleimide,DSPE-PEGMaleimide,DOPE-PEG-Maleimide and DLPE-PEG-Maleimide nanoscale polymer micelles were co-incubated with AntiGOLPH2 antibody for coupling,then co-incubated with SNU-423 cells. The positive rate of GOLPH2 antigen on SNU-423 cell membrane surface was determined by FCM,and the relative coupling efficiencies of polymer nanomicelles with AntiGOLPH2 antibody were determined. Results The positive rate of GOLPH2 antigen on SNU-423 cell surface was 1. 23%.The relative coupling efficiency of DLPE-PEG-Maleimide and Anti-GOLPH2 antibody was the highest in various nanoscale polymer micelles,which was positively correlated to the antibody concentration. Conclusion FCM for determination of antibody coupling efficiency on nanoscale polymer micelle surface reflected the coupling of antibody to nanoscale polymer micelle at a certain degree,of which the result was of a certain reference significance.

    2019 09 v.32 [Abstract][OnlineView][Download 311K]

  • Analysis of recombinant human erythropoietin isoform content by two capillary electrophoresis

    LI Xiang;SHI Xin-chang;YU Lei;ZHOU Yong;RAO Chun-ming;Key Laboratory of the Ministry of Health for Research on Quality and Standardization of Biotech Products,Division of Recombinant Biological Products,National Institute of Food and Drug Control;

    Objective To analyze recombinant human erythropoietin(rh EPO) isoform content by whole column imaging detection-capillary isoelectric focusing electrophoresis(WCID-CIEF)and capillary zone electrophoresis(CZE). Methods The contents of isoforms in five batches of bulk of rh EPO were determined by WCID-CIEF and CZE respectively. In WCID-CIEF,the mixture of 4 mol/L urea,0. 35% methylcellulose and 4% pharmalyte was used as sample buffer,while the focusing voltage was set as 3 k V,the prefocusing time as 1 min,the focusing time as 7 min,and the detection wavelength as 280 nm. However,in CZE,the mixture of 0. 01 mol/L sodium chloride,0. 01 mol/L tricine,0. 01 mol/L sodium acetate,7 mol/L urea and 2. 5 mmol/L putrescine was used as running buffer(pH 5. 55),while the pressure for sample injection was set as 3. 45 kPa,while the time for injection as 10 s,separation voltage as 15. 4 kV,the separation time as 80 min,and detection wavelength as 214 nm. Results Both WCID-CIEF and CZE showed 5 ～ 8 isoforms in five batches of bulk of rh EPO. The statistic results showed no significant difference between the contents of isoforms determined by the two methods(P > 0. 05). However,the determination results by the two methods showed significant correlation(r = 0. 999), and the difference in the percentages of the corresponding peak areas was less than3%. Conclusion Though the meanings of WCID-CIEF and CZE spectra are slightly different,the determination results by the two methods are basically in agreement,both of which meet the requirement for quality control of rhEPO isoforms.

    2019 09 v.32 [Abstract][OnlineView][Download 230K]

  • Development of a method for evaluation of influence of virus inactivation/removal procedure on biological activity of anti-CD20 monoclonal antibody

    LIU Chun-yu;ZHANG Feng;YU Chuan-fei;WU Gang;CUI Yong-fei;HUANG Jing;WANG Lan;Division of Monoclonal Antibody,National Institutes for Food and Drug Control,Key Laboratory of the Ministry of Health for Research on Quality and Standardization of Biotech Products;

    Objective To develop a method for evaluation of the influence of virus inactivation/removal procedure on biological activity of anti-CD20 monoclonal antibody(mAb). Methods The biological activity of anti-CD20 mAb was determined by complement dependent cytotoxicity(CDC) test with CCK-8 reagent,using Raji cells as target cells. The method was verified for systemic suitability. The relative potencies in percentage of test samples before and after virus inactivation/removal by low pH inactivation and nanofiltration were calculated by using anti-CD20 mAb reference,and the result was analyzed by using statistic software. Results The activity of anti-CD20 mAb was dose-dependent,of which the determination result complied with the following four-parameter equation:y =(A-D)/[1 +(x/C)B]+ D. The method possessed good stability and effectiveness with dose-response curves paralleling to each other. The relative potencies of samples before and after virus inactivation by low pH incubation were(97. 22 ± 2. 49)% and(87. 84 ± 5. 60)%respectively,of which the quadratic mean of relative variation was 10. 57%. However,the relative potencies before and after virus removal by nanofiltration were(87. 65 ± 4. 31)% and(89. 43 ± 7. 33)% respectively,of which the quadratic mean of relative variation was 4. 17%. No significant differences were observed in the data of one-way analysis of variance(ANOVA) among 3 tests or in those of paired t-test between samples before and after virus inactivation/removal procedure(P > 0. 05). Conclusion A method for evaluation of the influence of virus inactivation/removal procedure on the potency of anti-CD20 mAb was developed,which showed good parallelism,stability and effectiveness. It provided an experimental basis for the quality control of other therapeutic mAb drugs.

    2019 09 v.32 [Abstract][OnlineView][Download 250K]

  • Effect of anion exchange chromatography on human immunoglobulin(pH 4)for intravenous injection products

    ZHANG Xue-cheng;MA Xiao-wei;AN Wen-qi;LIANG Xue-shuang;LUO Jian;WANG Cheng-xu;WU Jing-juan;LI Guang-fei;Hualan Biological Engineering Inc.;

    Objective To analyze the effect of anion exchange chromatography(AEX) on quality of human immunoglobulin(pH 4)for intravenous injection products based on low temperature ethanol protein separation. Methods Human immunoglobulin(pH 4)for intravenous injection prepared by low-temperature ethanol protein separation method alone and in combination with AEX were determined for molecular weight distribution,anti-complement activity(ACA),anti-HBs titer,diphtheria antibody titer,IgA content,antibody Fc fragment activity,IgG subtype distribution,N-glycosylation type,secondary and tertiary structures of protein,and the results were compared and analyzed. Results Based on the low temperature ethanol protein separation method,the introduction of AEX decreased the IgA content in human immunoglobulin(p H 4) for intravenous injection products significantly,while was beneficial to removal of proteins with tertiary structure damaged,which showed no significant impact on other quality indicators of the product. Conclusion Based on the low temperature ethanol protein separation method,the introduction of AEX improved the quality of human immunoglobulin(pH 4)for intravenous injection products.

    2019 09 v.32 [Abstract][OnlineView][Download 327K]

  • Development of determination range of IgG against pneumococcal capsular polysaccharide of 11 serotypes in quality control serum 09CS

    SHI Gang;LI Hong;GUO Li-na;LU Xu;MAO Qi-qi;CHEN Xiao-hang;WANG Xing-ru;CHEN Cui-ping;YE Qiang;Key Laboratory of the Ministry of Health for Research on Quality and Standardization of Biotech Products,Division of Respiratory Bacterial Vaccines,National Institutes for Food and Drug Control;

    Objective To develop the determination range of IgG against capsular polysaccharide of pneumococcus(Pn)of 11 serotypes(2,8,9 N,10 A,11 A,12 F,15 B,17 F,20,22 F and 33 F) in quality control serum 09 CS. Methods Cell wall C polysaccharide(CPS) was mixed with polysaccharide of serotypes of Pn22 F and Pn25 to prepare sample absorbent solutions CPS + Pn22 F and CPS + Pn25 respectively,with which 09 CS serum was diluted and determined for IgG levels of 13 serotypes in 13 valent pneumococcal conjugate vaccine(13 PCV)to verify the consistency of test results with two absorbent solutions. The 09 CS was diluted with CPS + Pn25 and determined for 52 times by ELISA serving the second generation of international reference serum 007 sp as standard serum(diluted with the absorbent solution containing only CPS)and the first generation of international reference serum 89 SF as the quality control serum. The GMCs of IgG of11 serotypes as well as the SD,CV(%) and the normal distribution range in 99% confidence interval of U test were calculated. Results The determination results of 13 serotypes of IgG in 09 CS serum were consistent(r2 = 0. 983 5),which showed no significant difference(P > 0. 05). All the abnormal rates of effective calibration numbers of the 11 serotypes were less than 20%. The abnormal rate of Pn20 was the highest,which was 17. 3%. The CVs of determination results of various serotypes were 7. 55% ～ 12. 86%,all of which were less than 15%. The determination ranges of IgG levels for quality control of serotypes Pn2,Pn8,Pn9 N,Pn10 A,Pn11 A,Pn12 F,Pn15 B,Pn17 F,Pn20,Pn22 F and Pn33 F were 18. 56 ～ 31. 45,13. 35 ～ 26. 61,11. 90 ～ 23. 63,14. 23 ～ 25. 74,5. 96 ～ 8. 84,3. 70 ～ 6. 47,23. 89 ～37. 50,10. 68 ～ 16. 90,15. 97 ～ 24. 31,10. 63 ～ 17. 61 and 24. 24 ～ 47. 55 μg/mL respectively. Conclusion The determination range of IgG against capsular polysaccharide of Pn of 11 serotypes in quality control serum 09 CS was developed,which might be used for clinical serum assay of IgG level against capsular polysaccharide in Pn vaccine.

    2019 09 v.32 [Abstract][OnlineView][Download 224K]

  • Progress in research on biological drugs targeting IL-6 signal pathway

    JIA Chun-cui;RAO Chun-ming;YU Lei;National Institutes for Food and Drug Control;

    Interleukin-6(IL-6) is a multifunctional cytokine that functions by formation of a complex with its receptors and signal transduction. In view of the important role of IL-6 in various diseases,with the deepening of research on IL-6 signal pathway system,drug screening and drug design targeting IL-6 and its receptors IL-6 R,gp130 and sIL-6 R will provide new approaches for treatment of the diseases. This paper reviews the progress in research on biological drugs targeting IL-6 signal pathway.

    2019 09 v.32 [Abstract][OnlineView][Download 220K]


@2012 Editorial Department of "Chinese Journal of Biologicals"