2019年03期目次

  • Biological characteristics of mice infected with herpes simplex virus type-Ⅰmutant strain M3 and live attenuated varicella-zoster virus vaccine Oka strain

    LIU Lei;XU Xing-li;FENG Min;HE Yu-feng;WANG Li-chun;ZHANG Ying;LI Qi-han;Yunnan Key Laboratory of Vaccine Research and Development on severe Infection Disease,Institute of medical Biology,Chinese Academy of Medical Science and Peking Union Medical College;

    Objective To compare the biological characteristics of BALB/c mice infected with herpes simplex virus type-Ⅰ(HSV-Ⅰ)mutant strain M3 and live attenuated varicella-zoster virus(VZV)vaccine Oka strain. Methods Mice were infected with HSV-ⅠM3 strain at various dosages(103,104 and 105 PFU)and VZV Oka strain at vaccine dosage respectively by various(intraperitoneal,intramuscular,nasal and foot pad)routes,of which the biological characteristics such as acute clinical reaction,body weight,virus proliferation,histopathologic lesion and virus reactivation were compared.Results No obvious clinical symptoms of acute reactions such as dorsal bow,inverted hair and blindness of unilateral eye were observed in the mice in HSV-ⅠM3 and VZV Oka groups,while the bodyweights increased gradually. The virus copy numbers in organ and tissue of mice in HSV-ⅠM3 and VZV Oka groups were small,which showed no significant difference(P > 0. 05).The brain tissue of mice showed mild local inflammatory cell infiltration,hemorrhage,colloidal nodule or no abnormality,which showed little difference in HSV-ⅠM3 and VZV Oka groups. However,no obvious abnormalities were observed in spinal cord tissue of various groups. No CPE was observed in the co-culture of nerve tissue and Vero cells. Conclusion The biological characteristics of mice infected with HSV-ⅠM3 strain and VZV Oka strain showed no significant difference.

    2019 03 v.32 [Abstract][OnlineView][Download 515K]

  • Effect of VP1 proteins of enterovirus 71 and coxsakievirus A 16 on expression of natural immunity-associated signaling molecules and immune response of T cells in human bronchial epithelial cells

    LI Hao;ZHANG Ying;WANG Li-chun;JIANG Li;FAN Sheng-tao;YANG Er-xia;DONG Cheng-hong;JIANG Guo-run;ZHANG Xiao-long;LIAN Ya-ru;LI Qi-han;Institute of Medical Biology,Chinese Academy of Medical Sciences and Peking Union Medical College,Yunnan Key Laboratory of Vaccine Research and Development on Severe Infectious Diseases;

    Objective To investigate the effect of VP1 proteins of enterovirus(EV)71 and coxsakievirus A 16(CA16)on expression of natural immunity-associated signaling molecules and the immune response of T cells in human bronchial epithelial cells. Methods The VP1 protein sequences of EV71 and CA16 were amplified by RT-PCR and inserted into vector pcDNA3. 1(+). The constructed recombinant plasmids pcDNA-EV71-VP1 and pcDNA-CA16-VP1 were transfected to human bronchial epithelial cell line 16 HBE respectively. The cells were collected 24,48 and 72 h after transfection,and determined for the expression of natural immunity-associated signaling molecules and the proliferation of T cells. Results Restriction analysis and sequencing proved that recombinant plasmids pcDNA-EV71-VP1 and pcDNA-CA16-VP1 were correctly constructed. Compared with those in negative control group,the expression levels of IFNγ,OX40 L,RANKL,TNF-α and IL-2 in both EV71-VP1 and CA16-VP1 groups increased,while those in CA16-VP1 group showed a small increase. Both the intracellular and extracellular extracts of 16 HBE cells in EV71-VP1 and CA16-VP1 groups stimulated the proliferation of T cells secreting IFNγ significantly(P < 0. 05). Conclusion The VP1 proteins of EV71 and CA16 expressed in 16 HBE cells induced the differential expressions of natural immunity-associated signaling molecules and similarly nonspecific T cell proliferation.

    2019 03 v.32 [Abstract][OnlineView][Download 382K]

  • Glycan binding pattern of norovirus GⅡ.13P protein

    CONG Xin;LI Han-bo;WANG Peng-fei;JIN Miao;LI Dan-di;DUAN Zhao-jun;National Institute for Viral Disease Control and Prevention,Chinese Center for Diseases Control and Prevention;

    Objective To analyze the binding pattern between the P protein of GⅡ.13 strain(09 N3145)and histo-blood group antigens(HBGAs). Methods The P protein of G Ⅱ.13 09 N3145 was expressed and purified,of which the binding feature to various glycans was evaluated by saliva and glycan binding assays. A homology modeling of G Ⅱ.1309 N3145 P protein was built based on the complex structure of P protein of G Ⅱ.13/2010-Lea. Results G Ⅱ.1309 N3145 P protein showed binding to the types A,B and O+ secretor as well as O-non-secretor saliva. However,the P protein showed binding to Lec disaccharide antigens and lactose,while no significant binding to oligosaccharides of types A,B,H and Lewis. The homology modeling indicated that P protein bound to galactose ring using a potential glycan binding site. Conclusion The binding pattern between P protein of G Ⅱ.13 09 N3145 and HBGAs was determi-ned.These findings provided a basis for elucidating the mechanism of evolution,epidemiology and natural antiviral strategy of human norovirus(huNoV).

    2019 03 v.32 [Abstract][OnlineView][Download 225K]

  • Breeding of new high bacteriocin-producing Lactobacillus plantarum by complex mutagenesis

    AN Yu;WANG Ying;ZUO Zhao-hang;YI Hua-xi;ZHANG Dong-jie;College of Food Science,Heilongjiang Bayi Agricultural University;

    Objective To isolate high bacteriocin-producing Lactobacillus platarum from traditional fermented meat products in Northeast China,breed by complex mutagenesis and identify the obtained strain. Methods L. plantarum M1 strain with ability of decreasing cholesterol was subjected to four rounds of complex mutagenesis,consisting of microwave mutagenesis,nitroso-guanidin(NTG) mutagenesis,atomospheric room temperature plasma(ARTP) mutagenesis and ultraviolet mutagenesis,to obtain the L. plantarum strain producing broad-spectrum bacteriocin. The obtained strain was analyzed for stability,microbial morphological characteristics as well as physiological and biochemical characteristics,and identified by PCR. Results A high bacteriocin-producing L. plantarum was obtained by four rounds of complex mutagenesis,which showed genetic stability,excellent fermentation characteristics and antibacterial effect,was identified as L. plantarum and named as M1-UVs300,with an accession number of CGMCC No.7972. The bacteriocin secreted by the strain was named as M1-UVs301. Conclusion The L. plantarum M1-UVs300 with broad bacteriostatic spectrum as well as cholesterol lowering and antiseptic effects were obtained by four rounds of complex mutagenesis,which was worthy of popularization in industrial production.

    2019 03 v.32 [Abstract][OnlineView][Download 410K]

  • Paracrine effect of human amnioticepithelial cells and its influence on wound surface angiogenesis of diabetic rats

    LIU Chun-xiang;FU Yin-sheng;SUN Pei-ying;LU Hui-ying;SHI Yu-zheng;ZHANG Yi;National and Local Joint Stem Cell Research & Engineering Center for Aging Diseases;

    Objective To investigate the paracrine status of human amnioticepithelial cells(hAECs)and its role in wound healing and angiogenesis in diabetic skin lesions. Methods The phenotype of hAECs was analyzed by flow cytometry.The paracrine factor content in culture supernatant of hAECs was determined by ELISA. The wound healing effect of conditioned medium of h AECs on human umbilical vein endothelial cells(HUVECs)was evaluated by wound healing test.The skin lesion model of diabetic rats was established. The model rats were treated with hAECs,of which the CD34 expression and Cyclin D1 mRNA transcription levels in skin were determined. Results CD29,CD73 and CD105 as surface markers of mesenchyma stem cells(MSCs) and SSEA4 as surface marker of embryonic stem cells were highly expressed in hAECs,while CD44 and CD90 as surface markers of MSCs and CD45,CD34 and HLA-DR as surface markers of ematopoietic stem cells were low expressed. Vein endothelial growth factor(VEGF) was secreted in a large quantity,of which the expression level 72 h was significantly higher than those 24 and 48 h after culture and those in medium control group(P < 0. 05). Compared with the basal medium,the conditional media at two concentrations showed strong healing effects on HUVECs 24 and 48 h after scratch(P < 0. 01). However,the migration rates of HUVECs in conditional media at two concentrations showed no significant difference(P > 0. 05). Both the expression level of CD34 and transcription level of Cyclin D1 mRNA in skin of diabetic rats after treatment with hAECs were significantly higher than those in control group under the same condition(P < 0. 05). Conclusion The hAECs secreted VEGF in a large quantity and promoted the in vitro migration and proliferation of HUVECs as well as the skin vascular regeneration of diabetic rats through paracrine effect.

    2019 03 v.32 [Abstract][OnlineView][Download 588K]

  • Analysis of differentially expressed genes in MDBK cells infected with bovine viral diarrhea virus

    MA Li-li;CHEN Yue-ji;ZHAI Jun-jun;NI Hong-bo;College of Animal Science and Veterinary Medicine,Heilongjiang Bayi Agricultural University;

    Objective To analyze the transcriptomics of MDBK cells infected with bovine viral diarrhea virus(BVDV)by high-throughput sequencing(Illumina platform),and classify the function and investigate the relevant molecular mechanism of differentially expressed genes screened. Methods MDBK cells were infected with BVDV,of which the total RNA was extracted 12,24,48 and 72 h later respectively and analyzed for transcriptomics. The differentially expressed genes were subjected to function classification and enrichment analysis by using Kyoto Encyclopedia of Genes and Genomes(KEGG)and Gene Ontology(GO). Results There were 56,390,487 and 164 differentially expressed genes in the MDBK cells 0 ～12,13 ～ 24,25 ～ 48 and 49 ～ 72 h after infection as compared with those before infection respectively. However,there were 9 the same differential genes between the cells 12 and 24 h,while 55 between those 24 and 48 h,and 270 between those 48 and 72 h,after infection. KEGG and GO analyses showed the differential genes 24,48 and 72 h after infection were mainly enriched in P53,FoxO,NF-kB and PI3 K-AKT signaling pathways. The immunity-associated differential genes screened,such as P21,FOXO1 and GABARAPL1,were mainly concentrated in the signal pathways associated with cell cycle,apoptosis and immunity. Conclusion The differentially expressed genes screened were preliminarily classified,which provided a basis for molecular study on infection and intracellular replication of BVDV in MDBK cells.

    2019 03 v.32 [Abstract][OnlineView][Download 415K]

  • Karyotype analysis of engineered cells with recombinant human growth hormone Fc fusion protein

    ZHANG Yu;YU Lu;ZHNEG Quan-li;LIU Han;LIU Yun-nan;LIU Yu-lin;GUO Sheng-cang;XU Jia-hui;WANG Yu;Department of Cytokines,Changchun Institute of Biological Products Co.,Ltd.;

    Objective To analyze the karyotype of engineered CHO cells with recombinant human growth hormone(rhGH)Fc fusion protein(rhGH-CHO cells). Methods The cells in metaphase were treated with 0. 1 μg/m L colchicines,then fixed,stained and observed microscopically,and the visual fields with good morphology and dispersity of chromosomes were photographed. The images were processed by using Image-Pro Plus 6. 0(IPP)software,and the lengths of long and short arms of chromosomes in 50 CHO-k1 cells and 50 rhGH-CHO cells,each containing 22 chromosomes,were selected randomly and measured by manual measurements,based on which the relative lengths(the ratios of lengths of various chromosomes to that of the longest chromosome) and ratios of lengths of short arm/long arm of various chromosomes were calculated,and the B-Spline curve was plotted. Results Both the chromosome numbers of rhGH-CHO and CHO-k1 cells were 22,while the characteristics of B-Spline curves were in agreement. Conclusion The rhGH-CHO cells were derived from CHO-k1 cells,which were not contaminated with other cells.

    2019 03 v.32 [Abstract][OnlineView][Download 177K]

  • Comparative study on pathogenicity in mice and carriage of virulence genes of Proteus mirabilis isolates from various origins

    YIN You-qin;HAN Chun-hua;CUI Yi-long;SHI Yun;HUO Xiao-wei;MA De-hui;College of Animal Science and Technology,Inner Mongolia university for Nationalities;

    Objective To compare the pathogenicity in mice and carriage of virulence genes of Proteus mirabilis isolates from various origins in Tongliao Region,Inner Mongolia university for Nationalities,China. Methods Four strains of Proteus mirabilis from various origins,SPM,GPM,BPM-1 and BPM-2,were subjected to biochemical identification,drug sensitivity test and pathogenicity test in animals,of which the eight virulence genes(ureC,zapA,mrpA,ucaA,rsbA,pmfA,atf A and atfC) were detected by PCR. The 16 S r RNA gene sequences of four strains were analyzed for homology,based on which a phylogenetic tree was plotted. Results All the four strains were identified as P. mirabilis,to which the highly sensitive drugs included amikacin,meropenem,ceftazadine,cefotaxime,cefepime,taxoeillin and aztreonam. However,gentamicin,imipenem,cefazolin,ampicillin,clavulanic acid,colysin,sulfamethoxazole,chloramphenicol,ciprofloxacin and tetracycline were insensitive to any of the four strains. In the turn of median lethal dose,the four strains were BPM-2,BPM-1,SPM and GPM,while the virulence of GPM was the highest. The P. mirabilis strains showed different virulence gene profiles. All the eight virulence genes were detected in SPM,GPM and BPM-2,while ucaA gene was undetected in BPM-2. BPM-2 and BPM-1 belonged to the difference branches of the same clade.However,SPM and GPM belonged to difference clades. Conclusion P. mirabilis strains from different origins showed minor difference in pathogenicity to mice. The phenotype of pathogenic virulence gene might be correlated to the distribution of the gene.

    2019 03 v.32 [Abstract][OnlineView][Download 291K]

  • Effect of artemisinin on proliferation and apoptosis of breast cancer cells and relevant mechanism

    FAN Cong-li;WANG Hua;ZHANG Dao-yu;ZHANG Sheng;AN Xing-lan;LI Zi-yi;College of Veterinary Medicine,Jilin University;

    Objective To explore the effect of artemisinin(ART) on the proliferation and apoptosis of human breast cancer MDA-MB-231 and MCF-7 cells as well as the relevant mechanism. Methods The cells were divided into control and test groups,and treated with 1 μL/mL DMSO and ART at a final concentration of 300 μmol/L respectively. The colony formation abilities of human breast cancer cell lines and human breast epithelial cell line MCF-10 A after treatment with ART for 48 h were determined by plate cloning test. The expression of Ki-67 protein as a marker of cell proliferation was determined by immunofluorescent staining,while the apoptosis of human breast cancer and human breast epithelial cell lines by flow cytometry. The expressions of DNMTs and cancer suppressor genes FHIT,CDH1 and PTEN in human breast cancer cell lines were determined by RT-qPCR,while those of BAX,BCL-2,DNMT1 and DNMT3 A proteins by Western blot. The DNA methylation levels of FHIT and CDH1 genes were determined by bisulfite genomic sequencing method. Results ART significantly inhibited the proliferation and induced the apoptosis of breast cancer cells(P < 0. 01).ART also significantly induced the apoptosis of normal human epithelial cells(P < 0. 01). However,ART decreased DNMT1 expression and increased DNMT3 A expression(P < 0. 05). The DNA methylation levels of FHIT and CDH1 genes decreased in breast cancer cells after treatment with ART. Conclusion ART inhibits the proliferation and induces the apoptosis of breast cancer cells by changing the expressions of DNMT1 and DNMT3 A,resulting in reduced methylation and increased expression level of some anti-cancer genes such as FHIT and CDH1. The safety of ART in the treatment of breast cancer should be further evaluated.

    2019 03 v.32 [Abstract][OnlineView][Download 677K]

  • Prokaryotic expression of glutamate dehydrogenase of Clostridium difficile and development of a colloidal gold immunochromatography assay

    REN-Yong-xin;SUN Bin;XU Guang-xian;College of Animal Science and Veterinary Medicine,Heilongjiang Bayi Agriculture University;

    Objective To express the glutamate dehydrogenase(GDH)of Clostridium difficile(CD) in prokaryotic cells and develop a colloidal gold immunochromatography assay for GDH. Methods GDH gene was amplified by PCR using the whole genome of CD(ATCC BAA-1382)as a template and cloned into vector p ET-30 a(+). The constructed recombinant plasmid pET-30 a-GDH was transfected to E. coli BL21(DE3)and induced with IPTG for expression of recombinant GDH.The final concentration of IPTG(0,0. 2,0. 4,0. 6,0. 8 and 1. 0 mmol/L),time for induction(0,2,4 and 6 h)and temperature for induction(31,34 and 37 ℃) were optimized,and the expressed product was purified by Ni-NTA purification system. Rabbits were immunized with the purified GDH,and the obtained polyclonal antibody was used as the gold-labeled antibody to develop a colloidal gold test strip which was verified for sensitivity,specificity,precision and stability. Results Restriction analysis and sequencing proved that recombinant plasmid p ET-30 a-GDH was constructed correctly. The optimal temperature,final IPTG concentration and time for induction were 37℃,0. 8 mmol/L and 4 h respectively. The obtained immune serum reached a titer of 1 ∶ 51 200. The prepared colloidal gold strip,with a sensitivity of 60 ng/mL,showed no cross reactions with other intestinal bacteria while showed high stability,by which the precision test results showed no significant difference in intra-and inter-assays. Conclusion Recombinant GDH was expressed in prokaryotic cells,which showed high sensitivity,specificity,precision and stability,and was suitable for clinical application.

    2019 03 v.32 [Abstract][OnlineView][Download 561K]

  • Safety of bivalent oral live attenuated poliomyelitis vaccine in Dalian City,Liaoning Province,China

    YANG Yue;HAN Yi-nan;SUN Bo-yu;ZHOU Ling;Center for Disease Control and Prevention of Dalian;

    Objective To compare the incidence rates of Adverse Events Following Immunization(AEFI)of bivalent oral live attenuated poliomyelitis vaccine(bOPV),trivalent oral live attenuated poliomyelitis vaccine(t OPV)and inactivated poliovirus vaccine(IPV)and evaluate the vaccine safety. Methods Cases and doses of AEFI caused by all the vaccines(including those of classesⅠandⅡ)reported from 2009 to 2017 in Dalian City,Liaoning Province,China were collected and subjected to statistical analysis by descriptively epidemiologic method. Results Nineteen cases of AEFI caused by bOPV was reported,8 in males and 11 in females,indicating an incidence of 6. 88/100 000,which were significantly lower than the reported mean incidence of AEFI caused by all the vaccines but significantly higher than that by t OPV(each P < 0. 05),and showed no significant difference with that by IPV-Salk(P > 0. 05). Eleven cases appeared within1 d,while 7 cases 1 d and one case 15 d after vaccination,including 18 cases of general reaction and one case of abnormal reaction. The reported incidence rate of severe abnormal reaction was 0. 36/100 000,which showed no significant difference with those caused by all the vaccines and by t OPV(P > 0. 05). Conclusion The incidence rate of AEFI caused by bOPV was higher than that by t OPV and lower than that by all the vaccines,indicating a high safety.

    2019 03 v.32 [Abstract][OnlineView][Download 189K]

  • Change of epidemiological characteristics of mumps before and after implementation of the Expanded Program on Immunization in Liaoning Province,China

    FANG Xing;CHANG Lin;YAO Wen-qing;Liaoning Provincial Center for Disease Control and Prevention;

    Objective To analyze the change trend of epidemiological characteristics of mumps before and after the implementation of the Expanded Program on Immunization(EPI) in Liaoning Province, China and evaluate the effectiveness of introduction of mumps vaccine into the EPI so as to put forward suggestions on the prevention and control of mumps. Methods The epidemiological characteristics of mumps in Liaoning Province before(2005 ～ 2010)and after(2011 ～ 2016) implementation of EPI were analyzed and compared by epidemiological methods. Results Before and after the implementation of EPI,the annual average incidences of mumps were 24. 83/100 000 and 18. 38/100 000 respectively,which showed significant difference(P < 0. 05). Cases were reported in each month during 2005 ～ 2016,while the peak of incidence appeared in January,May,June and December. The proportions of cases in the months in which the peaks of incidence appeared before and after implementation of EPI were 45. 70% and 44. 30% respectively.The annual average incidences of mumps in 10 prefecture-level cities(71. 43%) decreased at a certain extent after implementation of EPI. The top three incidences appeared in students,children in nurseries and kindergartens and scattered children both before and after implementation of EPI,which accounted for 90. 08% and 84. 63% of total cases respectively(P < 0. 05). However,the proportions of cases in children at ages of less than 15 years were 78. 25% and73. 30% before and after implementation of EPI,which showed significant difference(P < 0. 05). Conclusion After the implementation of EPI,the incidence of mumps in Liaoning Province decreased significantly,though the trend of continued decline was not obvious. However,the incidence in children at ages of less than 14 years remained at a high level. It is suggested that two doses of measles,mumps and rubella combined vaccine(MMR)should be inoculated to the children at school age,and comprehensive measures should be taken to further decrease the incidence of mumps by eliminating measles.

    2019 03 v.32 [Abstract][OnlineView][Download 188K]

  • Optimization of purification procedure for phycocyanin from cyanobacteria from Chaohu Lake

    HE Tao;SU Yu;ZHANG Fa-yu;YU Jin-wei;WU Kang;WANG Jia-quan;School of Resources and Environmental Engineering,Hefei University of Technology;

    Objective To optimize the purification procedure for phycocyanin(PC)from cyanobacteria from Chaohu Lake.Methods Fresh cyanobacterial mud salvaged from Chaohu Lake was subjected to freeze-thawing for breakage,and the obtained crude extract of PC was purified by two-step salting-out and Cellfine A-500 anion exchange chromatography.The molar concentrations ammonia sulfate in the steps 1(0.8,1.0,1.2,1.4 and 1.6 mol/L)and 2(1.6,1.8,2.0,2.2 and 2.4 mol/L)of salting-out,pH value of buffer in anion exchange chromatography(6.0,6.5,7.0,7.5 and 8.0),sample concentration for loading(0.418,0.726,1.044,1.288 and 2.090 mg/L),elution rate(0.5,1.0,1.5,2.0,2.5 and 3.0 cm/min)and salt gradient of eluent[(0.08,0.10,1.0),(0.08,0.15 and 1.0),(0.08,0.20 and 1.0),(0.08、0.25、1.0),(0.08,0.30and 1.0)mol/L]were optimized using purity and recovery rate as indicators.Three batches of PC were purified under the optimal condition.Results The optimal molar concentrations of ammonia sulfate in steps 1 and 2 of salting-out were 1.0and 1.8 mol/L respectively,while optimal pH of buffer,sample concentration for loading,elution rate were 7.0,1.0～2.0 mol/L and 2 cm/min respectively.However,the concentration gradient of eluent for anion exchange chromatography was 0.08,0.20 and 1.0 mol/L.The purity of three batches of purified PC reached more than 4.0,while the recovery rate was more than 11%.Conclusion The purification procedure for phycocyanin from cyanobacteria from Chaohu Lake was optimized successfully,and the phycocyanin of reagent grade(with a purity of more than 4.0)was obtained.

    2019 03 v.32 [Abstract][OnlineView][Download 227K]

  • Establishment and verification of fluorescence quantitative PCR assay for Bacillus cereus in milk

    YANG Jian;HOU Ting-ting;ZUO Zhao-hang;GUO Yu;YAO Di;College of Food Science,Heilongjiang Bayi Agricultural University;

    Objective To establish and verify a fluorescence quantitative PCR assay for rapid detection of Bacillus cereus in milk. Methods Specific primers were designed according to the gyr B gene sequence of B. cereus in GenBank and the requirement of fluorescence quantitative PCR,based on which the bacterial DNA was extracted,and the target gene was cloned. Fluorescence quantitative PCR assay was performed using recombinant plasmid as a template,and a standard curve was plotted. Meanwhile,the fluorescence quantitative PCR assay was performed using DNA template of B. cereus,and the condition for amplification was determined. The established method was verified for specificity and sensitivity.Results The established fluorescence quantitative PCR assay showed no cross reactions with three common pathogenic bacteria in B. cereus,E. coli,Shigella shigae and salmonella. The minimum detection limit of the developed method was1 × 10-7 ng/L. The B. cereus at a concentration of 10. 4 CFU/mL in milk was detected by the method. However,by the method in GB 4789. 14-2014,only the B. cereus at a concentration of 1. 04 × 102 CFU/mL was detected.Conclusion The developed fluorescence quantitative PCR assay showed high specificity and sensitivity,which was suitable for rapid and accurate detection of B. cereus in milk. It provided a reference for rapid detection of pathogenic bacteria in laboratory.

    2019 03 v.32 [Abstract][OnlineView][Download 322K]

  • Optimization of production procedure for inactivated turbot Vibrio anguillarum vaccine

    SUN Cheng-wen;LI Jie;LAI Ying-tiao;JIANG Xiao-yan;MO Zhao-lan;HUANG Zhi-bin;TAO Jia-fa;River Fisheries Research Institute,Chinese Academy of Fisheries Sciences;

    Objective To optimize the production procedure for inactivated vaccine against turbot Vibrio anguillarum VAM003 strain.Methods The medium,(TSB and LB),sodium chloride concentration(1.5%,2.0%,2.5%,3.0%and 3.5%),inoculum size(5%,10%and 15%)and fermentation time(6～12 h)for fermentation of V.anguillarum VAM003 strain of turbot were optimized.Fermentation liquid was prepared under the optimized condition,and inactivated with formaldehyde at various final concentrations(0.1%,0.2%,0.3%and 0.4%)for 24,48 and 72 h to optimize the condition for inactivation.Two batches of inactivated vaccines were prepared with VAM003 strain by the optimized procedure and tested for safety and potency.Results The optimal fermentation condition for VAM003 strain of V.anguillarum was as follows:the strain was inoculated at an inoculum size of 10%into TSB medium containing 2.5%sodium chloride and cultured for 10～12 h until the viable cell count reached more than 5×109CFU/mL.However,the optimal final formaldehyde concentration for inactivation of V.anguillarum VAM003 strain was 0.2%,while the optimal inactivation time was 24 h.All the turbots injected with the inactivated vaccine survived without clinical symptoms,of which the food intake,swimming posture and body color were normal,and no redness and swelling in injection site or pathological change in organs were observed.The protective rate of vaccine was more than 90%.Conclusion The production procedure for inactivated vaccine against V.anguillarum VAM003 strain was successfully optimized,and the prepared inactivated vaccine showed high safety and potency and might be used for the prevention of Vibrio disease in turbots.

    2019 03 v.32 [Abstract][OnlineView][Download 227K]

  • Isolation,purification and identification of recombinant human lysozyme

    DENG Han;SHI Jin;HAO Dong;WANG Wei;ZHAO Jin-li;Shaanxi Huikang Bio-tech Co.,Ltd;

    Objective To develop a method for isolation and purification of recombinant human lysozyme(hLYZ)and identify the purified product. Methods The hLYZ was isolated and purified by solid-liquid separation,membrane filtration,hydrophobic chromatography and ion exchange chromatography,and determined for purity by SDS-PAGE and HPLC,for potency according to the method in Chinese Pharmacopoeia(VolumeⅡ,2015 edition),for specificity by Western blot,and for relative molecular mass by matrix-assisted laser desorption/ionization time of flight mass spec-trometry(MALDITOF). Results Recombinant hLYZ with a single component was isolated by the developed method,of which the purity reached 99% by SDS-PAGE and 98. 8% by HPLC. Purified hLYZ showed specific binding to human anti-LYZ antibody,of which the activity was 1. 2 × 105 U/mg,and the relative molecular mass was 14 697. Conclusion The developed method for isolation and purification of h LYZ was easy to handle and of low cost. The purity of obtained hLYZ was more than 98%. The method may be used for the large-scale production of hLYZ.

    2019 03 v.32 [Abstract][OnlineView][Download 235K]

  • Determination of arginine hydrochloride in human fibrinogen by high performance liquid chromatography with external standard

    ZHANG Xiao-jie;ZHENG Xia;ZHANG Jie;WANG Yu;ZHAO Qi;Shandong Taibang Biological Products Co.,Ltd;

    Objective To develop an external standard method for determination of arginine hydrochloride in human fibrinogen by high performance liquid chromatography(HPLC). Methods HPLC was performed on amino bond column(250 mm × 4. 6 mm × 5 μm)using the mobile phase consisting of acetonitrile-phosphate buffer(1 ∶ 1)at a flow rate of1. 0 mL/min. The detection wavelength was 205 nm,while the column temperature was 40 ℃,and the injection volume was 20 μL. The content of arginine hydrochloride in human fibrinogen was calculated by area external standard method.The developed method was verified for systemic suitability,linear range,reproducibility,stability,accuracy and limit of quantitative(LOQ),by which the arginine hydrochloride contents in three batches of human fibrinogen were determined,and the results were compared with those by the method in Chinese Pharmacopoeia(Volume Ⅲ,2015 edition). Results Single chromatographic peak without other interference was observed. The separation degree of glycine and arginine hydrochloride was 10. 54. The arginine hydrochloride at a concentration range of 0. 116 2 ～ 0. 536 1 mg/m L showed good linear relationship to the peak area,of which the linear equation was y = 7 476. 695 68 x + 72. 481 815,with a correlation coefficient(R value)of 0. 999 24. The relative standard deviation(RSD)of determination results of arginine hydrochloride content in the same samples in six repeat tests was 0. 22%. However,the RSD of determination results of the sample after storage at 4 ℃ for 0,4,8,12,16 and 20 h was 0. 57%. The mean recovery rate of samples at high,moderate and low concentrations was(101. 36 ± 1. 53)%,with a RSD of 1. 51%. The LOQ was 0. 581 μg/m L. The arginine hydrochloride contents in three bathes of samples determined by two methods were 25. 9 ～ 26. 3 g/L,which showed no significant difference(P > 0. 05). Conclusion The HPLC with external standard is simple,reproducible,stable and accurate,which may be used for the determination of arginine hydrochloride content in human fibrinogen.

    2019 03 v.32 [Abstract][OnlineView][Download 182K]

  • Effect of macromolecular crowding on enzyme protein and DNA

    QIN Ya-lan;TONG Jin;Department of Respiratory Medicine,The Second Affiliated Hospital of Chongqing Medical University;

    Macromolecular crowding is caused by the remarkable reduction of practically achievable space of any macromolecule due to the impermeability of macromolecules in the environment formed by proteins,polypeptides,nucleic acids and polysaccharides,which is a ubiquitous physiological phenomenon caused by spatial exclusion. Since enzyme plays an effective and specific catalytic role in organism,the change of molecular environment may cause the change of enzyme activity,thus affects the steady-state of cell and organism. Meanwhile,macromolecular crowding may influence the DNA synthesis. This paper reviews the influence of macromolecular crowding on enzyme(including the property,activity and optimal temperature of enzyme)and DNA as well as the possible mechanism.

    2019 03 v.32 [Abstract][OnlineView][Download 184K]

  • Progress in research on role of CircRNA in autoimmune diseases

    LEI Bo;XUAN Xiu-yun;FAN Wei-ping;Department of Microbiology and Immunology,Shanxi Medical University;

    Circular RNA(CircRNA)is a newly discovered,long chain and noncoding endogenous circular RNA molecule,which participates in the regulation of transcriptional and post transcriptional levels of genome and competes for endogenous RNA to bind miRNA specifically thus involves in the development,apoptosis and many diseases by regulating the expression of miRNA downstream target gene. The latest research has confirmed that CircRNA regulates the occurrence of autoimmune diseases such as rheumatoid arthritis(RA) systemic lupus erythematosus(SLE) and multiple sclerosis(MS),and is expected to be a diagnostic marker or therapeutic target for such diseases. This article reviews the current biological characteristics of CircRNA and progress in research on its role in autoimmune diseases.

    2019 03 v.32 [Abstract][OnlineView][Download 156K]

  • Advance in study of Rift Valley fever

    BI Jin-hao;YAN Fei-hu;HUANG Pei;ZHAO Yong-kun;YANG Song-tao;College of Animal Science,Jilin Agricultural University;

    Rift Valley fever(RVF)is a fever amphixenosis caused by Rift Valley fever virus(RVFV). It leads to massive death of ruminants and fatal hemorrhagic fever in humans. The virus was restricted to the eastern region of Africa initially,while is in spreading at present. The first imported case was diagnosed in China in 2016,suggesting a threat to nonepidemic countries or regions. The epidemic of RVF not only seriously influencs the health of humans and the development of animal husbandry,but also might be used as a potential biological warfare,thus has been listed to the notifiable epidemics by the OIE. This paper reviews the epidemiology,molecular biology and detection technology of RVF as well as the progress in vaccine research,so as to provide a reference for further research on RVF.

    2019 03 v.32 [Abstract][OnlineView][Download 205K]

  • Research status of type Ⅲ interferon

    YAN Jia-li;WANG Jian;National Vaccine and Serum Institute;

    Objective The earliest discovered cytokines are interferons(IFNs),which are known to interfere with the replication and infection of viruses. Type Ⅲ interferons with characteristics of interferons and interleukins were discovered in 2003 and called IFNλs(including IL-28 A,IL-28 B and IL-29). The receptor complex of type Ⅲ interferons is formed by a distinct heterodimer consisting of IL-28 Rα and IL-10 Rβ. Type Ⅲ interferons have antiviral,anti-tumor and immune regulation activities through JAK/STAT and other signal pathways. This paper reviews the progress in research on type Ⅲinterferons,especially that on biological activities.

    2019 03 v.32 [Abstract][OnlineView][Download 175K]

  • Production process and quality control of influenza vaccine from different matrixes

    LI Guang-pu;ZHANG Yan;ZHANG Fei-qi;BAI Peng-xun;Development Center of Drugs,Jilin Yatai Group Co.,Ltd.;

    Several millions of people are infected with influenza virus worldwide. The infection may cause severe symptoms even death. At present,vaccination is the most effective measure to control influenza. Because of the high mutation rate of influenza virus,the virus strain used for vaccine production should be changed every year to make the two major antigens,hemagglutinin(HA)and neuraminidase(NA),of the prepared vaccine match to those of epidemic virus strains and to ensure the effectiveness of vaccine. This paper reviews the marketing status,production process and quality control of influenza vaccine prepared with chick embryos and cells as matrixes.

    2019 03 v.32 [Abstract][OnlineView][Download 181K]


@2012 Editorial Department of "Chinese Journal of Biologicals"