2023年05期目次

  • Immune persistence following intradermal vaccination with fractional dose of diphtheria-tetanus-acellular component pertussis and Sabin-derived inactivated poliovirus vaccine

    LI Jingyan;CAI Lukui;MA Yan;WEN Jiana;HU Wenzhu;LIAO Hongwei;SHI Li;YANG;JingsiInstitute of Medical Biology,Chinese Academy of Medical Science and Peking Union Medical College;

    Objective To evaluate the immune persistence following intradermal(ID) vaccination with diphtheria-tetanusacellular component pertussis and Sabin-derived inactivated poliovirus vaccine(DTacP-sIPV).Methods 40 wistar rats were randomly assigned into four groups.Two test groups were injected intradermally with fractional-doses(1/5 and 1/10dose) of DTacP-sIPV(1/5D ID and 1/10D ID group);The positive control group was intramuscularly injected with full dose of DTacP-sIPV(full-dose IM group);The negative control group was injected with PBS intradermally.Wistar rats were immunized 3 times at 0,1 and 2 months and the blood samples were collected via tail vein 12 months after the last immunization and the serum samples were isolated.The titer of neutralizing antibody against poliovirus was detected by micro-neutralization test,and the titers of IgG antibodies against diphtheria toxin(DT),tetanus toxin(TT),pertussis toxin(PT),filamentous hemagglutinin(FHA) and pertactin(PRN) in rat serum were detected by indirect ELISA.The geometric mean titer(GMT)and positive rate of antibody were calculated.The rats were challenged with aerosolized B.pertussis for 30 min 12 months after the last immunization and determined for the white blood cell(WBC) count and colony-forming unit(CFU) in lung,trachea and nose at day 2,5 and 14 after challenge.Results Compared with the full-dose IM group,there was no significant difference in the positive rates of poliovirus type Ⅰ,Ⅱ and Ⅲ neutralizing antibodies between 1/5D ID and 1/10D ID groups(each P> 0.05) and the positive rates of all types of antibodies in the control group were 0.The positive rates of IgG antibodies against DT,TT,PT,FHA and PRN in 1/5D ID,1/10D ID and full-dose IM groups were all 100%,and those in control group were all 0.Compared with 2 d after challenge,the WBC counts of rats in control group increased significantly 5 d after aerosol challenge with B.pertussis(F=3.48,P <0.05),and then began to decrease,while those in other groups remained stable with time(F=0.14～1.30,P> 0.05).After aerosol challenge,the CFU in lungs of rats in control group was significantly higher than that in the other three groups(F=19.00～206.00,P<0.05),and B.pertussis was still detected 14 d after challenge;Except for the control group,the bacterial load in lungs of rats in the other three groups reached the peak 5d after challenge,the B.pertussis was basically cleared on the 14d,and there was no significant difference among the groups at each time point(F=1.14～1.25,P> 0.05).The bacterial load of trachea and nose in the control group was slightly higher than that in other groups at each time point,but the difference was not significant(F=0.71～3.54,P> 0.05).Except for the control group,the bacterial load in the trachea and nose of the other three groups were similar,and no significant difference was observed(F=0.75～3.41,P>0.05).Conclusion ID immunization with1/5 dose of DTacP-sIPV induced persistent protective antibodies against various components of the vaccine in rats.This study provided an experimental basis for the formulation of immunization strategy of ID immunization with fractional dose of DTacP-sIPV.

    2023 05 v.36 [Abstract][OnlineView][Download 873K]

  • Immune effect of electric charge modified influenza vaccine liposomes

    LI Piao;LIU Yanhong;LIU Xiaolin;YANG Ai;ZHANG Qian;LU Weidong;School of Pharmaceutical Science and Yunnan Key Laboratory of Pharmacology for Natural Products,Kunming Medical University;

    Objective To explore the physical and chemical properties of electric charge modified liposomes and the immune enhancement effect as influenza vaccine adjuvants.Methods Four kinds of electric charge-modified liposomes were prepared with soybean lecithin,cholesterol,N-1-(2,3-dioleyloxy) propyl-N,N,N-trimethyllam-moniumchloride(DOTAP)and 1,2-dioleoyl-sn-glycero-3-phosphocholine(DOPC)(DOTAP and DOPC mass ratio 1:4,2:3,3:2 and 4:1).The tetravalent influenza vaccine was mixed with the four liposomes according to the volume fraction of 1.4:1.0,which was prepared into cationic modified influenza vaccine liposome freeze-dried powder by freeze-thaw freeze-drying method.The liposome freeze-dried powder was redissolved with PBS,the particle size and Zeta potential were measured by laserparticle size analyzer,and the encapsulation efficiency of liposome was measured by Lowry method.231 female KM mice were randomly divided into negative control group(PBS),positive control group(tetravalent vaccine bulk),cationic liposome group(four electric charge modified influenza vaccine liposomes) and neutral liposome group(non-electric charge modified influenza vaccine liposomes),which were injected intraperitoneally at 7,14 and 28 d,0.2 mL per mouse.The cellular immune effect was evaluated by MTT assay and T lymphatic surface labeling,and the humoral immune effect was evaluated by hemagglutinin inhibition(HI) at 3,7,14,28,42 and 56 d of immunization.Results When the mass ratio of DOTAP to DOPC was 4:1,the particle size and Zeta potential of cationic modified liposomes reached the maximum,which were 529.65 nm and 12.05 mV respectively,and the encapsulation efficiency was also high,which was 81.82%.Stimulus index(SI) and CD4~+/CD8~+value of spleen cells of mice in the four cationic liposome groups were significantly higher than those in the negative control group(F=4.651～25.866,each P<0.05),among which,the two indexes in 4:1 cationic liposome group were higher than those in the other three groups;mice in this group produced early humoral immune effect 3 d after immunization,and the antibody titer reached the highest 42 d after immunization and maintained a high level during the whole immunization cycle.Conclusion Cationic liposomes with different electric charge modifications improved the immune effect of influenza vaccine,the immune enhancement effect was the best and the immune effect lasted the longest when the mass ratio of DOTAP to DOPC was 4:1.

    2023 05 v.36 [Abstract][OnlineView][Download 846K]

  • Prokaryotic expression and immunogenicity of hepatitis A virus capsid proteins VP1 and VP3

    ZHAO Danying;ZHOU Yongfei;XU Yanling;CHEN Ziyang;TANG Jianguang;CHANG Dongying;WANG Yibo;GUAN Shiyu;CHANG Junliang;CAO Yufeng;Changchun Institute of Biological Products Co.,Ltd.;

    Objective To express the capsid proteins VP1 and VP3 of hepatitis A virus(HAV) in prokaryotic cells and evaluate their immunogenicity.Methods VP1 and VP3 gene fragments were amplified by PCR,cloned into vector pETG28a to construct recombinant expression plasmids pET-G28a-VP1 and pET-G28a-VP3,which were transformed into competent E.coli BL21(DE3),induced by IPTG,and then analyzed by 12% SDS-PAGE.The target protein was purified by ion exchange chromatography,renatured and combined with several adjuvants to immunize mice.The mice were divided into VP1-MF59,VP1-AH,VP1-AP,VP1-AP-10CpG,VP1-AP-50CpG,VP3-AP,VP3-VP1-AP and PBS control groups,five for each group.Serum IgG antibody titers of mice in various groups were detected by ELISA,and serum neutralizing antibody titers of mice in VP1-AP,VP3-VP1-AP and PBS control groups were detected by rapid fluorescence focus immunosuppression experiment.Results Colony PCR and sequencing showed that the recombinant plasmids pET-G28a-VP1 and pET-G28a-VP3were constructed correctly.The recombinant proteins VP1 and VP3,with relative molecular masses of about 37 000 and26 000 respectively,mainly existed in the form of inclusion bodies,the expression levels were 18.6% and 32.4%,and the purity was 86.3% and 84.7%,respectively.The recombinant proteins VP1 and VP3 reacted specifically with rabbit anti-HAV antiserum and mouse anti-HAV-VP3 antiserum respectively.The serum IgG antibody titer of mice in VP1-AP-50CpG group was significantly higher than that in VP1-AP group(q=22.05,P <0.01),and the serum IgG antibody titer of mice in VP3-VP1-AP group was significantly higher than that in VP1-AP group and VP3-AP group(q=22.05 and 22.49 respectively,each P <0.01).Compared with PBS control group,the serum neutralizing antibody titer of mice in VP1-AP and VP3-VP1-AP group increased significantly(q=7.79 and 25.11 respectively,P<0.01) Conclusion Prokaryotic HAV capsid proteins VP1 and VP3 showed high purity and good immunogenicity,and the addition of CpG in the preparation was beneficial to enhance the immunogenicity of antigens.This study laid a foundation of the development of HAV recombinant subunit vaccine.

    2023 05 v.36 [Abstract][OnlineView][Download 902K]

  • Analysis of evolution and receptor binding characteristics of GⅡ.4 Sydney[P31]norovirus GZ19 strain

    WANG Jindong;MA Yalin ;DUAN Zhaojun;Department of Pathogen Biology,School of Basic Medicine,Weifang Medical University;

    Objective To analyze the evolutionary characteristics of GZ19 strain of G Ⅱ.4 norovirus(NoV) in China,and clarify its ability and mode of binding to receptors of histo-blood group antigens(HBGAs).Methods According to the sequence of ORF2 region in GZ19 strain,the evolutionary tree was constructed and the amino acid sequences at HBGA binding sites(HBSs) and key blocking epitopes were analyzed.P particles were expressed by prokaryotic expression system and purified.The obtained protein was identified by SDS-PAGE and indirect ELISA,and analyzed for the receptor binding characteristics of P particles by saliva binding and oligosaccharide binding assays.Results The GZ19 strain belonged to G Ⅱ.4Sydney [P31] lineage,of which the amino acid sequences of receptor binding sites and blocking epitopes were relatively conservative.It showed high homology with other G Ⅱ.4 Sydney [P31] strains in recent five years,while significant difference from G Ⅱ.4 Sydney 2012 original strain and G Ⅱ.4 Sydney [P16] strains.P particles only combined with A,B,O,AB secretory saliva and H-di oligosaccharide.Conclusion GZ19 strain represented the current evolutionary direction of G Ⅱ.4Sydney [P31] NoV.The successful expression of P particles and analysis of the binding characteristics with HBGA receptors laid a foundation of the research of epidemic evolution dynamics and vaccine development of G Ⅱ.4 NoVs in China.

    2023 05 v.36 [Abstract][OnlineView][Download 1029K]

  • Pharmacokinetic analysis of mouse short interspersed nuclear element B1 antisense RNA

    WU Peiyuan;WANG Xiaodie;LIU Xin;WU Chongguang;JI Ning;WANG Xiufang;LV Zhanjun;Department of Genetics,Hebei Medical University,Hebei Key Lab of Laboratory Animal;

    Objective To investigate the biological distribution of B1 antisense RNA(Blas RNA) of mouse short interspersed nuclear element in blood and tissues of normal mice after vein injection and detect the cell uptake efficiency of B1 as RNA using cultured normal mouse embryo cells after transfection.Methods Six 8～12-week-old BALB/c mice,three males and three females,were injected with 20 μg Blas RNA via tail vein,and blood samples were collected at different times after injection.54 BALB/c mice of 8～12-weeks were injected with 20 μg Blas RNA via tail vein,of which six mice,three males and three females,were euthanized at different times after injection,and various tissues,including the heart,liver,spleen,lung,kidney and thymus were harvested.Blas RNA was transfected into cultured mouse embryonic cells,and a certain amount of cells were taken at different time after transfection.The biological distribution of B1as RNA in mouse blood and different tissues and the persistence of Blas RNA in cultured embryo cells were detected by RT-qPCR.30naturally senescent BALB/c mice(≥ 14 months old) were divided into three groups:treatment group(20 μg Blas RNA injected via tail vein,once a week),irrelevant RNA control group(20 μg LacZ3F3R RNA injected via tail vein,once a week) and saline control group(injected with the same volume of saline),with 10 mice in each group,and a young control group(normal young 8～12-week-old BALB/c mice,five males and five females) was set.Four weeks after administration,mice in each group were euthanized,the liver tissues were taken,and the expression levels of aging-related genes(Sirtl,p21,p16~(Ink4a),p15~(Ink4b),p19~(Arf)) were detected by RT-qPCR.Results After tail vein injection,Blas RNA was available in the blood of mice for approximately 30 min,persisted for approximately 2～4 h in most detected tissues and persisted for approximately 48 h in lungs.The efficiency of cellular uptake of Blas RNA was approximately 400 molecules per mouse embryo culture cell 45 min after transfection with B1as RNA.Compared with the saline control group,Blas RNA treatment significantly down-regulated the mRN A expression of p21,p16~(In4a),p15~(In4b) and p19~(Arf) genes(t=10.01,4.461,4.420 and 5.309 respectively,each P <0.05),and significantly up-regulated the mRNA expression of Sirt1 gene(t=4.579,P <0.05).Conclusion Blas RNA was efficiently taken up by cells after transfection.After intravenous injection,Blas RNA stayed in the blood and tissues for a certain period of time and regulated the expression of aging-related genes in aged mice so as to make them approach to the expression level of young normal mice.

    2023 05 v.36 [Abstract][OnlineView][Download 976K]

  • Effect of siRNA interference with C-C chemokine ligand 5 on biological characteristics of laryngeal carcinoma cells

    NIU Li;HAN Rui ;HE Long ;WANG Ying ;GUO Huina ;HUANG Fuhui;The First Clinical College,Shanxi Medical University;

    Objective To investigate the expression of C-C chemokine ligand 5(CCL5) in head and neck squamous cell carcinoma(HNSCC),and explore the effect of CCL5 on the biological characteristics of laryngeal carcinoma cells.Methods Gene Expression Profiling Interactive Analysis(GEPIA) database was used to investigate the expression of CCL5 in HNSCC.The laryngeal carcinoma cells TU177 were transfected with siRNA(siRNA group),and the control(NC) group was set up.The cell proliferation,migration,cycle and apoptosis of each group were detected by CCK8 assay,cell scratch test and flow cytometry respectively.RT-PCR and Western blot were used to detect the knock-down efficiency of CCL5 and the mRNA transcription and protein expression of multidrug resistance protein 2(MRP2) and bcl-2-associated x protein(Bax).Results The expression of CCL5 in HNSCC was higher than that in normal tissues(P <0.05).Compared with NC group,siRNA showed higher knock-down efficiency(t=12.898 and 22.656 respectively,each P <0.01);siRNA interference with CCL5 inhibited the proliferation and migration of laryngeal carcinoma cells,and promoted the late apoptosis of laryngeal carcinoma cells and the expression of apoptosis protein Bax(t=2.600～11.667,each P <0.05).Conclusion CCL5 was highly expressed in HNSCC,while siRNA interference with CCL5 inhibited the proliferation,migration and promoted apoptosis of laryngeal carcinoma cells TU177 by up-regulating the expression of Bax,which laid a foundation of the possibility of CCL5 as a new target for the treatment of laryngeal carcinoma.

    2023 05 v.36 [Abstract][OnlineView][Download 1024K]

  • Topological analysis of IFN-induced transmembrane proteins in porcine peripheral blood lymphocytes and changes of transcription level after infection with porcine reproductive and respiratory syndrome virus

    MENG Xiaogang;SONG Yang;LU Huijun ;HUO Xiaowei;LV Xiangyu;LIU Kai;WEN Shubo;College of Animal Science and Technology,Inner Mongolia Minzu University;

    Objective To analyze the topology of IFN-induced transmembrane(IFITM) protein in porcine peripheral blood lymphocytes(PBMCs) and detect the change of IFITM mRNA transcription in PBMCs after porcine reproductive and respiratory syndrome virus(PRRSV) infection in vitro.Methods PRRSV,porcine circovirus 2(PCV2) and Japanese encephalitis virus(JEV) negative anticoagulant blood of piglets were collected aseptically and isolated for PBMCs.Porcine IFITM CDS sequence was amplified by PCR,sequenced and analyzed for topology.PBMCs were infected with PRRSV in vitro.Cell samples were collected at 12,24,36 and 48 h after infection,detected for PRRSV infection by RT-PCR,and detected for mRNA transcription level changes of IFITM1,IFITM2 and IFITM3 by RT-PCR.Results The porcine PBMCs were successfully isolated and the full-length sequence of IFITM CDS derived from PBMCs was cloned.The porcine IFITM protein might have two topological structures.PBMCs inoculated with PRRSV for 24 h produced obvious cytopathic effect.PRRSV was replicated in PBMCs.The transcription levels of IFITM1,IFITM2 and IFITM3 mRNA in PBMCs were significantly up-regulated at the early stage of PRRSV infection,and reached the peak at 12h after infection,and then gradually decreased;The transcription level of IFITM1 mRNA increased at 36 h after virus infection and then declined rapidly.Conclusion PRRSV infection in vitro significantly up-regulated the transcription level of IFITM mRNA in PBMCs,indicating that IFITM was involved in the antiviral immune response of PBMCs.This study provided a reference for revealing the natural immune response against PRRSV in vivo.

    2023 05 v.36 [Abstract][OnlineView][Download 1340K]

  • Expression of recombinant human interleukin-29-Fc fusion protein in HEK293F cells and analysis of its anti-tumor activity in vitro

    YAN Jiali;ZUO Qun;QI Fangbing;LIU Mingyang;LIU Jianwei;LI Suzhen;LIU Sen;WANG Jian;The Fourth Laboratory of National Vaccine and Serum Institute;

    Objective To express recombinant human interleukin-29-Fc(rhIL-29-Fc) fusion protein in human embryonic kidney 293-F(HEK293F) cells and analyze its anti-tumor activity in vitro.Methods The recombinant expression plasmid UCOE-IL-29-Fc was constructed and transiently transfected into HEK-293F cells.After expression and purification,rhIL-29-Fc fusion protein was obtained and identified by SDS-PAGE and Western blot;Female Japanese white rabbits were immunized with rhIL-29 and rhIL-29-Fc protein subcutaneously in the left ear respectively,2 rabbits in each group,0.5 mg per rabbit.Blood samples were collected from the vein of right ear,and the serum was separated.The half-life was measured by ELISA and the anti-proliferation effect of rhIL-29-Fc protein on human colon cancer HT-29,human colon cancer HCT-116,human Burkkit lymphoma Daudi,human non-small cell lung cancer NCI-H1975,human small cell lung cancer NCI-H209,human esophageal cancer EC109 and human pancreatic cancer PANC-1 cells in vitro was detected by CCK-8 assay,and the inhibitory concentration 50(IC_(50)) was calculated.Results The recombinant expression plasmid UCOE-IL-29-Fc was constructed correctly as identified by double digestion and sequencing.After transient transfection into HEK-293 cells for 6 d,the culture supernatant was harvested.The relative molecular mass of the purified rhIL-29-Fc fusion protein was consistent with the expectation.The protein showed a specific binding reaction with mouse anti-human IL-29 monoclonal antibody with a concentration of 1.5 mg/ml and a purity of 93%.RhIL-29-Fc protein had a half-life of 25 h and showed different inhibitory effects on the proliferation of 7 kinds of tumor cells,and the IC_(50) on different cells was also different.Conclusion The rhIL-29-Fc fusion protein was successfully expressed in HEK-293F cells,and the half-life of the fusion protein was 20 h longer than that of rhIL-29.According to the different anti-tumor proliferation activity in vitro and IC_(50) results on 7 kinds of tumor cells,it was found that the anti-tumor activity of rhIL-29-Fc fusion protein was higher than that of rhIL-29.This study laid a foundation of the development of IL-29 protein in the treatment of tumors.

    2023 05 v.36 [Abstract][OnlineView][Download 984K]

  • Effect of a disintegrin and metalloprotease 17 deletion on production of reactive oxygen species and mitochondrial function in nasopharyngeal carcinoma cells

    YIN Yongbo;LI Xueshen;QIU Jinxia;JI Yuqing;CUI Jing;Xingtai People's Hospital,Affiliated Hospital of Hebei Medical University;

    Objective To investigate the effects of a disintegrin and metalloproteinase 17(ADAM17) deletion on the production of reactive oxygen species(ROS) and mitochondrial function in nasopharyngeal carcinoma(NPC) cells.Methods Three groups of ADAM1 7 interfering plasmid ADAM17 shRNA and empty plasmid ADAM17-shRNA-NC were transfected into NPC cell line(CNE1) and detected for the interference efficiency by RT-PCR and Western blot to select shRNA with the best interference effect for the follow-up experiments.The cell proliferation was detected by CCK-8 assay,while the cell growth by clone formation test,the apoptosis and changes in mitochondrial membrane potential(MMP) by flow cytometry,the level of mitochondrial oxidative damage product ROS by fluorescence microscope,the contents of oxidative stress markers MDA and SOD by malondialdehyde(MDA) kit and superoxide dismutase(SOD) kit and the expression of mitochondrial damage markers Bax/Bcl-2,cleaved-caspase 9/caspase 9,cleaved-caspase 3/caspase 3 and c-Myc by Western blot.Results ADAM17-shRNA2 group showed the best interference effect.Compared with shRNA-NC group,the proliferation rate of cell in ADAM17-shRNA 2 group decreased significantly(t=8.964,P=0.036);the number of colonies were significantly reduced(t=10.351,P=0.014);the number of apoptosis increased significantly(t=11.25,P=0.008);the fluorescence intensity representing ROS level in cells increased obviously;the mitochondrial membrane potential decreased significantly(t=9.233,P=0.013);the SOD content decreased(t=7.233,P=0.034) and MDA content increased(t=7.415,P=0.038) significantly;the levels of Bax/Bcl-2,cleaved-caspase 9/caspase 9 and cleaved-caspase 3/caspase 3 significantly increased(t=8.985,9.021 and 7.789,P=0.023,0.011 and 0.031,respectively),while the expression of c-Myc proteins significantly decreased(t=10.352,P=0.004).Conclusion Interfering with ADAM1 7 induced SOD decrease and MDA increase by promoting oxidation,thereby alleviating oxidative damage of cell membrane,which also promoted the expression level of ROS in mitochondrion,reduced MMP,inhibited cell proliferation in vitro,and promoted apoptosis.

    2023 05 v.36 [Abstract][OnlineView][Download 1045K]

  • Culture of human sapovirus GⅠ.1 in vitro and preparation of polyclonal antibody against capsid protein VP1

    DU Wenjing;LIU Dan ;CONG Xin ;PANG Lili ;MAO Tongyao ;DUAN Zhaojun;College of Public Health,Gansu University of Traditional Chinese Medicine;

    Objective To culture human sapovirus(HuSaV) GⅠ.1 in vitro and prepare polyclonal antibody against the capsid protein VP1.Methods HuSaV GⅠ.1 positive stool specimens preserved in diarrhea department of National Institute for Viral Disease Control and Prevention were inoculated with HuTu-80 cells supplemented with different bile acid salts[glycine chenodeoxycholic acid(GCDCA) and glycine cholic acid(GCA)],and the infection,proliferation and passage of the virus were determined by PCR and RT-qPCR.The VP1 gene was amplified by PCR and cloned into prokaryotic expression vector pGEX-6P-1.The constructed recombinant expression plasmid pGEX-6P-1-VP1 was transformed into E.coli BL21(DE3) and induced by IPTG.Two female New Zealand white rabbits were immunized with the purified recombinant VP1 protein for 4 times.The blood samples were collected 18,28,38 and 48 d after immunization,and the serum titers were detected by ELISA.Results HuTu-80 cells were effectively infected by HuSaV GⅠ.1 in the presence of bile acid salt GCA,and the proliferated virus were stably and continuously transmitted for three generations in HuTu-80 cells.The expressed recom-binant GST-VP1 protein showed a relative molecular mass of about 86 000,and about 60 000 after purification(GST tag excision).The titer of polyclonal antibody against HuSaV VP1 protein was over 1:12 800.Conclusion HuSaV was successfully isolated and cultured in vitro using HuTu-80 cells supplemented with bile acid salt,and polyclonal antibody with high titer against HuSaV VP1 protein was prepared,which laid a foundation of in-depth research of HuSaV identification,infection and pathogenesis.

    2023 05 v.36 [Abstract][OnlineView][Download 914K]

  • Expression of long non-coding RNA FRAPT in triple-negative breast cancer and its biological significance

    HE Sai;LIU Zhifang ;LI Ruiwei;Thyroid Breast Surgery,Third People's Hospital of Yunnan Province;

    Objective To investigate the expression of long non-coding RNA FRAPT(lncRNA FRAPT) in triple-negative breast cancer(TNBC) cells and analyze its effects on proliferation,cell cycle and drug resistance of TNBC cells.Methods Using scRNASeqDB and TCGA on-line bioinformatics database,the expression of lncRNA FRAPT in TNBC and its correlation with prognosis were detected;TNBC cell line MDA-MB-231 was transfected with si-lncRNA FRAPT and si-Ctrl(negative control) by using Lipofectamine~(TM) 2000 respectively,of which the proliferation and cell cycle changes after transfection were detected by SRB test and flow cytometry,and the drug resistance to chemotherapy drug paclitaxel(PTX)was detected.Results The expression of lncRNA FRAPT was up-regulated in TNBC tissues and cells,and its high expression was positively correlated with the poor prognosis of TNBC patients(Hazard Ratio=1.66,P=0.047).Silencing lncRNA FRAPT significantly inhibited the proliferation of TNBC cells,with the cell cycle arrested in G1 phase,while increased the sensitivity of TNBC cells to PTX.Conclusion LncRNA FRAPT was highly expressed in TNBC and related to tumor prognosis-Silencing lncRNA FRAPT inhibited proliferation and cell cycle of TNBC,and increased the sensitivity to chemotherapy drug PTX.

    2023 05 v.36 [Abstract][OnlineView][Download 949K]

  • Preparation of national reference panel of HBV for nucleic acid testing donor screening

    LI Kejian;HAO Xiaotian;ZHOU Cheng;The First Department of Infectious Disease Diagnosis,National Institutes for Food and Drug Control;

    Objective To prepare the national reference panel of hepatitis B virus(HBV) for nucleic acid testing(NAT)donor screening.Methods A number of plasma samples from donors positive for HBV antibody and patients with HBV infection collected from blood centers,plasma stations and biological products companies in Shanghai,Gansu,Henan,Hunan,Hubei and other regions were tested for HBV DNA viral load agent,and negative and positive reference candidates were screened;The HBV DNA national standard was diluted to 10~3 IU/ml with human negative plasma,as a candidate for limit of detection(LOD).National negative and positive reference candidates of HBV for NAT donor screening and LOD reference to be calibrated were distributed to 8 enterprise laboratories for joint detection of HBVHCVHIV NAT donor screening.The homogeneity and stability of the national reference panel were investigated.Results A total of 8 negative samples with HBV viral load of 0 were screened as negative references and 9 positive samples with viral load of 10~3～10~4 IU/mL were used as positive references;One LOD reference was calibrated with WHO HBV DNA standard,and the virus content was 1.0 × 10~3 IU/ml.The national reference panel showed good stability and the homogeneity inspection met the requirements.Conclusion The national reference panel of HBV DNA for NAT donor screening was prepared,which provided a basis for the quality control and standardization of HBV DNA reagents for donor screening.

    2023 05 v.36 [Abstract][OnlineView][Download 837K]

  • Etiological analysis of clinical cases of live attenuated varicella vaccine

    QUAN Yaru;YUAN Liyong;CHEN Zhen;QIU Ping;LI Changgui;Division of Respiratory Virus Vaccines,National Institutes for Food and Drug Control;

    Objective To analyze the etiology of clinical cases of live attenuated varicella vaccine.Methods 64 samples of varicella vesicle fluid from 49 patients clinically diagnosed as varicella cases in phase Ⅲ clinical trial of live attenuated varicella vaccine in enterprises(the test site was Henan Province) were collected,extracted for DNA,and distinguished for wild-type strains and vaccine strains by PCR and restriction fragment length polymorphism(PCR-RFLP).Genotype was analyzed using the genotyping method recommended at the international(varicella-zoster virus,VZV) nomenclature meeting2008.Results 64 vesicle fluids were all wild-type strains(Pst Ⅰ~+Sma Ⅰ~-BssH Ⅱ~-Nae Ⅰ~-),and no vaccine-related cases occurred.All 49 isolates belonged to Clade 2.Additionally,compared with Clade 2,a synonymous mutation(T→C) in SNP18 082 was detected in all 49 isolates,and a mutation(C→A) in SNP790 was detected in one isolate.Conclusion In the clinical trial of live attenuated varicella vaccine in Henan Province,all the clinical cases were caused by infection of wild-type strain which belonged to Clade 2 genetic branch.

    2023 05 v.36 [Abstract][OnlineView][Download 816K]

  • Reporter gene assay for detection of biological activity of human growth hormone

    YU Lu;LIU Yulin;ZHU Qiumei;ZHANG Yu;LI Jihong;LIU Ying;LIU Jinghui;Recombinant Protein Drug Research Laboratory,Changchun Institute of Biological Products Co.,Ltd.;

    Objective To develop and verify a reporter gene assay(RGA) for the detection of biological activity of human growth hormone(hGH).Methods The biological activity of hGH was evaluated by the expression of luciferase(Luc)activated by hGH binding to hGH receptor(hGHR) on HEK293/GH-Luc cell membrane.The developed detection conditions were as follows:the initial concentration of sample was 1 μg/mL;the cell inoculation amount was(2.45～2.66) × 10~4 cells/well;the sample was of 3-fold serial dilution,with a total of 8 dilutions and the incubation time was 18～24 h.The relative biological activity of the sample was calculated by measuring Luc intensity and comparing it with the national standard by four-parameter fitting.The developed method was verified for specificity,repeatability,intermediate precision,relative accuracy,linear range and durability.Results The excipient components in product and the serum components in culture medium showed no effect on the activity detection results;The geometric coefficient of variation(GCV) of relative titer of one batch of samples in six repeated detections was 6.794%,much lower than 20%.The relative titer GCV detected by two experimenters in different batches of samples at different times were both lower than 20%;The relative deviations of the relative titer determination values of samples at different concentrations were within ±12%,the slope of linear regression equation was 0.982,the linear range was 0.6～1.6 μg/mL,and the coefficient of determination(R~2) was 0.997;The GCV of three batches of stock solutions and one batch of finished products were 4.758%,4.430%,7.294% and 2.771% respectively under the conditions of different cell generation,cell density and sampling location,all of which were less than 20%.Conclusion The developed RGA showed good specificity,repeatability,intermediate precision,relative accuracy,linear range and durability,which met the application requirements and was expected to replace the traditional in vivo biological activity detection methods for the activity evaluation and quality control of hGH.

    2023 05 v.36 [Abstract][OnlineView][Download 846K]

  • Development and verification of capillary isoelectric focusing-wholecolumn imaging detection method for isoelectric point analysis of recombinant human brain natriuretic peptide

    ZHANG Jing;ZHOU Zhiyan;LI Dandan;AN Likang ;WANG Yanbo ;ZOU Wei;Hebei Jizhaolian Science and Technology Development Co.Ltd.;

    Objective To establish and verify a capillary isoelectric focusing-whole column imaging detection(CIEFWCID) method for the analysis of isoelectric point(pI) of recombinant human brain natriuretic peptide.Methods The ampholyte,space-occupying agent,protein concentration,focusing time were optimized by CIEF-WCID method,and the best condition for the detection of recombinant human brain natriuretic peptide was obtained.The repeatability,precision and durability of the developed method were verified,and three batches of recombinant human brain natriuretic peptide produced continuously were analyzed for pI.Results HR AESlyte 8-10.5 was selected as ampholyte,while 25 mmol/L sodium hydroxide as the space-occupying agent;The final concentration of the sample was 87.5 μg/mL and the focusing time was 8min.The relative standard deviation RSD of pI detection was 0.1% after six consecutive injections of the same sample;The RSD of pI detection of six samples was 0.1%;The pI RSD of the main peak was 0.1% at different final concentrations of the sample,and the pI RSD of the sample was 0.1% at different storage time,while the pI markers could not be changed arbitrarily.The pI was detected in three consecutive batches of recombinant human brain natriuretic peptide samples.Conclusion The developed CIEF-WCID method for pI analysis of recombinant human brain natriuretic peptide had good repeatability and precision and might be used for follow-up quality control of recombinant human brain natriuretic peptide.

    2023 05 v.36 [Abstract][OnlineView][Download 999K]

  • Research progress on viral subunit vaccines

    SU Jinxian;BAI Jialin;ZHANG Haixia;Key Laboratory of Biotechnology and Bioengineering of State Ethnic Affairs Commission,Biomedical Research Center,Northwest Minzu University;

    Subunit vaccines are developed from the immunologically active fragments of pathogens and do not have genetic materials,which have been the center of research attention,guiding the direction of the development for new generations of vaccines.Given their safety advantages and high immunological activities,subunit vaccines play important roles in prevention of the spread of pathogenic viruses.This paper mainly introduces the research progress and applications of the subunit vaccines in common viral diseases,aiming to establish a theoretical basis for the development of the subunit vaccines on new viral diseases.

    2023 05 v.36 [Abstract][OnlineView][Download 934K]

  • Research progress on function and molecular mechanism of transcription factor ELK1 of Ets

    ZHAO Yuanshen;ZHAI Yanhui;SHI Lijing ;LI Ziyi;Key Laboratory of Organ Regeneration and Transplantation of Ministry of Education,Bethune First Hospital,Jilin University;

    Ets transcription factor ELK 1,a member of the ternary complex factor(TCF) subfamily in the Ets family,is directly downstream signal of MAPK/ERK pathway and is activated by phosphorylation to execute the function of ERK signal.ELK1,which is highly expressed and phosphorylated in stem cells and tumor cells,plays a role in promoting proliferation,inhibiting apoptosis and differentiation in stem cells.In tumor cells,ELK1 has shown to promote proliferation,migration,and inhibit apoptosis.In neural cells,ELK1 is involved in long-term memory,learning,addiction and other functions.In this paper,the research progress on the main structure and basic biological characteristics of ELK1,the function and mechanism of ELK 1 in tumor cells,stem cells and nerve cells are reviewed in order to provide new ideas for the follow-up research.

    2023 05 v.36 [Abstract][OnlineView][Download 865K]

  • Research progress on combined vaccines based on diphtheria-tetanus and acellular pertussis vaccine

    QU Xucheng;WANG Tingting;LIU Jiankai;ZHENG Haifa;Beijing Minhai Biotechnology Co.,Ltd.,Beijing Engineering Technology Research Center for New Combined Vaccine;

    Combined vaccine is a single vaccine preparation made by mixing two or more different organism or purified antigens by physical methods.The use of combined vaccine can reduce the number of immunization shots and improve the compliance of children and acceptance of parents;reduce the costs of transportation,storage and management;avoid missing vaccination and improve vaccination rate.At present,a variety of combined vaccines have been licensed abroad,which have good safety and immunogenicity;some combined vaccines have been put on the market in China,and many combined vaccines are in clinical trials.In recent years,with the successful development of component pertussis vaccine and inactivated poliovirus vaccines of Sabin strain Ⅰ,Ⅱ and Ⅲ in China,the combined vaccines based on diphtheria-tetanus-acellular pertussis vaccine(DTaP) have been greatly developed.In this paper,the research progress on combined vaccines based on DTaP,which have been licensed and in clinical trials at home and abroad,was reviewed,in order to provide ideas for the development of related combined vaccines in China.

    2023 05 v.36 [Abstract][OnlineView][Download 868K]

  • Research progress on receptor activity-modifying proteins and their application prospects in disease prevention

    QIAN Rui;WANG Liqun;YAO Yuqin;FANG Aiping;West China School of Public Health and West China Fourth Hospital,Sichuan University;

    Receptor activity-modifying proteins(RAMPs) are type I transmembrane proteins,which are activity-modifying proteins of a variety of G-protein-coupled receptors(GPCRs).RAMPs are related to physiological and pathological phenomena such as neurological diseases,cardiovascular diseases,renal function,skeletal development and obesity,and of great significance for disease prevention,which are potential targets for the treatment of various diseases and also closely related to the prognosis of diseases.At present,there are few studies on proteins that may regulate the expression of RAMPs.This paper focuses on the sterol regulatory element-binding factor-2(SREBF-2) that may regulate the transcription and expression of RAMP3, and reviews the research progress of RAMPs in biology,pathology and pharmacology,providing a reference for the further research on RAMPs and the prevention and detection of related diseases.

    2023 05 v.36 [Abstract][OnlineView][Download 842K]

  • Opportunities,challenges and suggestions for development of cell culture media industry in China

    CHEN Qi;GUO Wei;YANG Zhe;YU Zhenhang;SHEN Jianzhong;China National Center for Biotechnology Development;

    Cell culture medium is one of the essential raw materials in the field of life and health.In recent years,the performance and quality of domestic cell culture media have been improving,and the market share of domestic vendors has steadily increased from 19.2% in 2017 to 33.7% in 2021.However,there are also some problems and shortcomings in industrial development,mainly including:technology and process accumulation need to be strengthened;product quality need to be improved;lack of industry standards and norms.Based on literature research,special topic discussion and expert interview,this paper reviews the development history of cell culture medium,deeply analyzes and systematically combs the opportunities and challenges faced by the industry development from the basic situation,current situation and trend of the development of cell culture medium industry in China,and puts forward relevant countermeasures and suggestions.

    2023 05 v.36 [Abstract][OnlineView][Download 825K]


@2012 Editorial Department of "Chinese Journal of Biologicals"