2023年09期目次

  • Construction and identification of recombinant adenovirus expressing S protein receptor binding domain and N protein of SARS-CoV-2 Delta variant

    SONG Zexin;ZHOU Yan;LIN Xiaochen;ZHAO Yongmei;SHI Peng;WANG Xuefu;ZHANG Chaowu;LI Hongjun;Key Laboratory of Vaccine Development for Major Infectious Diseases in Yunnan Province,Institute of Medical Biology,Peking Union Medical College,Chinese Academy of Medical Sciences;

    ObjectiveTo construct and identify a recombinant adenovirus expressing S protein receptor binding domain(RBD)and N protein of severe acute respiratory symptom coronavirus 2(SARS-CoV-2)Delta variant.MethodsThe RBD and N gene fragments of SARS-CoV-2 were cloned into pcDNA3.0BA vector respectively to construct recombinant plasmid pcDNA3.0BA-RBD-N. The RBD-CMV-N fragment was amplified by PCR and inserted into shuttle vector pShuttle-CMV. The shuttle plasmid pShuttle-RBD-N was then homologously recombined with pAdeasy-1 to obtain recombinant plasmid pAdeasy-1-RBD-N,which was transfected into HEK293 cells for recombinant adenovirus Ad-RBD-N packaging. The transcription of RBD and N genes of recombinant adenovirus in HEK293 cells was detected by RT-PCR,while the expre-ssion of RBD and N proteins by Western blot and immunofluorescence assay. 12 female BALB/c mice were immunized with Ad-RBD-N by intramuscular injection at a dose of 5 × 109copies per mouse. Blood samples were collected 14 d after immunization,and the serum antibody titers were measured by ELISA.ResultsThe RBD and N genes of recombinant adenovirus were transcribed normally in HEK293 cells,and the RBD and N proteins were expressed normally in MA104 cells. Mice immunized with the recombinant adenovirus produced specific IgG antibodies against RBD and N proteins.ConclusionThe recombinant adenovirus expressing S protein RBD and N protein of SARS-CoV-2 Delta variant was succe-ssfully constructed,which laid a foundation of the follow-up research on Delta variant vaccines.

    2023 09 v.36 [Abstract][OnlineView][Download 936K]

  • Effects of overexpression of carbapenem OXA-48 on drug resistance,adaptability of bacterial strain and Toll-like receptor signaling pathway of host cell

    PAN Yafei;ZHAO Zhijun;DANG Tiantian;KANG Yuting ;JIA Wei;Laboratory Medical Center, General Hospital of Ningxia Medical University;

    Objective To investigate the effects of overexpression of OXA-48 on drug resistance,adaptability of bacterial strain and Toll-like receptor(TLR)signaling pathway of host cells. Methods The recombinant plasmid pET32a(+)-OXA-48was transformed into E.coli BL21(DE3),and the recombinant strain pET32a(+)-OXA-48-BL21(DE3)was identified by colony PCR and sequencing. Taking A_(600)(0. 3,0. 5 and 0. 7),IPTG final concentration(0. 4,0. 6 and 0. 8mmol/L)and induction time(2,4 and 6 h)as variables and mRNA transcription level as response value,an orthogonal experiment with three factors and three levels was designed to optimize the induced expression conditions of the plasmid. The drug resistance of recombinant strain pET32a(+)-OXA-48-BL21(DE3)to Imipenem(IPM),Meropenem(MEM),Ceftriaxone(CRO)and Cefepime(FEP)was detected by disk diffusion method;The adaptability was detected by biofilm formation test and serum resistance test. Mouse alveolar macrophages(MH-S)were infected with pET32a(+)-OXA-48-BL21(DE3),pET32a(+)-BL21(DE3)and E.coli BL21(DE3)strains,respectively. The mRNA transcription levels of TLR2,TLR4 and NF-кB(p65)genes were detected by qRT-PCR method,and the expressions of Interleukin-6(IL-6),IL-10,tumor necrosis factor-α(TNF-α)and transforming growth factor-β(TGF-β)were detected by ELISA. Results The recombinant strain pET32a(+)-OXA-48-BL21(DE3)was constructed correctly as identified by colony PCR and sequencing. The optimum induction conditions were as follows:A_(600)of 0. 3,IPTG final concentration of 0. 6 mmol/L and induction time of 2 h. Compared with pET32a(+)-BL21(DE3)strain,the resistance of recombinant strain pET32a(+)-OXA-48-BL21(DE3)to IPM,MEM,CRO and FEP significantly decreased(t = 7. 14～22. 32,P < 0. 05),the biofilm formed significantly increased(t = 15. 69,P < 0. 05),and the survival rate in serum significantly increased(t = 10. 60,P < 0. 05);The mRNA transcription level of TLR2 gene in MH-S cells infected with pET32a(+)-OXA-48-BL21(DE3)significantly increased 24 h after infection(t = 5. 77,P < 0. 05),while the mRNA transcription level of TLR4 and NF-кB(p65)genes(t = 3. 71～10. 06,P < 0. 05)and the expression level of IL-6 significantly increased 12 and 24 h after infection. Compared with the normal group,the expression of IL-6and TNF-α in MH-S cells infected with pET32a(+)-OXA-48-BL21(DE3)increased significantly at 6,12 and 24 h after infection(t = 7. 90 ～ 13. 44 and 5. 40～6. 32 respectively,each P < 0. 01),while the expression of IL-10 decreased significantly(t = 3. 15～4. 08,each P < 0. 05). There was no significant difference in the expression of TGF-β(t = 0. 013～1. 41,each P > 0. 05). The expression of IL-6 was significantly higher than that in pET32a(+)-BL21(DE3)group at 12 and 24 h after infection(t = 2. 92 and 3. 79 respectively,each P < 0.05) Conclusion Overexpression of OXA-48 can reduce bacterial drug resistance,improve bacterial adaptability and the transcription level of factors related to TLR signaling pathway in host cells,and affect the expression level of downstream cytokines in host cells.

    2023 09 v.36 [Abstract][OnlineView][Download 982K]

  • Construction of CeRNA network of differential LncRNAs in THP-1 cells infected by Dengue virus type Ⅲ and its antibody dependent enhancement

    LONG Mingwang;WANG Han;ZHANG Li;JIA Fan;LIU Yanhui;NING Xuelei;CHEN Junying;PAN Yue;SUN Qiangming;Institute of Medical Biology,Chinese Academy of Medical Sciences & Peking Union Medical College;

    ObjectiveTo establish models of Dengue virus type Ⅲ（DENV-3,DV-3）infection and antibody dependent enhancement(ADE)infection at the acute monocytic leukemia cells(THP-1),investigate the differential expression of long non-coding RNAs(LncRNAs),map the competitive endogenous RNA(CeRNA)regulatory network and predict the translation function of LncRNAs.MethodsThe culture supernatant was harvested 6 d after C6/36 cells were infected with DENV-3,the virus titer was determined by CCID50,and the type and full-length genome amplification were identified by PCR;The DENV-3 standard plasmid was amplified,identified by PCR,and the standard curve was drawn;THP-1 cells were divided into negative control group(THP-1),direct infection group(DV-3),ADE group and blank control group[1640(-)]. After 48 h of infection,the total RNA was extracted and the copy number of intracellular virus nucleic acid was measured;Through the whole transcriptome sequencing technology,the CeRNA regulatory network was constructed for the top five up-regulated and down-regulated LncRNAs in THP-1 vs DENV3,THP-1 vs ADE,DENV3 vs ADE groups,and the functions of their coding proteins were analyzed.ResultsC6/36 cells infected with DENV-3 for 3 d showed obvious cell fusion,vacuoles and abscission;The virus had a titer of about 1. 0 × 104. 64PFU/mL and was identified as DENV-3 by PCR specific primers,of which the complete gene sequence was obtained;The number of viral nucleic acid copies in ADE group was significantly higher than those in DV-3 group and blank control group;In THP-1 vs DENV-3,the expression of cytohesin interacting protein(CYTIP)was predicted to be up-regulated;In THP-1 vs ADE,the expression of kinesin family5A(KIF5A)was predicted to be down-regulated;In DENV-3 vs ADE,the expression of cluster differentiation antigen 9(CD9)and insulin like growth factor 2(IGF2)was predicted to be up-regulated. All of these differential LncRNAs had open reading frames(ORFs). Except Lnc-SH3BP1 and Lnc-RPL41,all of the other LncRNAs had internal ribosome binding site(IRES).ConclusionIn DENV-3 infection of THP-1 cells and ADE infection mediated by DENV-3,the expression of LncRNAs has changed significantly,and may regulate the process of infection through a variety of biological functions,which is helpful for a deeper understanding of the mechanism of ADE infection.

    2023 09 v.36 [Abstract][OnlineView][Download 1046K]

  • Evaluation of biological activity of an eukaryotic expressed anti-H5N1-M1 cell entry single molecule antibody

    SUN He;WANG Yu;MAN Hongyang;QIU Yue;TIAN Yuan;LI Zehong ;YUE Yuhuan;Biotechnology Application Research Laboratory,Chinese Academy of Agricultural Sciences Changchun Veterinary Institute;

    Objective To evaluate the biological activity of a eukaryotic expressed anti-H5N1-M1 cell entry single molecule antibody(TAT-ScFv-mFc). Methods The immune binding activity and affinity of TAT-ScFv-mFc to H5N1-M1 protein were detected by Western blot and localized surface plasmon resonance(LSPR)respectively;The inhibitory effect of TAT-ScFv-mFc on influenza virus H1N1 was detected by CCK-8 assay;The membrane penetration ability of TAT-ScFv-mFc to MDCK cells was verified by immunofluorescence assay. A total of 30 female BALB/c mice were injected with TAT-ScFv-mFc via tail vein,200 μL per mouse. Blood samples were collected at 5,60,120,240 and 360 min after injection. Serum samples were separated and detected for the titers by ELISA,and the half-life of TAT-ScFv-mFc was calculated according to the half-life curve drawn by Origin 2021 software. Results TAT-ScFv-mFc showed specific binding to H5N1-M1 protein with a binding rate constant of 6. 67 × 10~4[1/（M*s）]. The survival rate of MDCK cells infected by H1N1 increased gradually with the increase of TAT-ScFv-mFc concentration in a dose-dependent manner,which obviously inhibited the replication of H1N1. TAT-ScFv-mFc penetrated the cell membrane of MDCK cells in a short time,entered the cell and bound to virus M1protein,thus inhibiting virus replication and assembly. The half-life of TAT-ScFv-mFc in mice was 212 min. Conclusion TAT-ScFv-mFc has good immune binding activity and affinity with H5N1-M1,can effectively inhibit the replication of H1N1,has good penetration ability to MDCK cell membrane,and has a long half-life in mice,which lays a foundation of the drug treatment,vaccine research and preventive treatment of H5N1 infection.

    2023 09 v.36 [Abstract][OnlineView][Download 1020K]

  • High level expression of recombinant human serum albumin in CHO cells and optimization of its culture technology

    NIU Yuhui;LI Hongshan;LI Dianyu;WU Bei;LI Xiangrong;FENG Ruofei;Key Laboratory of Biotechnology & Bioengineering of State Ethnic Affairs Commission,Biomedical Research Center,Northwest Minzu University;

    ObjectiveTo achieve efficient expression of human serum albumin(HSA)in Chinese hamster ovary(CHO)cells and optimize its culture technology,so as to lay a foundation of the large-scale production of HSA.MethodsThe eukaryotic expression vector of HSA was constructed by gene recombination technology,and then electrotransfected into fully suspended CHO cells. The monoclonal cell lines with stable and high expression of HSA were screened by G418 and limited dilution method. By adding glucose,sodium butyrate and supplementalmedium to the basal medium,the cell culture process was optimized to improve the expression of HSA. Finally,the scale-up culture verification was carried out in a 5 L bioreactor.ResultsThe recombinant expression vector pcDNA3.1-HSA was successfully constructed and expressed in fully suspended CHO cells. After two monoclonal screening,the secondary monoclonal cell lines CHO-rHSA-7H2A9 and CHOrHSA-7H2D12 were obtained with high HSA expression of 29. 37 mg/L and 25. 26 mg/L respectively. The HSA expression level reached about 100. 00 mg/L by optimizing the culture process and wasfinally increased to 166. 16 mg/L in the 5 L bioreactor,which was about 30 times higher than that in the supernatant of the first monoclonal cells.Conclusion The high level expression of HSA in CHO cells was achieved,which laid a foundation of the further large-scale production of HSA in the field of biological products and solving the market supply problems.

    2023 09 v.36 [Abstract][OnlineView][Download 1069K]

  • Effect of activation of RIG-Ⅰ signaling pathway by LRRC23 on replication of influenza virus

    ZHU Yanhui;YANG Fangfang ;WANG Xin ;XING Yaling ;LIU Yalin;Henan University of Chinese Medicine;

    ObjectiveTo investigate the effect of activation of RIG-Ⅰsignaling pathway by leucine rich repeat containing23(LRRC23)on replication of influenza virus.MethodsOverexpression and knock-down of LRRC23 were performed in A549 cells to investigate its effect on influenza virus replication. A549 cells were transfected with pcLRRC23 plasmid or siLRRC23(small interfering LRRC23)for 24 h and then infected with influenza virus A/jingfang/1/86(H1N1). The virus titer and HA protein expression level in the cell supernatant were determined by plaque assay and ELISA respectively.The expression of LRRC23,RIG-Ⅰ,MAVS,M1 and HA at gene and protein levels were determined by qRT-PCR and Western blot respectively. The interactions between LRRC23 and RIG-Ⅰwere analyzed by co-immunopre-cipitation(Co-IP)and immunofluorescence assay(IFA). IFN-β-luc and NF-κB-luc activities were determined by dual-luciferase reporter assay.ResultsThe LRRC23 overexpression significantly decreased the influenza virus titer and inhibited the expression of HA protein in the supernatant of A549 cells,while enhanced the NF-κB and IFNβ activations by activation of RIG-Ⅰ-MAVS signaling pathway,resulting in the inhibition of expressions of M1 gene and HA protein. Conversely,the knock-down of LRRC23 increased the protein expression level of HA in the supernatant of A549 cells,up-regulated the relative expression level of M1 gene and down-regulated those of RIG-ⅠmRNA and MAVS mRNA.ConclusionLRRC23 plays an essential role in innate antiviral response by inhibiting influenza virus replication through activation of RIG-Ⅰsignaling pathway.

    2023 09 v.36 [Abstract][OnlineView][Download 1031K]

  • Complete genome comparative analysis of Coxsackievirus A6 causing hand,foot and mouth disease and herpangina in Yunnan Province in 2018

    TANG Jiaolian;ZHANG Xiaolin;ZHANG Zhilei;NAN Weiwei ;XUE Li ;JIANG Haiyan ;ZHANG Zhen ;SUN Qiangming ;JIANG Hongchao;Kunming Children's Hospital(The Affiliated Children's Hospital of Kunming Medical University);

    ObjectiveTo analyze the genome-wide characteristics of 17 strains of Coxsackievirus A6(CVA6)that cause hand,foot and mouth disease(HFMD)and herpangina(HA)in Yunnan Province in 2018,and understand the genetic differences between different pathogenic CVA6.MethodsA total of 1 909 stool samples clinically diagnosed as HFMD and HA in Kunming Children′s Hospital in 2018 were randomly selected for detection using enterovirus group A universal primers and screening of CVA6 positive samples. The CVA6 whole genome sequence was amplified with CVA6 whole genome primers,spliced by BioEdit splicting software,and analyzed for the whole genome characteristics by BioEdit,MEGA 7.0,Simplot,Heml 1.0 and Phyre2softwares.ResultsA total of 929 CVA6 positive samples were screened,and 17CVA6 complete gene sequences were obtained(9 of which were clinically diagnosed as HFMD and 8 were clinically diagnosed as HA). All 17 CVA6 strains were in type IV clade on the whole phylogenetic tree. No significant recombination occurred in HA and HFMD representative strains,while mutations occurred in non-structural protein 3D region. HFMD and HA representative strains showed differences in VP1 loci S597T,Q705L and Q663L. Online predictive analysis showed that the secondary structure of VP1 was consistent with that of CVA6 with no change.ConclusionThe 17 CVA6 strains causing HFMD and HA had high genomic homology,as well as nucleotide and amino acid differences,which may affect the replication and adaptability of CVA6.

    2023 09 v.36 [Abstract][OnlineView][Download 1400K]

  • Role of ELP30-intein-tag in prokaryotic expression of recombinant lipoprotein-associated phospholipase A2

    RUAN Yao;DING Ning ;YU Jun;Clinical Experimental Centre,Xi'an International Medical Centre Hospital;

    Objective To investigate the role of ELP_(30)-intein-tag in the prokaryotic expression of recombinant lipoproteinassociated phospholipase A_2 (LP-PL A_2 ). Methods The recombinant plasmid pIG6-ELP_(30)-intein-LP-PL A_2 was transformed into E.coli W3110. Using the recombinant protein LP-PL A_2 without ELP_(30)-intein-tag as negative control,the expression of recombinant ELP_(30)-intein-LP-PL A_2 in periplasm under different temperatures(20,25 and 30 ℃)and induction methods(IPTG and automatic induction)was analyzed by Western blot. The target protein was purified and isolated via inverse transition cycling(ITC)based on elastin-like polypeptide(ELP)-tag,and then detected by SDS-PAGE and Western blot.Results The recombinant LP-PL A_2 protein without ELP_(30)-intein-tag was expressed only intracellularly and not located in the periplasmic space. Under the same induction temperature,the amount of target protein induced by automatic induction was much lower than that induced by IPTG. Compared with 20 ℃,the overall expression level of target protein induced by IPTG increased at 25 ℃ and 30 ℃. The target protein ELP_(30)-intein-LP-PL A_2 was successfully isolated and purified by ITC technique,and the recombinant protein LP-PL A_2 was obtained with the purity of about 70% by inducing intein self-cleavage,which showed specific binding to the anti-LP-PL A_2 antibody. Conclusion The ELP_(30)-intein-tag can promote the expression of recombinant protein LP-PL A_2 in the periplasmic space of E.coli,and the recombinant protein LP-PL A_2 isolated by non-chromatographic purification has high purity,which provides a new method for the rapid,low-cost and effective production of recombinant protein LP-PL A_2 .

    2023 09 v.36 [Abstract][OnlineView][Download 832K]

  • Preparation of the second generation internal control reference for anti-SARS-CoV-2 IgG antibody and evaluation of its applicability in ELISA detection method

    ZHOU Zhijun;PENG Yan;YUE Shenglan;CHEN Limei;LUO Juan ;FENG Lu;JI Deming;LI Pu;DENG Kun;LI Cesheng;LI Taojing ;HU Yong;Sinopharm Wuhan Plasma-derived Biotherapies Co.,Ltd.;

    Objective To prepare the second generation internal control reference(B2)for Ig G antibody against severe acute respiratory symptom coronavirus 2(SARS-CoV-2)and evaluate its applicability in ELISA detection method. Methods Among the volunteers vaccinated with SARS-CoV-2 inactivated vaccine(BBIBP-Cor V)produced by Beijing Institute of Biological Products Co.,Ltd.,19 Ig G antibody positive plasma samples with ELISA-Ig G dilution ratio of 20 ～ 60 were screened,and the Ig G antibody,IgM antibody and neutralizing antibody were detected by ELISA,B2 was prepared from nonlipid plasma with ELISA-Ig G dilution ratio of 32 ～ 45,IgM negative and similar neutralizing antibody inhibition rate. The neutralizing antibody potency of the first generation internal control reference(B1)and B2 detected by ELISA was calibrated with the first generation WHO international standard of anti-SARS-CoV-2 immunoglobulin(NIBSC 20/136),and the accelerated stability(storage at 2 ～ 8 ℃ for 5,8,14,20,and 30 d respectively),the service stability(storage at 18 ～25 ℃ for 1,2,and 3 h respectively),the freeze-thaw stability(1,2 and 3 times)and the long-term stability(storage at-25 ℃ for10 months)of B2 were tested. B2 was used as standard to detect plasma after single vaccine immunization and mixed plasma was prepared according to different ELISA-Ig G dilution ratio. The correlation and linear regression analysis between ELISA-Ig G dilution ratio and neutralizing antibody potency of pseudovirus in mixed plasma were carried out. Results Among 19 plasma samples,5 samples were non-lipid plasma with ELISA-Ig G dilution ratio of 32 ～ 45,IgM negative and similar neutralizing antibody inhibition rate. B2 was prepared by mixing every plasma in equal volume fraction,and the dilution ratio of ELISA-Ig G was assigned to 32. The neutralizing antibody potency of B1 calibrated with NIBSC 20/136 was 133. 38 EIU/m L and that of B2 was 122. 14 EIU/m L. The recovery rates of accelerated stability,service stability,freeze-thaw stability and long-term stability of B2 were all in the range of(100 ± 15)%. The ELISA-Ig G dilution ratio of the mixed plasma from the same source was significantly correlated with the neutralizing antibody potency of pseudovirus.(each R~2> 0. 99,each P < 0. 000 1).Conclusion B2 prepared from plasma immunized with SARS-CoV-2 inactivated vaccine can replace B1 prepared from plasma of COVID-19 convalescent patients.

    2023 09 v.36 [Abstract][OnlineView][Download 872K]

  • Effect of cryopreservation on biological activity of national standard of human granulocyte colony-stimulating factor after reconstitution

    ZHU Liuqiang;SHI Xinchang;LIU Lan;PEI Dening;YU Lei;QIN Xi;ZHOU Yong;WANG Junzhi;NHC Key Laboratory of Research on Quality and Standardization of Biotech Products,National Institutes for Food and Drug Control;

    ObjectiveTo investigate the effect of cryopreservation on the biological activity of the national standard of human granulocyte colony-stimulating factor(hG-CSF)after reconstitution,so as to provide a reference for the use of the national standard of hG-CSF.MethodsThe biological activity of the standard was determined according to the general rule 3525 of Chinese Pharmacopoeia(Volume Ⅲ,2020 edition);The reconstituted hG-CSF national standard was aliquoted and stored at-80 ℃,-40 ℃ and-20 ℃,and then sampled at 1,2,3,5 and 6 months to detect the biological activity. The standards reconstituted before use were used to quantify the standards stored at -80 ℃ for different time durations,and the standards stored at -80 ℃ were defined as the reference of 100% activity to quantify the relative biological activity of the other samples.ResultsThe Eyrlng equation fitted by the thermal acceleration stability experiment was:ln {k(t)} = 6. 75-3 772. 20/T + ln(T),R~2= 0. 969. The biological activity of hG-CSF national standard was predicted to decrease by 5% after about 93. 4 months storage at -80 ℃；The biological activity of reconstituted standards frozen at -80 ℃ decreased by about 24%.ConclusionThe aliquoted reconstituted hG-CSF national standard can be stored at -80 ℃ stably for more than half a year. However,freezing and thawing will cause the activity value to drop by more than 20%,so it is not recommended to reuse after reconstitution.

    2023 09 v.36 [Abstract][OnlineView][Download 782K]

  • Expression of PLCH1 gene in esophageal squamous cell carcinoma and its effect on cell proliferation and migration

    BIAN Lin;GAO Yingzhen;SHEN Liuyi;ZHANG Ling;Department of Pathology,School of Basic Medicine, Shanxi Medical University;

    ObjectiveTo detect the gene variation and expression of PLCH1 in esophageal squamous cell carcinoma(ESCC),analyze the function of PLCH1 gene in ESCC and explore its mechanism.MethodsThe copy number variation of PLCH1 in ESCC was analyzed by GISTIC,and the expression of PLCH1 in ESCC and normal esophageal tissues was detected by TCGA database and immunohistochemistry method. The expression of PLCH1 in ESCC cell lines was detected by real-time fluorescence quantitative PCR(qPCR) and Western blot,and the effects of PLCH1 silencing on the proliferation and migration of ESCC cells were detected by MTT assay,colony formation assay and Transwell assay.Results There was significant copy number amplification of PLCH1 in ESCC(G-scores > 0. 1,P < 0. 05),and the expression levels of PLCH1 mRNA and protein in ESCC were significantly higher than those in normal tissues(F = 36. 00 ～ 1 101. 00respectively,each P < 0. 000 1). After PLCH1 silencing,the ability of proliferation,clone formation and migration of ESCC cells KYESE180 and TE-9 decreased significantly(F = 35. 49 ～ 634. 00 respectively,each P < 0. 001).Conclusion PLCH1 plays an oncogenic role in ESCC,which is of great significance for the metastasis and proliferation of ESCC,and can be used as a potential target for the treatment of ESCC.

    2023 09 v.36 [Abstract][OnlineView][Download 1272K]

  • Safety and immunogenicity of SARS-CoV-2 inactivated vaccine(Vero cells)in people aged 60 and above with hypertension and/or diabetes

    CHEN Xianhong;ZHANG Ruizhi ;MU Qiuyue ;FENG Jun ;WEI Shaofeng;LEI Shiguang;School of Public Health,the Key Laboratory of Environmental Pollution Monitoring and Disease Control,Minstry of Education,Guizhou Medical University;

    Objective To evaluate the safety and immunogenicity of SARS-CoV-2 inactivated vaccine(Vero cells)for people aged 60 and above with hypertension and/or diabetes. Methods In Songtao Miao Autonomous County,Guizhou Province,hypertensive and diabetic patients aged 60 and above and healthy people were selected as the research objects. They were divided into hypertension group,diabetes group,combined disease group(suffering from hypertension and diabetes simultaneously)and healthy people control group,and injected intramuscularly with SARS-CoV-2 inactivated vaccine(Vero cells)through the lateral deltoid muscle of upper arm at 0 and 21 d respectively,with a dose of 0. 5 mL each time. Adverse events in ≤ 30 min,0 ～ 7 d and 8 ～ 21 d after immunization were recorded and followed up to 6 months after vaccination. Venous blood samples of 5. 0 mL were collected before vaccination and 28 d after vaccination. The sera were separated,detected for neutralizing antibody levels by cytopathic assay and calculated for the geometric mean titers(GMTs)of neutralizing antibody and antibody seroconversion rate. Blood pressure of patients was measured in fixed time before vaccination and 30 min and 1 ～ 7 d after vaccination in hypertension group and combined disease group respectively;The fasting blood glucose was measured before vaccination and 2 h postprandial blood glucose was measured once on the day of vaccination. The fasting blood glucose and 2 h postprandial blood glucose were measured on 1,3,5 and 7 d after vaccination respectively. Results There was no significant difference in the incidence of solicited adverse events among hypertension group,diabetes group,combined disease group and healthy control group（χ2= 1. 790,P = 0. 617）;There was no significant difference in the incidence of non-solicitation adverse events(P = 0. 412). A total of 5 serious adverse events occurred,all of which were judged to be unrelated to vaccines. No abnormal fluctuation of blood pressure and blood glucose was observed. The lower limits of 95% CI of the rate difference of hypertension,diabetes and combined disease groups were all greater than-10%,and the lower limits of 95% CI of the neutralizing antibody GMT ratio were greater than 0. 67,all of which were not inferior to the healthy control group. Conclusion SARS-CoV-2 inactivated vaccine(Vero cells)has good safety and immunogenicity for people aged 60 and above with hypertension and/or diabetes.

    2023 09 v.36 [Abstract][OnlineView][Download 920K]

  • Analysis of measles epidemic regularity in Jilin Province from 2009 to 2022

    SHAN Yuanchun;WANG Shuang;WU Donglin;TIAN Xin;CHENG Tao;FU Simei;GUO Shuying ;YANG Xianda;Jilin Provincial Center for Disease Control and Prevention;

    ObjectiveTo analyze the epidemiological regularity of measles in Jilin Province from 2009 to 2022,so as to provide evidence for measles prevention and control measures.MethodsThe representative strains of measles from 2009 to2022 in Jilin Province were sequenced,the dominant epidemic strains and their variation were analyzed by bioinformatics software Bioedit and Mega 11,and the morbidity,immunization history and age distribution of the confirmed cases were analyzed by descriptive epidemiological method.ResultsA total of 6 560 cases of measles were reported in Jilin Province from 2009 to 2022,of which 50. 17% and 27. 58% were cases with zero doses of measles vaccine and unknown immu-nization history,respectively. Children aged 0 ～ 23 months accounted for 47. 29% of the total number of cases,adult group aged ≥15 years accounted for 37. 41%. The reported incidence reached the elimination level of < 1 per million population in the last 3 years due to the impact of immunization strategies and COVID-19 pandemic. The dominant genotype of measles virus in Jilin Province was still H1a genotype. Molecular epidemiological analysis showed that two large transmission chains continued to be prevalent together,one of which was blocked in 2015.ConclusionFrom 2009 to 2022,the reported incidence of measles in Jilin Province showed a downward trend,and the age of the cases showed a "two-way shift" distribution,which was concentrated in the population with no immunization history and unknown immunization history. The basic vaccination rate should be increased continuously,the targeted enhanced immunization should be carried out,and the molecular epidemiological surveillance should be strengthened to block transmission chains in time.

    2023 09 v.36 [Abstract][OnlineView][Download 1280K]

  • Epidemiological investigation of Lyme disease in animals in Xinjiang Uygur Autonomous Region,China

    YE Feng;DU Qiuli ;LIU Liya;MA Xiaojing;XIE Caiyun;GU Wenxi;MA Junjie;YI Xinping;Institute of Veterinary Medicine,Xinjiang Academy of Animal Sciences;

    ObjectiveTo investigate the epidemiology of Lyme disease and analyze the genotypes of Borrelia burgdorferi in five kinds of host animals of ticks in four cities of Xinjiang Uygur Autonomous Region,China.MethodsA total of 814 serum samples were collected from cattle,sheep,goats,horses and dogs in Urumqi,Ili,Changji and Kashgar Cities,and determined for IgG antibody against Lyme disease. A total of 135 ticks in the above-mentioned cities were collected,from which DNAs were extracted and used as template for amplification of the 5S-23S rRNA spacer fragments of B. burgdorferi by nested PCR. The positive samples were sequenced,and the results were compared with those of 5S-23S rRNA spacer of B. burgdorferi reported in GenBank by BLAST,and a phylogenetic tree was constructed by MEGA X software.ResultsThe IgG antibody positive rates against Lyme disease in Urumqi,Ili,Changji and Kashgar Cities were 23. 6%,2. 4%,2. 7% and 0 respectively,which showed significant difference（χ2= 48. 481,P < 0. 001）. However,the positive rates in cattle,sheep,goats,dogs and horses were 1. 1%,4. 4%,18. 7%,60. 5% and 0 respectively,which showed sig-nificant difference（χ2= 129. 03,P <0. 001）. Of the 135 tick DNA samples,24 were positive for B.burgdorferi,indicating a carrier rate of 17. 78%. Phylogenetic analysis showed that the genotypes B.garinii accounted for 75%,while B.afzelii accounted for 16. 67%,and B.burgdorferi accounted for 8. 33%.ConclusionThere are Lyme diseases in cattle,sheep,goats and dogs in Urumqi,Kashgar and Ili Cities of Xinjiang Uygur Autonomous Region,China,of which the major genotype is B.garinii. The study provides a scientific basis for prevention and control of Lyme disease in Xinjiang.

    2023 09 v.36 [Abstract][OnlineView][Download 862K]

  • Development and verification of multiplex real-time RT-PCR assay for classing and identification of HPIV1,HPIV2 and HPIV3

    CHENG Zien;LIU Zhining;CAO Pengcheng ;ZHAO Yaqi ;HOU Guangzheng ;LIU Qiqi ;LIU Xin;The First Affiliated Hospital of Jinzhou Medical University;

    ObjectiveTo develop and verify a multiplex real-time RT-PCR assay for simultaneous identification of human parainfluenza virus type 1(HPIV1),type 2(HPIV2)and type 3(HPIV3).MethodsThe whole genome sequences of HPIV1,HPIV2 and HPIV3 were downloaded from the database for alignment analysis,and the conserved regions were selected. Specific primers and probes were designed for the three viruses respectively to develop a multiplex real-time RTPCR assay with human ribonuclease P(RNase P)as theinternal quality control. The method was verified for the sensitivity,specificity and precision and used to detect 192 clinical samples.ResultsAfter optimization,the multiplex real-time RTPCR reaction system was determined to be 30 μL,including 10 × NeoscriptRTase/UNG Multi mix 3 μL,5 × Neoscript RT Premix Multi Buffer 6 μL,upstream and downstream primers of HPIV1,HPIV2 and HPIV3(100 μmol/L)0. 1 μL respectively,HPIV1,HPIV2,HPIV3 probes(100 μmol/L)0. 05 μL respectively,RNase P upstream and downstream primers(50 μmol/L)0. 06 μL respectively,RNase P probe(50 μmol/L)0. 03 μL respectively,template 15 μL,and ddH2O supplemented to 30 μL. The reaction conditions were 50 ℃ 20 min,95 ℃ 3 min and 45 cycles of 95 ℃ 15 s and54 ℃ 30 s. Fluorescence signals were collected during annealing in each cycle. The minimum detection limits of HPIV1,HPIV2 and HPIV3 were all 500 copies/mL by the multiplex real-time RT-PCR assay;The method showed no cross-reaction with influenza A virus,influenza B virus,respiratory syncytial virus andnovel coronaviruses. The coefficients of variation(CVs)in intra-and inter-groups of the recombinant plasmid standard mixture with three different concentrations were all less than 3%. HPIV1,HPIV2 and HPIV3 were detected in 192 clinical samples,and the positive rates were7. 81%,0. 05% and 3. 1%,respectively.ConclusionThe multiplex real-time RT-PCR assay for detection of HPIV1,HPIV2 and HPIV3 developed in this study has good sensitivity,specificity and precision,which has a high clinical application prospect in the field of rapid diagnosis and identification of HPIV.

    2023 09 v.36 [Abstract][OnlineView][Download 835K]

  • Preparation of Chinese national potency reference for hepatitis E vaccine

    GAO Fan;BIAN Lianlian;MAO Qunying;LIANG Zhenglun;WU Xing;National Institutes for Food and Drug Control,NHC Key Laboratory of Research on Quality and Standardization of Biotech Products,NMPA Key Laboratory for Quality Research and Evaluation of Biological Products;

    ObjectiveTo prepare the hepatitis E vaccine national potency reference for the quality control of hepatitis E vaccine.MethodsA batch of hepatitis E vaccine was selected as the row material,and the screened lyoprotectant was added. After aliquot and freeze-drying,it was prepared as candidate reference material,and the homogeneity and stability were investigated,which was distributed to four laboratories for collaborative calibration and applicability study.Results The 5% trehalose + 2% dextran was selected as the lyoprotectant to prepare the candidate reference material. The aliquot accuracy of candidate reference material was 0. 7%;The coefficient of variation(CV)of antigen content homogeneity was9. 0%;The CV of aluminum content homogeneity was 4. 0%. Moisture content was 1. 7%. The candidate reference material showed good acceleration stability and reconstituted stability. A total of 18 valid data were obtained from the collaborative calibration. The results showed that the average value of median effective dose(ED50)was 0. 15 μg(95% confidence interval of 0. 12 ～ 0. 18 μg)with the intra-laboratory CV of 30. 8% ～ 64. 2%,and the inter-laboratory CV of 32. 6%. Two hepatitis E vaccines produced by two laboratories themselves and the candidate reference material showed good dose-response relationship,of which the seroconversion rate decreased with the decrease of vaccine antigen content and the curve change trend was similar.ConclusionThe first Chinese national potency reference for hepatitis E vaccine(300031-201801)was prepared,which can be used for the quality control of potency ED50detection of hepatitis E vaccine.

    2023 09 v.36 [Abstract][OnlineView][Download 841K]

  • Research progress of phage display vaccine

    WANG Cai;ZHAO Zihong;LI Weifeng;ZHANG Yutuo;Institute of Pathogenic Biology and Immunology,Hebei North University;

    Bacteriophages are viruses that infect microorganisms such as bacteria,fungi,actinomycetes and spirochetes.Because of the inherent immunogenicity,genetic plasticity,stability,safety and many other advantages,it has unique potential in vaccine research and development. At present,there are countless researches using it to construct vaccine delivery platforms,mainly including three forms,phage display vaccine,phage DNA vaccine and hybrid phage DNA vaccine,of which the phage display vaccine is the most widely studied. Phage display technology is a novel vaccine preparation technology,which is a molecular biology technology using phage as carrier,integrating foreign polypeptide or protein genes into phage genes and displaying them on the surface of phage in the form of fusion protein. This review mainly elaborated the immunological basis of phage display vaccine,the display system and its application in disease prevention,so as to provide a reference for the development and application of phage display vaccine.

    2023 09 v.36 [Abstract][OnlineView][Download 827K]

  • Status and challenge of vaccine research for Clostridiodes difficile in clinical trials

    TAO Wei;FU Ting;HONG Yan;School of Laboratory Medicine and School of Bioengineering,Hangzhou Medical College;

    Clostridiodes difficile(C.difficile)is the most common causative agent of antibiotic-associated diarrhea(ADD)in the world. In recent years,with the emergence of highly resistant and virulent strains,the outbreaks of C.difficile infection have occurred around the world. The incidence,recurrence and mortality of C.difficile infection are on the rise worldwide,and bring great challenges to clinical treatment. Pathogenic strains mainly produce two homologous glycosylation toxins A and B,which can cause symptoms ranging from diarrhea to highly lethal toxic megacolon. In view of the malignant consequences caused by C.difficile infection,disease prevention is still an important way worth exploring. Until now there is no approved vaccine against C.difficile. Therefore,this review assessed the status and challenge of clinical trials of vaccine research for C.difficile.

    2023 09 v.36 [Abstract][OnlineView][Download 799K]

  • Research progress on vesicle-associated membrane protein 8

    ZHANG Xin;HOU Xueyang;ZHANG Rui;TANG Jingfeng;School of Food and Biological Engineering,Hubei University of Technology;

    Membrane fusion is essential for all life activities of eukaryotes and the fusion process requires the interaction of different vesicular transporters,which are highly conserved in eukaryotes,to coordinate specifically and facilitate the fusion of different biofilms. Vesicle associated membrane protein 8(VAMP8)mainly locates in vesicular as well as lysosomal membranes,and plays a vital role in the fusion of many different biofilms. This paper focused on the molecular structure,transcriptional regulation,post-translational modification and biological function of VAMP8 as well as the research progress on its correlation with human diseases,so as to provide new ideas for the treatment of related diseases and the development of effective therapeutics targeting VAMP8.

    2023 09 v.36 [Abstract][OnlineView][Download 853K]

  • Common problems and considerations in challenge tests of different animal models of SARS-CoV-2 vaccine

    WU Shuang;WANG Yin;SUN Tao;Center for Drug Evaluation,National Medical Products Administration;

    Animal challenge test is an effective means to study the efficacy of severe acute respiratory symptom coronavirus 2(SARS-CoV-2)vaccine. The appropriate animal models and reasonable experimental design play an important role in obtaining efficacy and safety information,as well as supporting clinical trials. At present,common non-clinical animal models include transgenic mice,non-human primates,hamsters,ferrets and so on. This paper reviews the common animal models used in non-clinical trials and the problems encountered in their application.

    2023 09 v.36 [Abstract][OnlineView][Download 791K]
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@2012 Editorial Department of "Chinese Journal of Biologicals"