2024年04期目次

  • Preclinical safety and immunogenicity evaluation of pre-sealed syringe inactivated tick-borne encephalitis vaccine

    CHEN Nana;LIU Chenyang;XING Kunpeng;GAO Qiang ;ZHANG Shuo;WANG Ying;SUN Hongliang;LI Jingliang;Changchun Institute of Biological Products Co.,Ltd.;

    Objective To evaluate the preclinical safety and immunogenicity of 0. 5 mL/dose of inactivated tick-borne encephalitis(TBE)vaccine with pre-sealed syringe.Methods The safety of the new packaged TBE inactivated vaccine was evaluated by using rabbit muscle irritation test and guinea pig systemic active anaphylaxis test,and compared with that of the original package(1. 0 mL/bottle,cillin bottle). Thirty Kunming mice with 10-12 g body mass were immunized i.p.with TBE inactivated vaccines of new package and original package twice at an interval of 7 d,respectively. At 14 d after the initial immunization,the experimental group was challenged with virus(TBEV of“Senzhang”strain)of 10~(-2)-10~(-6) dilution,while the control group with the virus of 10~(-6)-10~(-10) dilution by intraperitoneal administration. The results were observed continuously for 21 d,and the immunogenicity of the TBE inactivated vaccines was evaluated by calculating the titer using Reed-Muech method.Results No death or dying condition was observed in all the groups of animals in the rabbit irritation test,and no abnormal reaction was observed in all groups in the guinea pig systemic active allergic reaction test. The immuno-genicity(immune protection index)of three batches of TBE inactivated vaccines with original package was 1. 0×10~8,1. 3×10~8,1. 0×10~8,and that of three batches of TBE inactivated vaccines with new specification were 1. 1×10~8,1. 0×10~8 and 1. 3×10~8,respectively,which met the requirements of greater than 1. 0×10~5 in Chinese Pharmacopoeia(Volume Ⅲ,2020 edition),with no significant difference in immunogenicity between the vaccines of new and original specifications(F = 0. 063,P > 0. 05).Conclusion The 0. 5 mL/dose of pre-sealed syringe TBE inactivated vaccine has good preclinical safety and immunogenicity.

    2024 04 v.37 [Abstract][OnlineView][Download 882K]

  • Identification of candidate antigens of Proteus mirabilis vaccine and evaluation of immune protection effect

    ZHENG Hua;BAI Yunchuan ;YANG Rui;FU Lizhi;WANG Xiaoyou;ZHANG Suhui;SHEN Kefei;Chongqing Academy of Animal Sciences;

    Objective To identify the candidate antigens of Proteus mirabilis vaccine and evaluate their immune protection effect in mice. Methods The membrane proteins of P.mirabilis were extracted by bacterial membrane protein extraction kit,and were detected by Western blot and analyzed by mass spectrometry. The genes encoding outer membrane protein F(OmpF)and serine hydrolase(SH)were synthesized by chemical method,cloned into vector pET-28a,and induced to express in E.coli BL21(DE3). The recombinant proteins were detected by Western blot,purified by Ni Sepharose High Performance and then injected into the leg muscle of 15 female Kunming mice at a dose of 100 μg/(0. 2 mL per mouse)for three times with an interval of two weeks. The blood samples were collected through the anterior orbital vein 15 d after the last immunization,the serum was separated,and the specific antibody level was detected by indirect ELISA. The mice were intraperitoneally inoculated with a lethal dose of P.mirabilis. One month after inoculation,the survival of mice was observed,and the immune protection rates of antigens were calculated. Results The extracted membrane proteins showed specific binding with the antiserum of mice immunized with P.mirabilis,and the homology with APB87305(OmpF)and WP＿004247605(SH)was 100% as identified by mass spectrometry. The recombinant proteins OmpF and SH expressed in prokaryotic cells displayed specific binding with the antiserum of mice immunized with P.mirabilis. The immunized mice induced high levels of specific antibodies,and the protection rates against fatal P. mirabilis infection were 100% and 86. 67% respectively. Conclusion OmpF and SH are host immune protective antigens and promising new targets for P.mirabilis vaccines.

    2024 04 v.37 [Abstract][OnlineView][Download 1073K]

  • Immunogenicity of recombinant hepatitis B virus-like particles in mice

    WANG Weixiao;ZHAO Danying;CHEN Ziyang;CHANG Junliang;Changchun Institute of Biological Products Co.,Ltd.;

    Objective To evaluate the immunogenicity of recombinant hepatitis B virus-like particles(HBV VLPs)in mice.Methods A series of doses of NLMS vaccine,namely HBV VLPs containing NL,M and S proteins(0. 2,0. 1,0. 05,0. 025,0. 012 5 μg/mL)and marketed vaccine(4,2,1,0. 5,0. 25 μg/mL)were used to immunize female BALB/c mice intraperitoneally in a single dose,0. 5 mL/mouse,20 mice for each group. Four weeks later,the blood samples were collected from the inner canthal vein of mice,and the serum was separated. The positive conversion rate of serum HBsAb was detected by HBsAb detection kit,and the half effective dose(ED_(50))was calculated. The mice were immunized intraperitoneally with NLMS vaccine(1 μg/mL)and the marketed vaccine(20 μg/mL)at 0 and 2 weeks,0. 5 mL/mouse,separately. At 2,3,4,5,6 and 7 weeks after the initial immunization,the blood samples were collected from the inner canthus vein of mice,and the serum was separated,which was detected for the serum S antibody level by corresponding kit;the antibody levels of preS1 and preS2 and the contents of IFNγ,IL-2 and IL-6 in serum were detected by corresponding kits 7 weeks after the initial immunization. Results After 4 weeks of immunization,the ED_(50)of the NLMS vaccine was0. 039 μg/mL,with a 95% confidence interval of 0. 032-0. 049 μg/mL,and the ED_(50)of the marketed vaccine was1. 095 μg/mL,with a 95% confidence interval of 0. 881-1. 406 μg/mL. The difference in ED_(50)between the two groups was significant(χ~2= 34. 0,P < 0. 05). After two doses of immunization,the serum antibody titers in mice of both groups reached the highest at the 7th week after the initial immunization. The S antibody titer of NLMS vaccine group was 1∶4 096,the preS1 antibody titer was 1∶64,and the preS2 antibody titer was 1∶128;the S antibody titer in the marketed vaccine group was 1∶1 024,and the preS1 and preS2 antibodies were below the limit of detection. There were significant differences in the titer of three antibodies between the two groups(t =-2. 025,17. 433 and-7. 844,respectively,each P <0. 05). Both vaccines stimulated different levels of IFNγ,IL-2 and IL-6 in spleen lymphocytes of mice,which were significantly higher than those in negative control group(F = 250. 1,425. 6 and 652. 8,respectively,each P < 0. 05). Conclusion NLMS vaccine can induce stronger humoral immune response in mice,induce S,preS1 and preS2 antibodies at the same time,and can also induce corresponding cellular immune response.

    2024 04 v.37 [Abstract][OnlineView][Download 869K]

  • Regulation of Ca2+/NLRP3/Caspase-1 signaling pathway in atherosclerotic endothelial dysfunction

    FAN Jianghong;GUO Rui;GAO Jia;ZHAO Jing;WANG Jing;LIU Caihong;XIE Yaoli;WANG Yajing;College of Basic Medicine,Shanxi Medicial University;

    Objective To explore the regulation of Ca~(2+)/NLRP3/Caspase-1 signaling pathway on endothelial dysfunction in atherosclerosis(AS).Methods 16 male ApoE~(-/-)mice were randomly divided into a normal diet group and a high-fat diet group and measured for the weight on an empty stomach every week;The mice were fed continuously for 20 weeks and then sacrificed,of which the arterial tissues were collected;Serum cholesterol(TC),triglyceride(TG),low density lipoprotein(LDL-C)and high density lipoprotein(HDL-C)were detected by biochemical analyzer;The thickness of aortic luminal plaque and intima was assessed by oil red O staining. Human umbilical vein endothelial fusion cells EA.hy926 were divided into control group,high-sugar and high-fat group,and Ca~(2+)inhibitor group,which were detected for Ca~(2+)content by flow cytometry at different time periods,for the expression of NLRP3 by immunofluorescence,and for the protein expression levels of NLRP3,Caspase-1/p10/p20,VCAM-1 and ICAM-1 in aortic tissue and cells by Western blot.Results Compared with the normal diet group,the weight of the mice in high-fat diet group increased significantly(F = 3. 333,P =0. 026),the serum TC,TG and LDL-C significantly increased,while HDL-C significantly decreased(F = 1. 852,2. 410,1. 920 and 2. 917,P = 0. 000,0. 004,0. 002 and 0. 003,respectively);The aortic plaque surface area/aortic surface area increased significantly(F = 3. 256,P = 0. 000),plaque cross-sectional area/aortic lumen area increased significantly(F = 6. 433,P = 0. 008),and NLRP3,Caspase-1/p10/p20,VCAM-1 and ICAM-1 protein expressions increased significantly(F = 8. 997,4. 664,4. 486 and 9. 949,P = 0. 036,0. 022,0. 005 and 0. 018,respectively). Compared with the control group,the Ca~(2+)content of endothelial cells in high-sugar and high-fat group increased in the 6 h,12 h,and 24 h(F = 0. 310,5. 649 and 5. 580,P = 0. 006,0. 009 and 0. 004,respectively),especially in the 6 h group;The expression of NLRP3 in high-sugar and high-fat group increased significantly;VCAM-1,ICAM-1 and Caspase-1/p10/p20 protein expression increased significantly(F = 10. 476,5. 310 and 9. 306,P = 0. 029,0. 030 and 0. 018,respectively). Compared with the high-sugar and high-fat group,the expression of NLRP3 and Caspase1/p10/p20 protein in EA.hy926 cells of Ca~(2+)inhibitor group decreased significantly(F = 2. 196,2. 882 and 0. 035,P < 0. 01,< 0. 05 and < 0. 05,respectively).Conclusion NLRP3 inflammasome induced dysfunction of vascular endothelial cells and aggravated the progression of AS,which might be closely related to Ca~(2+)and downstream signaling pathways.

    2024 04 v.37 [Abstract][OnlineView][Download 1035K]

  • Inhibitory effect of miR-628-3p on progression of ovarian cancer by targeting STK17B gene

    YAO Huixin;GAO Dan;LI Huamin;YANG Bing;WANG Yuening;LI Huiying;SUN Zhe;LIU Feifei;Hongqi Hospital Affiliated to Mudanjiang Medical College;

    Objective To analyze the ability of miR-628-3p to inhibit the proliferation,migration and invasion of ovarian cancer(OV)by targeting serine/threonine kinase 17B(STK17B).Methods The expression level of miR-628-3p in OV tissues and prognosis were analyzed by bioinformatics. OV cells(SKOV3,HO8910)were transfected with miR-NC(miRNC group),miR-628-3p(miR-628-3p group)and miR-628-3p + STK17B(miR-628-3p + STK17B group). The expression level of miR-628-3p in OV cells after transfection was determined by RT-qPCR. Cells proliferation ability in each group was measured by CCK-8 and clone assays,the migration ability was detected by scratch assay,and the invasion ability was detected by Transwell invasion assay. The mass and volume of tumors were detected by tumor formation assay in nude mice. The target gene of miR-628-3p was predicted by Starbase databases,which was verified by dual luciferase reporter assay. The expression level of STK17B protein in each group was detected by Western blot.Results The expression level of miR-628-3p was low in OV tissues,and the disease-free survival(DFS)was short in patients. Compared with miR-NC group,the miR-628-3p expression was up-regulated in both SKOV3 and HO8910 cells of miR-628-3p group(t = 7. 789 and7. 862,respectively,each P < 0. 05),the activity(t = 8. 124 and 8. 209,respectively,each P < 0. 05),numbers(t =9. 012 and 9. 110,respectively,each P < 0. 05),migration(t = 10. 002 and 9. 983,respectively,each P < 0. 05)and invasion(t = 11. 245 and 11. 320,respectively,each P < 0. 05)abilities of OV cells were significantly decreased. Compared with miR-NC group,the volume and mass of transplanted tumors were significantly reduced in miR-628-3p group(t =8. 429 and 10. 367,respectively,each P < 0. 05). It was predicted by databases that STK17B was the target gene of miR-628-3p,which was verified by dual luciferase reporter assay. The levels of STK17B mRNA(t = 13. 852 and 13. 914,respectively,each P < 0. 05)and protein(t = 11. 524 and 11. 603,respectively,each P < 0. 05)in miR-628-3p group were lower than those in miR-NC group. GEPIA data analysis showed that STK17B was highly expressed in OV tissues.Compared with miR-628-3p group,the activity(t = 8. 692 and 8. 721,respectively,each P < 0. 05),numbers(t = 14. 112and 14. 093,respectively,each P < 0. 05),migration(t = 13. 805 and 13. 911,respectively,each P < 0. 05)and invasion(t = 14. 117 and 14. 207,respectively,each P < 0. 05)abilities of SKOV3 and HO8910 cells significantly increased in miR-628-3p + STK17B group.Conclusion The miR-628-3p inhibits the proliferation,migration and invasion of OV cells by down-regulating STK17B expression,thereby inhibiting the development of OV.

    2024 04 v.37 [Abstract][OnlineView][Download 1370K]

  • Establishment of chronic asthma mouse model induced by allergen ovalbumin and dynamic analysis on its immune response characteristics

    YAN Jiao;BAI Hongmei;YANG Xu;SUN Wenjia;LONG Qiong;MA Yanbing;Institute of Medical Biology,Chinese Academy of Medical Sciences & Peking Union Medical College;

    Objective To establish a chronic asthma mouse model induced by allergen ovalbumin(OVA),and analyze the immune response dynamic characteristics. Methods Twenty-two female BALB/c mice were sensitized intraperitoneally with OVA,and the airway response was stimulated by repeated infusion through nasal cavity. Samples were taken at different time points for analysis. The bronchoalveolar lavage fluid(BALF)of mice was collected and inflammatory cells were counted;the supernatant of lavage fluid was taken and detected for the levels of various related cytokines by ELISA. The lung tissue was taken to prepare sections,which was detected for the inflammatory cell infiltration by hematoxylin-eosin(HE)staining,for the goblet cell hyperplasia in airway epithelium by periodic acid-Schiff(PAS)staining,and for the collagen deposition by Masson′s Trichrome and Sircol staining. The venous blood of mice was collected,the serum was separated,and the levels of OVA-specific IgE,IgG1 and IgG2a in serum were detected by ELISA. The airway reactivity was measured using a FlexiVent small animal ventilator with low-frequency forced oscillation. Results Repeated challenges of OVA successfully induced typical asthma features such as inflammatory cell response in airway and lung tissues,inflammatory cytokine accumulation,airway goblet cell hyperplasia,lung fibrosis,allergen-specific IgE antibody response and airway hyperresponsiveness in mice,and different dynamic responsive characteristics appeared. With the repeated challenges of OVA,inflammatory cell response and goblet cell hyperplasia reduced,but collagen deposition developed,while airway hyperresponsiveness remained 4 weeks after the last stimulation. The expression of TGF-β1 was high during the inflammatory response,while the expression of TGF-β2 and IL-33 did not increase until 4 weeks after the last challenge. Conclusion This study successfully developed a chronic asthma model in mice,showing the immune response characteristics,laying a foundation for investigating immunopathological mechanisms of chronic asthma and developing therapeutic targets and strategies.

    2024 04 v.37 [Abstract][OnlineView][Download 1163K]

  • Effect of gene reassortment on growth characteristics of influenza B virus

    ZHANG Yao;ZHOU Jianfang ;LU Jian ;LI Xiyan ;ZENG Xiaoxu ;WANG Dayan;Microbiological Laboratory,Dongcheng District Center for Disease Control and Prevention;

    Objective To study the influence of various gene segments of influenza virus on its growth characteristics by using the reverse genetics system.Methods The growth curves of inter-lineage reassortment viruses B/FujianTongan/1565/2013 and B/BeijingHuairou/11764/2013 were determined. The nucleic acids of the two strains were extracted and all gene segments were amplified,then pHW2000 vector was used to construct recombinant plasmids of all segments.The reassortment B/FujianTongan/1565/2013 virus was constructed,then single or different combinations of segments were replaced by segments from B/BeijingHuairou/11764/2013 virus to construct new reassortment viruses. The growth characteristics of all reassortment viruses were analyzed to identify the influence of the gene segments.Results There was significant difference in growth characteristics between the two strains,and the peak value of the growth curve of B/FujianTongan/1565/2013 was about 2 times log higher than that of B/BeijingHuairou/11764/2013. However,there was no significant difference in growth characteristics between B/FujianTongan/1565/2013 and the 8 strains of reassortment viruses with single segment replaced by segment from B/BeijingHuairou/11764/2013,but when the gene combination of HA and NA(surface protein)or PB2,PB1 and PA(polymerase)or PB2,PB1,PA and NP(ribonucleoprotein,RNP)were replaced,the growth characteristics of the reassortment virus decreased significantly.Conclusion The growth characteristics of influenza B virus may be less affected by a single segment,and the synergistic effect of multiple segments is more significant. Therefore,the monitoring of reassortment variant viruses should be strengthened in the surveillance process.

    2024 04 v.37 [Abstract][OnlineView][Download 1201K]

  • Effect of myxobacteria metabolites on glioma

    LI Songyuan;LI Junda;ZHANG Xi;MA Tianyu;LIU Tao;LIU Huirong;College of Life Sciences,Inner Mongolia Agricultural University;

    Objective To isolate and purify some myxobacteria from Ordos,Inner Mongolia Autonomous Region,detect the inhibitory effect of their metabolites on human glioma cell line U87,and identify the types of the metabolites.Methods Using filter paper induction method,E.coli marking method and rabbit feces induction method,the soil samples of myxobacteria collected in Ordos area of Inner Mongolia Autonomous Region were isolated and purified. The purified strains were fermented,the inhibition rate of the fermentation broth on U87 cells was detected by CCK-8 assay,and the metabolites were qualitatively analyzed by chemical chromogenic method.Results Sixteen strains of myxobacteria were isolated and purified,belonging to five genera of myxobacteria. The fermentation supernatant had no inhibitory effect on U87 cells,while the methanol extract of macroporous resin had significant inhibitory effect on U87 cells. Except strain E19,the average highest inhibitory rate of all strains was 80%. There were significant differences in the types of secondary metabolites among different species of strains,among which the metabolites of Corallococcus contained the most species and exhibited the strongest inhibitory effect on the cells,with an inhibition rate of 97. 41%.Conclusion The natural products of myxobacteria can be used as a new anti-glioma drug source for in-depth study,and the related work can provide a reference for the development of new anticancer drugs.

    2024 04 v.37 [Abstract][OnlineView][Download 1013K]

  • Preparation and preliminary application of monoclonal antibody against biofilm-associated protein of Acinetobacter baumannii

    HOU Hao;LI Jiaji;ZHU Huihuang;KONG Mengmeng;WANG Yi;YANG Bo;HU Zheng;School of Food and Biological Engineering,Hubei University of Technology;

    Objective To prepare monoclonal antibody(mAb)against biofilm-associated protein(Bap)of Acinetobacter baumannii and to initially develop an immunological detection technology for A.baumannii. Methods The Bap gene sequence was synthesized chemically,expressed by prokaryotic expression system and then purified by affinity chromatography to obtain recombinant protein Bap-His. The spleen cells of two female BLAB/c mice immunized with Bap-His recombinant protein were fused with SP2/0 myeloma cells and screened for the positive hybridoma cell lines by indirect ELISA. The monoclonal antibodies were prepared,analyzed by SDS-PAGE and identified by Western blot. Based on indirect ELISA,the immunologicaldetection technology for A.baumannii was developed by using the obtained monoclonalantibodies,and the specificity and lowest detection limit were verified. Results Two hybridoma cell lines were screened,and two monoclonal antibodies,mAb-2F and mAb-7E,were prepared. Both monoclonal antibodies had the purity of over 90%,and showed specific binding with the natural protein of A.baumannii. The immunological detection technology developed by two monoclonal antibodies only cross-reacted with A.baumannii,but did not with other respiratory pathogens. The lowest detection limits were 1. 6×10~5 and 8. 0×10~4 cfu/mL respectively. Conclusion The prepared Bap monoclonal antibody has high purity and can provide biological materials for the study of Bap related functions;the initially developed immunological detection technology for A.baumannii has good specificity and sensitivity,which provides technical support for the detection of A.baumannii.

    2024 04 v.37 [Abstract][OnlineView][Download 1100K]

  • Construction and activity of respiratory syncytial virus nanobodies

    WANG Cenrong;ZHAO hui ;LU Jingcai ;YUAN Ruosen ;SONG Yueshuang ;WANG Yajun ;WANG Yudi ;JIANG Chunlai;WEI Wei ;WU Shanli;College of Life Sciences,Jilin University;

    Objective To screen nanobodies(Nbs)against respiratory syncytial virus(RSV)by immunizing llama and phage library construction,and to evaluate their binding activity and neutralizing ability.Methods Two llamas were immunized with RSV recombinant F protein to construct phage library,and the FR and CDR sequences of Nbs were screened.Three strains of RSV Nbs were constructed by sequence recombination. After purification by Ni-NTA affinity chromatography,the binding activity of the Nbs was detected by ELISA,and the neutralizing activity was evaluated by using RSV A2 strain.Results The three Nbs BF-42,BF-46 and Fh-24 had the relative molecular mass of about 17 000,with the purity of about 95% after purification. BF-42,BF-46 at the concentration of 0. 16 μg/mL and Fh-24 at the concentration of 32 ng/mL still exhibited binding activity,and the binding ability of the three was in a dose-dependent manner. At a concentration of 450 μg/mL,all the three Nbs protected 50% of Hep2 cells from virus infection,and 50% cytopathic effect(CPE)appeared,which effectively neutralized viruses.Conclusion The three Nbs constructed in this study have high binding activity and a certain neutralizing ability,which lays a foundation of the application of RSV Nbs.

    2024 04 v.37 [Abstract][OnlineView][Download 891K]

  • Effect of celastrol on proliferation and apoptosis of human esophageal squamous cell carcinoma KYSE180 cells and its mechanism

    LI Xiang;MA Yanchun;ZHANG Yuhan;HUA Yuyan;CHENG Xiaolong;Department of Translational Research of Esophageal Cancer,Shanxi Medical University;

    Objective To explore the effect of celastrol on the proliferation and apoptosis of human esophageal squamous cell carcinoma KYSE180 cells and the possible mechanism. Methods KYSE180 cells in logarithmic growth phase were added with 0. 5,1 and 2 μmol/L of celastrol respectively,and the blank control group was set up. MTT assay and colony formation test were used to detect the proliferation of cells;the apoptosis of cells was assessed by flow cytometry;Transwell assay was used to measure the migration and invasion ability of cells;the mRNA transcription levels of P53,FAS and DR5were determined by qRT-PCR;the expression levels of Caspase-3,Caspase-9,Bax,Bcl-2 and PARP proteins were detected by Western blot. Results Compared with the control group,cell proliferation in 2 μmol/L celastrol group was significantly inhibited(F = 10. 9,P < 0. 05),and the numbers of clones in 0. 5 and 1 μmol/L celastrol groups decreased significantly(F = 457. 9,P < 0. 05);the numbers of cell invasion and migration decreased significantly in 1 and 2 μmol/L celastrol groups(F = 751. 7-1 134. 0,each P < 0. 001);the apoptosis rates in 0. 5,1 and 2 μmol/L celastrol groups increased significantly(F = 243. 0-728. 0,each P < 0. 05);the mRNA transcription levels of P53,FAS and DR5 in 1 and 2 μmol/L celastrol groups significantly increased(F = 71. 0-539. 3,each P < 0. 01);the protein expression levels of Caspase-3,Caspase-9 and Bax in 1 and 2 μmol/L celastrol groups significantly increased(F = 142. 1,35. 3 and 347. 6,respectively,each P < 0. 01),the level of PARP in 2 μmol/L celastrol group significantly increased(F = 877. 6,P < 0. 01),and the expression of Bcl-2 in 0. 5,1 and 2 μmol/L celastrol groups decreased significantly(F = 59. 2,each P < 0. 01). Conclusion Celastrol can inhibit the proliferation and promote the apoptosis of KYSE180 cells,probably through the up-regulation of Caspase-3/9,Bax,PARP protein expression and the down-regulation of Bcl-2 protein expression.

    2024 04 v.37 [Abstract][OnlineView][Download 1099K]

  • Identification of chemical modification types of associated proteins in recombinant human interleukin-1 receptor antagonist preparations and precipitation

    YIN Tingting;ZHU Qiumei;LIU Bingying;ZHANG Yu;LIU Ying;LIU Jinghui;LIU Yulin;Recombinant Protein Drug Laboratory,Changchun Institute of Biological Products Co.,Ltd.;

    Objective To identify the types of protein chemical modifications associated with the precipitates of recombinant human interleukin-1 receptor antagonist(rhIL-1Ra)preparations and preparations obtained at room temperature acceleration.Methods rhIL-1Ra products and precipitates in rhIL-1Ra products obtained at room temperature acceleration were detected by reverse phase-high-performance liquid chromatography(RP-HPLC),and the percentage of associated proteins was determined by area normalization method. Then the rhIL-1Ra product and precipitates in rhIL-1Ra product were identified for the associated proteins by liquid mass spectrometry.Results RP-HPLC results showed that there were 6associated protein chromatographic peaks in rhIL-1Ra product,and the percentages were 0. 414%,0. 870%,0. 871%,0. 304%,0. 104% and 1. 182% respectively. There were 5 associated protein chromatographic peaks in rhIL-1Ra product precipitation,and the percentages were 0. 350%,3. 603%,1. 118%,0. 629% and 0. 866% respectively. The liquid mass spectrometry results showed that the rhIL-1Ra product had 6 protein chromatographic peaks,of which the chemical modification types were methionine oxidation,aspartic acid dehydration cyclization + methionine oxidation,methionine oxidation,methionine oxidation,aspartic acid dehydration cyclization,and aspartic acid dehydration cyclization. There were 5 associated protein chromatographic peaks in the precipitates generated by rhIL-1Ra product,and the chemical modification types were methionine oxidation,aspartic acid dehydration cyclization + methionine oxidation,mainly methionine oxidation,mainly aspartic acid dehydration cyclization + methionine oxidation,and mainly aspartic acid dehydration cyclization.Conclusion The modification types of associated proteins in rhIL-1Ra precipitation obtained at room temperature acceleration and rhIL-1Ra product were the same,which have three main types,namely methionine oxidation modification,aspartic acid dehydration cyclization,and aspartic acid dehydration cyclization + methionine oxidation. This study provides a reference for improving the quality of rhIL-1Ra preparation.

    2024 04 v.37 [Abstract][OnlineView][Download 935K]

  • Epidemic trend analysis of plague in Tibet Autonomous Region,2001-2018

    ZHAO Zhe;ZHAO Chenxi ;WU Xubin;DUOJI Ouzhu ;LIU Kun ;LONG Yong;Department of Public Health,Gansu University of Chinese Medicine;

    Objective To analyze the epidemiological characteristics of plague in Tibet Autonomous Region from 2001 to 2018,so as to provide data support for the formulation of plague prevention and control strategies.Methods Based on the data-center of China Public Health Science,the data of plague incidence and cases in 31 provinces,autonomous regions and municipalities from 2001 to 2018 were collected,and analyzed for the temporal trend of plague incidence by using the Joinpoint regression analysis model and Geographic Information Science(GIS).Results There was no significant difference in the change trend of Tibet Autonomous Region from 2001 to 2018(P > 0. 05). The average incidence rate of Tibet Autonomous Region was at a high level in both time periods(2001-2009 and 2010-2018),0. 04/100 000 and 0. 023/100 000 respectively. During the first period,Moran's I index of average incidence rate was 0. 123(Z = 2. 273,P < 0. 05),with a positive spatial autocorrelation. Local spatial autocorrelation analysis identified that Tibet Autonomous Region appeared a 'high-high' correlation model in the first time period. Getis' G analysis revealed that Tibet Autonomous Region was identified as a'hotspot'region in both periods.Conclusion From 2001 to 2018,plague in Tibet Autonomous Region was quiescent in most years,with occasional cases circulating in isolated years. It is recommended to pay attention to strengthening the local plague surveillance,strictly implement personalized plague prevention and control measures,and formulate plague emergency response plans.

    2024 04 v.37 [Abstract][OnlineView][Download 1145K]

  • Establishment of an improved model of oxygen glucose deprivation/reoxygenation of primary hippocampal neurons

    SHI Jinchao;MA Na;ZHANG Qian;CHEN Jing;ZHONG Jin;ZHOU Yang;LI Jianguo;Key Laboratory of Cellular Physiology,Ministry of Education,Shanxi Medical University;

    Objective To establish an improved model of oxygen glucose deprivation/reoxygenation(OGD/R)in primary hippocampal neurons. Methods Hippocampal neurons were isolated from brain tissue of SD neonatal rat,and neuron suspension was prepared. By inhibiting the number of glial cells,the neuron suspension was divided into three groups,and no inhibition,partial inhibition and complete inhibition,the cell morphology was observed under microscope to determine the optimal culture conditions for primary hippocampal neurons. Based on this condition,the hippocampal neurons were deprived of glucose and oxygen for 0. 5,1 and 1. 5 h,and then reoxygenated for 0,24,36 and 48 h,separately. The content of lactic dehydrogenase(LDH)in culture medium was detected by ELISA,and the survival rate of neurons was measured by CCK-8 assay. The condition with higher LDH content and less influence on survival rate was the optimal condition for OGD/R model of primary hippocampal neurons. Results When the glial cells were partially inhibited,the purity of neurons was high with uniform growth status. The ratio of glial cells to hippocampal neurons was about 1∶1,which preserved the structural and functional connection between glial cells and neurons. Compared with OGD 1 h group,the LDH content increased significantly at OGD 1 h/R 36 h(t = 4. 31,P < 0. 05),while there was no significant difference in neuronal survival rate(t = 1. 99,P > 0. 05). Conclusion Primary hippocampal neurons with about 1∶1 number of glial cells and neurons were obtained with good growth status. The OGD/R model established under the condition of OGD 1 h/R 36 h has relatively high survival rate with shortened dendrites and impaired vitality,which lays a foundation for the further study of the pathogenesis of related diseases.

    2024 04 v.37 [Abstract][OnlineView][Download 912K]

  • Development and verification of RP-HPLC for determination of preservative content in multi-dose bivalent HPV vaccine

    MIAO Chenyang;WANG Dekui;WEI Jian;Shanghai Zerun Biotech Co.,Ltd;

    Objective To develop a reverse phase-high-performance liquid chromatography(RP-HPLC)method for the determination of three candidate preservatives(methyl paraben,benzyl alcohol and m-cresol)in bivalent human papillomavirus(HPV)vaccine,and to verify and preliminarily apply the method.Methods Waters Acquity Peptide BEH C18 300 ?（150 mm × 2. 1 mm,1. 7 μm）chromatographic column was used;mobile phase A(0. 1% trifluoroacetic acid aqueous solution)and mobile phase B(0. 1% trifluoroacetic acid acetonitrile solution)were used for gradient elution;the flow rate was 0. 2 mL/min,column temperature was 40 ℃,and the detection wavelength was 254 nm. The specificity,linearity,accuracy,precision and stability of the developed method were verified,and the concentration of each preservative in bivalent HPV vaccine containing three preservatives was detected using this method.Results Under the selected chromatographic conditions,the peak times of methyl paraben,benzyl alcohol and m-cresol were different(14. 6 min,7. 2 min and15. 3 min respectively),which was not affected by other components in the sample and detection reagents. The linearity of methyl paraben was good in the range of 0. 005%-0. 020%(R~2= 0. 997 3),the average recovery was 99. 60%,and the RSD was 2. 31%(n = 5);the linear relationship of benzyl alcohol was good in the range of 0. 1%-0. 5%(R~2= 0. 999 9),the average recovery was 100. 46%,and the RSD was 2. 70%(n = 5);the linear relationship of m-cresol was good in the range of 0. 4%-2. 0%(R~2= 0. 999 4),the average recovery was 100. 12%,and the RSD was 0. 74%(n = 5). When methyl paraben and m-cresol were placed at 2-8 ℃ for 3 d and benzyl alcohol for 2 d,the RSDs of the detection results were all less than 5%,and the stability was good. The developed method was used to detect the concentration of three preservatives in bivalent HPV vaccine samples,all the results of which met the error range of ± 20%.Conclusion The developed RPHPLC method is simple for operation with good specificity,accuracy,precision and stability,which can effectively detect and control the quality of three preservatives,methyl paraben,benzyl alcohol and m-cresol,in bivalent HPV vaccines.

    2024 04 v.37 [Abstract][OnlineView][Download 837K]

  • Development and application of in vitro antiviral neutralizing activity detection method based on Vero cell line stably expressing firefly luciferase

    DING Ruxia;LI Xiaoyu;ZHAO Chenyan;LIU Qiang;YAO Jiawei ;HUANG Weijin;WANG Youchun;National Institutes for Food and Drug Control(NIFDC);

    Objective To develop a high-throughput detection method based on Vero cell line stably expressing firefly luciferase(fluc)to evaluate the antiviral neutralizing activity of samples in vitro.Methods Based on CRISPR/Cas9 targeted gene editing technology,a Vero cell line stably expressing fluc was constructed,and a cell viability detection method for poxvirus and respiratory syncytial virus(RSV)infection was developed. This method was used to evaluate the neutralizing activity of serum immunized by vaccinia virus and RSV monoclonal antibodies,and the result was compared with that by plaque suppression method and cell viability(adenosine triphosphate,ATP)assay.Results Vero-fluc cell line was successfully constructed,and there was a good correlation between the cell quantity and the expression of fluc(R~2=0. 994 8). Vero-fluc cells were used as infected cells to test the neutralizing activity of poxvirus,which had good stability and reliability[signal/background ratio(S/B)= 25],and met the requirements of high-throughput detection(Z'= 0. 9).Compared with traditional plaque suppression method,the consistency of this method was stronger(R~2= 0. 76),and the ID_(50) value of immune serum was twice higher than that of plaque suppression method on average. The neutralizing activity of anti-RSV monoclonal antibodies detected by the developed method was consistent with that by ATP activity kit. The inhibition rate of Vero-fluc cell method was significantly higher than that of ATP method at the titer level of 1∶10~3 to 1∶10~5.Conclusion The antiviral neutralizing activity detection method based on Vero-fluc cells has good correlation with traditional methods,and has higher sensitivity and detection throughput with reliable results,which is a universal in vitro antiviral neutralizing activity evaluation method.

    2024 04 v.37 [Abstract][OnlineView][Download 969K]

  • Development and verification of a double antibody sandwich ELISA method for determination of relative potency in vitro of inactivated SARS-CoV-2 vaccine(Vero cells)

    WANG Wenhui;LI Wei;WANG Kaiwen;GUO Jing;MENG Shengli;GONG Limeng ;WANG Zejun;GUO Jianghong ;SHEN Shuo;ZHANG Chengzhi;Wuhan Institute of Biological Products Co.,Ltd.,National Engineering Technology Research Center for Combined Vaccines;

    Objective To develop and verify a double antibody sandwich ELISA method for the determination of relative potency in vitro of inactivated SARS-CoV-2 vaccine(Vero cells). Methods Using cell fusion technology to prepare SARS-CoV-2 monoclonal antibodies,the sandwich ELISA method with the monoclonal antibodies as the coating antibody and SARS-CoV-2 polyclonal antibodies as the detection antibody was developed to detect the in vitro relative potency of inactivated SARSCoV-2 vaccine(Vero cells),and the linear range,specificity,precision,and durability of the method were verified. The in vitro relative potency of 20 batches of inactivated SARS-CoV-2 vaccine(Vero cells)was detected using the developed method,and the correlation with the corresponding in vivo potency results was analyzed. Results The monoclonal antibody 20D8 was selected for the development of the method,with a purified titer of up to 10~5 and a protein concentration of 2. 69 mg/mL. The reference of inactivated SARS-CoV-2 vaccine(Vero cells)showed a good linear relationship with A_(450/630) at the concentration of 3. 2 to 200 WU/mL. The linear equation was y = 0. 846 8x-1. 483,with an R~2 value of 0. 991 9. The developed method specifically detected inactivated SARS-CoV-2 vaccine(Vero cells)without cross-reactivity with other vaccines. The CVs of repeatability verification and intermediate precision verification were 9. 91% and 11. 03%,respectively. The in vitro relative efficacy CV of inactivated SARS-CoV-2 vaccine(Vero cells)sample was 8. 59% after post-dissociation at 37 ℃ for 22,24 and 26 h,and 6. 62% after post-dissociation at 36,37 and 38 ℃ for 24 h. The relative potency in vitro of20 batches of inactivated SARS-CoV-2 vaccine(Vero cells) was positively correlated with the results of potency in vivo(r =0. 7, P < 0. 05). Conclusion The developed sandwich ELISA method has good specificity,precision and tolerance,and is easy to operate,which can be used for the detection of the in vitro relative efficacy and the quality control of inactivated SARS-CoV-2 vaccine(Vero cells).

    2024 04 v.37 [Abstract][OnlineView][Download 908K]

  • Establishment and verification of headspace gas chromatography method for organic solvent residues in PEGylated recombinant Candida uricase for injection

    GAO Rui;WANG Fuxin;HUANG Hai;Liaoning Inspection Examination and Certification Centre;

    Objective To establish and verify a headspace gas chromatography method for the determination of residual solvents,isopropanol(IPA),dichloromethane(DCM)and toluene(TOL),in PEGylated recombinant Candida uricase for injection(SSS111). Methods A headspace gas chromatography was established using a capillary column DB-1301(30 m×0. 53 mm,1. 0 μm),with the detector temperature of 250 ℃,the injector temperature of 200 ℃,the carrier gas of nitrogen,the split ratio of three,the column flow rate of 3. 0 mL/min,the hydrogen flow rate of 60 mL/min,and the air flow rate of400 mL/min. The column temperature was programmed raising:the initial temperature was 50 ℃,maintaining for 12 min,and rose to 200 ℃ at the rate of 40 ℃/min,maintaining for 5 min. The equilibrium temperature of the headspace bottle was 120 ℃,and the equilibrium time was 20 min. The linear range,precision and accuracy of the method were verified,and the limit of detection(LOD)and limit of quantitation(LOQ)were determined. IPA,DCM and TOL residues in three batches of SSS111 samples were detected by using the established method. Results IPA,DCM and TOL showed a good linear relationship with the peak area in the range of 0. 001 982-7. 928,0. 002 118-8. 472 and 0. 041 960-8. 392 μg/mL respectively,with each r of more than 0. 99. The RSDs of IPA,DCM and TOL residues in the control solution in six repeated detections were all lower than 5%. The recoveries of IPA,DCM and TOL were all in the range of 80%-120% after control solution was added to SSS111 sample. The LODs of IPA,DCM and TOL were 1. 982,2. 118 and 41. 96 ng/mL,and the LOQs were6. 941,6. 236 and 126. 92 ng/mL,respectively. The residues of IPA in the three batches of samples were all lower than the LOD,the residues of DCM were about 1 ppm,and the residues of TOL were all less than 1 ppm. Conclusion The established headspace gas chromatography method has good precision and accuracy,simple operation and high sensitivity,and can be used for the determination of organic solvent residues in SSS111.

    2024 04 v.37 [Abstract][OnlineView][Download 861K]

  • Research progress on vaccines against fascioliasis

    LI Cihuai;CHEN Feng ;SUN Qiangming;School of Public Health,Dali University;

    Fascioliasis is a zoonotic disease caused by fasciola infection,which will cause acute and chronic damage to the body,seriously endanger the development of animal husbandry,and cause great economic losses. At present,the drugs used to treat fascioliasis are single with drug resistance occurring. Therefore,there is an urgent need for a more economical and feasible way to control Fasciola in livestock to reduce or avoid diseases in humans and animals. Vaccine is an economical and effective means to prevent pathogen infection,which may play an important role in the prevention of fascioliasis. In this paper,the antigenic immune response mechanism of fasciola and its vaccine research are reviewed,so as to provide scientific reference and theoretical basis for the control of fascioliasis.

    2024 04 v.37 [Abstract][OnlineView][Download 893K]

  • Research progress of antimicrobial peptides as potential biofilm inhibitors

    LU Zhangping;CHANG Yanbin ;LI Keke ;WEI Lianhua;Ningxia Medical University;

    Bacterial resistance to conventional therapies has become a major threat to human health,and the formation of biofilms has significantly increased the resistance of bacteria to antibiotics and the innate defense of the host to become the main virulence factor of microorganisms. Antimicrobial peptides(AMPs)have broad-spectrum antimicrobial properties and anti-biofilm activity,which have become potential therapeutic drugs. This review described biofilm formation,drug resistance and associated infections,with particular attention to the anti-biofilm potential of AMPs,highlighting their advantages,mechanism of action against planktonic bacteria,mechanism of anti-biofilm,and anti-biofilm activity in synergistic with antibiotics,so as to provide references for the application of AMPs in clinical biofilm-related drug-resistant bacterial infections.

    2024 04 v.37 [Abstract][OnlineView][Download 919K]
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@2012 Editorial Department of "Chinese Journal of Biologicals"