2023年06期目次

  • Optimization of culture conditions of SARS-CoV-2 four Omicron variants in Vero cells

    XIA Fei;DU Hongqiao;ZENG Yan;LI Yuwei;LI Jiali;LU Jia;LI Xinguo;Wuhan Institute of Biological Products Co.,Ltd.;

    Objective To optimize the culture conditions of four vaccine candidates of severe acute respiratory symptom coronavirus 2(SARS-CoV-2) Omicron variants BA.1,BA.1.1,BA.2 and BA.5 in Vero cells.Methods The harvest time(24,48,72 and 96 h) and MOI(0.01,0.001,0.0001 and 0.000 01) of four Omicron variants cultured in Vero cells were optimized by using cytopathic effect(CPE),viral nucleic acid copy number and viral titer as evaluation indexes.Results The optimum harvest time of the four Omicron variants BA.1,BA.1.1,BA.2 and BA.5 in Vero cells was 72 h,and the optimum MOI was 0.001～0.000 01,0.001～0.000 01,0.01～0.000 01 and 0.01～0.000 01,respectively.Conclusion The culture conditions of four Omicron variants in Vero cells were optimized,which laid a foundation of the development of SARS-CoV-2 Omicron variant inactivated vaccine based on Vero cells.

    2023 06 v.36 [Abstract][OnlineView][Download 1006K]

  • Screening and safety evaluation of heat-resistant protective agents for live classic swine fever vaccine

    ZHOU Fuchang;LIAO Changru;CHEN Weiwei;ZHOU Qi;ZHOU Jiafu;SHI Jiang;FU Xiaoyang;Research and Development Center,Royal(Wuxi)Bio-pharmaceutical Co.,Ltd.;

    Objective To screen the formulation of heat-resistant protective agent for live classic swine fever(CSF) vaccine in order to prolong the preservation time of vaccine under harsh conditions.Methods The heat-resistant protective agent prepared based on the formula containing gelatin,D-sorbitol,trehalose,D casein hydrolysate of different contents was mixed with the live virus antigen of CSF and then lyophilized.The finished products with qualified appearance were screened for routine detection and aging resistance test,and the over-dose(10 and 30 portions) of the products qualified in the aging resistance test were used to immunize piglets via subauricular neck,5 for each to evaluate the safety of the vaccines.Results Among the 6 batches of lyophilized live CSF vaccine,3 batches(20200301,20200302 and 20200303) met the requirements of the current Chinese Veterinary Pharmacopoeia.The lyophilized loss was within 0.04～0.33 titers,and the moisture contents were all less than 2%;20200301 and 20200302 batches of finished products passed the aging resistance test.No adverse clinical symptoms were observed in all piglets 30 min after vaccination;The body temperatures of piglets vaccinated with 10 and 30 doses of vaccine were normal for 14 d after vaccination.Conclusion The two formulations of heatresistant protective agent selected might be used for freeze-dried preparations of live CSF vaccine with good effect of aging resistance and safety to piglets.

    2023 06 v.36 [Abstract][OnlineView][Download 848K]

  • Absorption characteristics of aluminum hydroxide adjuvant with inactivated enterovirus 71

    YAN Jiao;ZHAO Yong;HE Lingyu;NA Ruixiong;LI Yadong;YUAN Mingcui;JIANG Xi;YI Li;Institute of Medical Biology,Chinese Academy of Medical Sciences;

    Objective To explore the absorption characteristics of aluminum hydroxide adjuvant with inactivated enterovirus 71(EV71).Methods The morphology,purity and particle size distribution of inactivated EV71 particles were analyzed by transmission electron microscope,size exclusion chromatography HPLC(SEC-HPLC) and dynamic light scatter(DLS),and the morphology of aluminum hydroxide adjuvant nanoparticles was observed by transmission electron microscope.Using inactivated EV71 antigen content(4 000,6 000,8 000,10 000,11 000,12 000,13 000,14 000,20 000,30 000 U/mL),aluminum hydroxide adjuvant concentration [0.35,0.25,0.17,0.085 mg/mL(aluminum content)],adsorption time(0.30 and 120 min),ionic strength(sodium chloride concentration of 0.15,0.75 and 1.25 mol/L)and phosphorus-aluminum molar ratio(P/Al,0.15,0.64,2.08 and 7.87) as variables,the adsorption characteristics of aluminum hydroxide adjuvant with inactivated EV71 antigen were investigated.Results Inactivated EV71 particles mainly existed in the form of intact virus particles with a diameter of about 30 nm;Aluminum hydroxide adjuvant showed the characteristics of nanocrystallization,and the particle size was distributed within 200～700 nm.The inactivated EV71 antigen at the concentration of no more than 11 000 U/mL was completely absorbed by aluminum hydroxide adjuvant of 0.35 mg/mL(aluminum content),and no free antigen was detected in the supernatant;while from 12 000 U/mL,the content of free antigen in the supernatant increased with the increase of antigen content;the inactivated EV71 antigen of 250 U/mL was completely absorbed by various concentrations of aluminum hydroxide adjuvant,and no free antigen was detected in the supernatant;the adsorption effect of aluminum hydroxide adjuvant consistent after incubation for different time,and no free antigen was detected in the supernatant.Under the conditions of single dose of vaccine with aluminum hydroxide content(0.35 mg/mL) and inactivated EV71 antigen content(250 U/mL),sodium chloride ion strength had no effect on the adsorption of inactivated EV71 virus,while phosphate ion concentration significantly effected the adsorption.Conclusion Aluminum hydroxide adjuvant in a single dose of vaccine completely absorbed inactivated EV71 antigen,and group replacement played an important role.

    2023 06 v.36 [Abstract][OnlineView][Download 894K]

  • Tandem expression and purification of tick-borne encephalitis virus E protein Domain Ⅲ and preparation of its polyclonal antibody

    FU Mingyue;WU Yue;CHEN Ziyang;CHANG Dongying;WANG Qingshuang ;CHANG Junliang;Changchun Institute of Biological Products Co.,Ltd.;

    Objective To express and purify the E protein Domain Ⅲ(ED Ⅲ) of tick-borne encephalitis virus(TBEV) in tandem and prepare the corresponding polyclonal antibody.Methods The TBEV RNA was extracted by Trizol method,and then reversely transcribed into cDNA,which was used as template to amplify ED Ⅲ gene fragment by PCR.Two ED Ⅲ gene fragments were ligated into fusion gene by the hydrophobic flexible polypeptide(G_4S)_3 using overlapping PCR,which was then linked to prokaryotic expression vector pET-28a(+) to construct the recombinant expression plasmid pET-28a-2ED Ⅲ.After sequencing,pET-28a-2ED Ⅲ was transformed into E.coli BL21(DE3) competent cells,induced by IPTG and purified by Ni~(2+) affinity chromatography.Female New Zealand white rabbits were immunized with the renatured recombinant protein to prepare polyclonal antibody.The antibody titer was detected by indirect ELISA and the specificity was identified by Western blot.The homology of ED Ⅲ amino acid sequence between TBEV and other flaviviruses was analyzed by DNAMAN software.Results The recombinant plasmid pET-28a-2ED Ⅲ was identified by sequencing,and the amplified sequence contained two genes consistent with the E sequence of TBEV "Senzhang" strain(JQ650523.1) included on GenBank,indicating that the recombinant plasmid was constructed correctly.The recombinant 2ED Ⅲ protein was expressed mainly in the form of inclusion bodies,with a relative molecular mass of about 21 000 and a purity of 97.5%.The titer of rabbit anti-2ED Ⅲ serum polyclonal antibody was 1:10~7,which reacted specifically with TBEV whole virus.DNAMAN software alignment showed that the homology of ED Ⅲ amino acid sequences between TBEV and Japanese encephalitis virus(JEV),yellow fever virus(YFV) and Dengue virus(DENY) was 36.56%,9.28% and 30.77%,respectively.Conclusion The TBEV envelope ED Ⅲ tandem recombinant expression plasmid pET-28a-2ED Ⅲ was successfully constructed.The expressed recombinant 2ED Ⅲ protein had good reactivity and immunogenicity,and the prepared polyclonal antibody had high titer.

    2023 06 v.36 [Abstract][OnlineView][Download 1065K]

  • Preparation of the first batch of national standard of recombinant trypsin

    WANG Lvyin;ZHANG Hui;LV Ping;LI Jing;LIANG Chenggang;Division of Hormone,National Institutes for Food and Drug Control,NHC Key Laboratory of Research on Quality and Standardization of Biotech Products;

    Objective To prepare the 1st batch of national standard of recombinant trypsin in order to standardize and improve the quality of recombinant trypsin.Methods Enzyme-substrate identification,HPLC identification,N-terminal sequencing and TOF-MS were used to confirm the property and structure of recombinant trypsin;the purity was determined by HPLC;according to the methods in Chinese Pharmacopoeia(Volume Ⅲ 3603,2020 edition),the specific activity of the candidate standard was determined,and the stability and uniformity were investigated.Results The structure of recombinant trypsin was confirmed,and the specific activity of the 1 st batch of national standard of recombinant trypsin was 5 169 U/mg,containing 80% β-trypsin and 9% α-trypsin.The RSD of purity of α,β-trypsin and retention time of 12 candidate standards were all less than 2.0%.The purity of α,β-trypsin showed no obvious decrease stored at 25 ℃ and relative humidity(RH) 80% for 10 d,while the purity of β-trypsin decreased slightly and the purity of α-trypsin increased slightly stored at 40℃ and RH 80% for 10 d.The purity of β-trypsin decreased slightly when exposed to light(4 000 lx) for 10 d.Conclusion The national standard of recombinant trypsin with accurate structure and high purity was prepared,which can be used for system suitability test of the purity determination.

    2023 06 v.36 [Abstract][OnlineView][Download 854K]

  • Effect of multi-target protein tyrosine kinase inhibitor Ponatinib on proliferation,adhesion and migration ability of human liver cancer cell line SK-Hep-1

    LIU Chang;PEI Jinhong;MU Xiuli ;YU Baofeng;Department of Biochemistry and Molecular Biology,Changzhi Medical College;

    Objective To investigate the effect of a multi-target protein tyrosine kinase inhibitor,Ponatinib,on proliferation,homogeneity adhesion and migration ability of human liver cancer cell line SK-Hep-1.Methods SK-Hep-1 cells were cultured routinely and added with 24 tyrosine kinase inhibitors such as Ponatinib respectively,and the effect of Ponatinib on the survival and proliferation of SK-Hep-1 cells was detected by MTT assay.SK-Hep-1 cells were cultured routinely until the fusion degree reached 90%,then added with 0.1,0.5 and 1.0 μmol/L Ponatinib respectively,and the control group(without Ponatinib) was set up.The effect of Ponatinib on adhesion ability of SK-Hep-1 cells was detected by cell slow aggregation assay and dissociation assay,while the effect on migration ability by scratch test,and the effect on E-cadherin protein expression in SK-Hep-1 cells by Western blot.Results All 24 tyrosine kinase inhibitors inhibited SK-Hep-1 cells,among which Ponatinib showed the strongest inhibitory effect with a IC_(50) of(0.288±0.044) μmol/L.Compared with the control group,the number of cell mass(t=16.143,44.002 and 44.853 respectively,each P <0.001) and N_(TC)/N_(TE) [ratio of single cell number(N) after digestion by trypsin containing EDTA(TE) and CaCl_2(TC)](t=4.276,10.625 and 27.571 respectively,each P <0.05) decreased significantly and E-cadherin protein expression increased significantly(t=-3.757,-4.561and-6.922 respectively,each P <0.05) in 0.1,0.5 and 1.0 μmol/L Ponatinib groups;Scratch migration rate significantly decreased in 0.5 and 1.0 μmol/L Ponatinib groups(t=6.272～16.733 respectively,each P <0.01),while there was no significant difference in 0.1 μmol/L Ponatinib group(t=0.473 and 0.872 respectively,each P> 0.05) after 24 h and 48 h of scratch.Conclusion Ponatinib inhibited proliferation and migration of SK-Hep-1 cells and promoted cell adhesion.

    2023 06 v.36 [Abstract][OnlineView][Download 1043K]

  • Effects of VALD-3,a derivative of o-vanilla Schiff base ligand,on proliferation,migration and apoptosis of colorectal cancer cells and its mechanism

    MENG Yuna;MA Shuping;DANG Chunyan ;XUE Li ;LI Hongling;Department of Hematology,School of Clinical Medicine,Ningxia Medical University;

    Objective To the effects of VALD-3,a derivative of o-vanilla Schiff base ligand,on proliferation,migration and apoptosis of colorectal cancer cells and evaluate its mechanism.Methods HT-29 and HCT116 cells were cultured in vitro,and the inhibitory effects of VALD-3(5,10,20 and 40 mg/L) on proliferation of the two kinds of cells were detected by MTT assay;The effects of VALD-3(10,20 and 40 mg/L) on the morphological changes of the cells were observed by inverted microscope,while the effects on the migration ability of HT-29 cells were detected by cell scratch test,and the effects on the apoptosis of HT-29 cells were detected by flow cytometry and Hoechst 33258 fluorescence staining;The effects of VALD-3(5,10,20 and 40 mg/L) on the expression of apoptosis-related proteins in HT-29 cells were detected by Western blot.Negative control groups were set up(with no VALD-3).Results Compared with the negative control group,the survival rates of HT-29 and HCT116 cells in 10,20 and 40 mg/L VALD-3 treated groups significantly decreased(t=7.717～2 006.148,each P <0.05);the number of HT-29 and HCT116 cells in 10,20 and 40 mg/L VALD-3groups decreased significantly with the increase of VALD-3 concentration,the cells appeared irregular morphology and gradually became round and smaller,and cell fragments increased;In 10,20 and 40 mg/L VALD-3 treated groups,the migration rate of HT-29 cell scratches decreased significantly(t=7.596～73.780,each P <0.01),the apoptosis rate increased significantly(t=7.092～8.057,each P <0.01),and the number of apoptotic cells increased significantly with strong bright blue fluorescence,chromatin concentration and nuclear fragmentation;The levels of cleaved caspase-3 and Bax protein in HT-29 cells treated with 5,10,20 and 40 mg/L VALD-3 significantly increased(t=2.998～24.901,each P <0.05),the level of Bcl-2 protein in 40 mg/L VALD-3 group decreased significantly(t=10.035,P <0.05),and the levels of cleaved caspase-8 in 20 and 40 mg/L VALD-3 group significantly increased(t=12.630 and 8.064 respectively,each P <0.01).Conclusion VALD-3 inhibited the proliferation and migration of colorectal cancer cells and induced apoptosis by regulating the expression of cleaved caspase-3,cleaved caspase-8,Bax and Bcl-2 proteins.

    2023 06 v.36 [Abstract][OnlineView][Download 1267K]

  • Evaluation of sensitivity of SARS-CoV-2 antigen-detecting rapid diagnostic cards to different strains

    SHI Jinrong;ZHOU Zhijun ;FENG Lu ;YUE Shenglan ;CHENG Xiaoling;ZHANG Xueting;HU Yong ;DUAN Kai;LI Cesheng;Wuhan Institute of Biological Products Co.,Ltd.;

    Objective To compare the sensitivity(dilution) of antigen-detecting rapid diagnostic cards for severe acute respiratory symptom coronavirus 2(SARS-CoV-2) at home and abroad to different strains.Methods Vaccine bulks of four SARS-CoV-2 strains(original strain,Beta,Delta and Omicron) produced by Wuhan Institute of Biological Products Co.,Ltd.were used as the sample panel for sensitivity assessment,of which a series of diluted samples were detected by using 21 batches of SARS-CoV-2 antigen-detecting rapid diagnostic cards from 17 domestic and foreign manufacturers and SARS-CoV-2 nucleic acid detection reagent from Shanghai GeneoDx Biotech Co.,Ltd,respectively.The sensitivity of antigendetecting rapid diagnostic cards and nucleic acid detection reagent was evaluated according to the dilutions.The results of SARS-CoV-2 antigen-detecting rapid diagnostic reagents and nucleic acid detection reagent were compared to determine the nucleic acid detection Ct value corresponding to the group of antigen-detecting rapid diagnostic reagent with the highest dilution,namely the highest sensitivity.Results The sensitivity of antigen detection cards for the vaccine bulks of original strain,Beta,Delta and Omicron was 1:10～1:8 × 10~4.1:10~3～1:2 × 10~5,1:10~2～1:4 × 10~4,and 1:10～1:4 × 10~5,respectively;The sensitivity of nucleic acid detection cards was 10~(-6),10~(-5),10~(-4) and 10~(-7),respectively.The Ct values of N gene which were reached by high sensitivity antigen-detecting rapid diagnostic cards were as follows:original strain(10~(-4)) of more than 31,Beta variant(10~(-5)) of more than 36,Delta variant(10~(-4)) of more than 34,Omicron variant(10~(-5)) of more than 33,meeting the requirements of domestic and European Union for SARS-CoV-2 antigen-detecting rapid diagnostic cards.Conclusion All the antigen-detecting rapid diagnostic cards detected the four virus strains,while the sensitivity of different reagents to different variants varies to some extent,among which the sensitivity to Omicron variant varies the most.

    2023 06 v.36 [Abstract][OnlineView][Download 854K]

  • Preparation of CD73/PD-L1 bispecific antibody and evaluation of its antitumor function in vitro

    CHE Yaojian;HU Yan;ZHU Yanyang;HU Li;Institute of Immunotherapy,Fujian Medical University;

    Objective To prepare bispecific antibody targeting cluster of differentiation 73(CD73) and programmed cell death-ligand 1(PD-L1),and evaluate its binding ability and killing ability in vitro.Methods Using genetic engineering method,PD-L1 single-chain fragment variable(scFv) was inserted into the hinge region of CD73 monoclonal antibody to construct anti-CD73/PD-L1 bispecific antibody(BS-21),which was screened by CHO GS expression system to obtain highly expressed cell line.After purified by Protein A and molecular sieve,the purity of antibody was detected by size exclusion chromatography-high performance liquid chromatography(SEC-HPLC),the binding ability of antibody in vitro was detected by flow cytometry,and the killing ability in vitro was detected by using peripheral blood mononuclear cell(PBMC) to kill Calu 1 lung cancer cells in vitro.Results High-yield cell lines were obtained by pressure screening.A bispecific antibody BS-21 with a purity of 99.6% was obtained by purification,which bound to CD73 and PD-L1 molecules simultaneously.Compared with anti CD73 and anti PD-L1 groups,BS-21 group significantly increased the killing rate of immune cells to Calu 1 tumor cells(F=30.36,each P<0.001).Conclusion Bispecific antibody BS-21 reduced the immunosuppressive effect of CD73 and PD-Ll on immune cells simultaneously,and showed good anti-tumor function.

    2023 06 v.36 [Abstract][OnlineView][Download 936K]

  • Epidemiological analysis of rubella and genetic characteristics of 1E-L2 subtype strains in Liaoning Province in 2019

    WANG Wensi;AN Xiaohui;FANG Xing;WANG Yan;Liaoning Province Center for Disease Control and Prevention;

    Objective To understand the epidemiological characteristics of rubella in Liaoning Province in 2019,and analyze the epidemiological characteristics of rubella virus genotypes and gene subtypes at molecular level.Methods By collecting the incidence data of rubella in Liaoning Province in 2019 from the national notifiable infectious diseases reporting system,the epidemiological characteristics of rubella were analyzed.At the same time,the measles/rubella laboratory network of Liaoning Province was used to collect throat swab samples from suspected rubella outbreaks and sporadic cases.After three generations of blind transmission of positive samples,rubella virus isolates were obtained.Viral nucleic acid was extracted,amplified and the 739 bp nucleotide fragment sequence of E1 gene of positive rubella virus isolates was determined.The phylogenetic tree was constructed with the genotype reference strain sequences recommended by WHO and the published gene subtype reference strain sequences.The genotypes and subtypes were compared and the amino acid variation sites were analyzed.Results The reported incidence of rubella in Liaoning Province in 2019 was 0.927/100 000,which showed an obvious trend of recovery after a significant decrease in the incidence of rubella from 2017 to 2018,and the age of rubella patients was mainly 15 to 19 years old.A total of 55 rubella virus strains were isolated from 7 cities in Liaoning Province in 2019.Sequence phylogenetic analysis showed that all rubella isolates belonged to 1E-L2 gene subtype,which was also the dominant gene subtype of rubella epidemic in China.The nucleotide and amino acid homology among the strains were 99.051%～99.864% and 98.780%～100% respectively.Compared with the BRD-Ⅱ vaccine strain,the rubella isolates mainly showed A333T mutation and showed highly conserved amino acid sequence.Conclusion The 2019 rubella isolates in Liaoning Province were all 1E-L2 gene subtypes,which led to the resurgence of rubella epidemic.Therefore,molecular epidemiological surveillance of rubella virus should be further strengthened to provide a basis for the formulation and elimination of rubella prevention and control measures in Liaoning Province.

    2023 06 v.36 [Abstract][OnlineView][Download 1127K]

  • Development and verification of whole-column imaging capillary isoelectric focusing electrophoresis analysis method for charge heterogeneity of human growth hormone Fc fusion protein

    MA Linlin;LIU Ying;YU Lu;ZHU Qiumei;LIU Han;BI Jiangkun;ZHANG Kaining;Recombinant Protein Drug Laboratory,Changchun Institute of Biological Products Co.,Ltd.;

    Objective To develop and verify a whole-column image capillary isoelectric focusing(iCIEF) electrophoresis method to analyze the charge heterogeneity of recombinant human growth hormone Fc fusion protein(Fc-rhGH).Methods The iCIEF analysis method of Fc-rhGH was developed by optimizing the target protein concentration,cosolvent(urea)concentration and focusing time.The target protein was simultaneously analyzed by this method and traditional flat plate isoelectric focusing(IEF) electrophoresis,and the results were compared;The specificity,accuracy,precision,limit of quantitation(LOQ) and durability of the developed method were verified.Results The optimized method was using the mixed solution of 8 mol/L urea,0.35% methyl cellulose(MC),4% amphoteric electrolyte and 0.5% isoelectric point marker as the sample buffer,and the focusing condition was 1 500 V 1 min,3 000 V 5.5 min.IEF was not suitable for analyzing the charge heterogeneity of Fc-rhGH solution.Using the optimized iCIEF for analysis,the target protein was significantly different from the unrelated protein,and the baseline of blank reagent was stable;The recovery rate of accuracy verification was within 90%～110%,and the linear range was 0.25～0.75 mg/mL(50%～150% of the target loading volume);The RSD of each isomer pI in the repeatability verification was less than 0.3%,and the RSD of peak area percentage was less than 5%;The LOQ was 0.04 mg/ml.The sample storage time durability,amphoteric electrolyte pharmalyte 3-10 durability and MC durability of this method were good.Using this method to analyze the charge heterogeneity of Fc-rhGH physicochemical reference substance,eight charge heterogeneities of the reference substance were effectively separated,and the pI ranged from 5.9 to 6.4.Conclusion The developed iCIEF method had good specificity,accuracy,precision and durability,and was more suitable for efficient analysis of charge heterogeneity of Fc-rhGH than traditional flat plate IEF,which was of great significance for the quality control of Fc-rhGH and other Fc fusion proteins.

    2023 06 v.36 [Abstract][OnlineView][Download 962K]

  • Development and verification of qPCR method for detection of residual host DNA in human rabies vaccine(Vero cells)

    LIU Liqiang;BAO Xiaohua ;ZHOU Huiming ;ZENG Zhi ;LIU Jianhua ;ZHANG Haiping ;CHI Baoqi ;WANG Shusheng;LIU Dawei;Jilin Agricultural University;

    Objective To develop and verify a qPCR method for the qualitative and quantitative analysis of residual host DNA in human rabies vaccine(Vero cells) stock solution.Methods The qPCR standard curve was established by using the Vero cell DNA quantitative national standard,and the residual host DNA was extracted using magnetic beads.The specificity,repeatability,intermediate precision,accuracy and durability of the method were verified,and the linear range and limit of quantification were determined.The residual DNA of three batches of human rabies vaccine(Vero cells) stock solution was quantitatively analyzed and the fragment size was qualitatively analyzed by using this method.Results The correlation coefficients(R~2) of Vero cell DNA quantitative national standard amplification standard curve were all more than 0.99 by qPCR,and the quantitative range was 0.3 pg/mL～30 ng/mL.The method showed good specificity and repeatability.In the verification of intermediate precision,accuracy and durability,the relative standard deviations(RSD)of detection results of the samples were all less than 10%.The residual DNA content of Vero cells in three batches of stock solution was 0.20～0.77 ng/dose,which met the relevant standard of Chinese Pharmacopoeia(Volume Ⅲ,2020edition).The residual DNA fragments greater than 154 bp accounted for 52%～63%.Conclusion The developed qPCR method for the detection of residual DNA in human rabies vaccine(Vero cells) stock solution had good specificity,repeatability,intermediate precision and durability,and qualitatively and quantitatively analyzed the residual DNA rapidly and accurately,which was of great significance for improving the detection and control of residual DNA content in the production process and final product of human rabies vaccine(Vero cells).

    2023 06 v.36 [Abstract][OnlineView][Download 1121K]

  • Verification and application of indirect ELISA for quantitative detection of content of antibody IgG against Shigella flexneri 2a and Shigella sonnei 0-specific polysaccharide in human serum

    LI Hong;SHI Gang;GUO Lina;CHEN Qiong;FU Hongbin ;WANG Guodong ;HU Xiaohua ;ZHU Weihua ;WANG Bin;YE Qiang;Key Laboratory of Method and Standardization for Quality Control of Biotechnical Products,National Institutes for Food and Drug Control;

    Objective To verify an ELISA method for quantitative detection of antibody IgG against Shigella flexneri 2a and Shigella sonnei O-specific polysaccharide in human serum and use it to detect human serum samples before and after immunization with flexneri and sonnei dysentery bivalent vaccine.Methods The reference serum and quality control serum were mixed in a certain proportion to prepare 10 serum samples of different concentrations(numbered as S1～S10),which was determined continuously for 6 d by the indirect ELISA quantitative detection method provided by the enterprise,and the recovery rate and coefficient of variation(CV) were calculated.The reference serum content with a recovery rate of CV less than 20% was used as the limit of quantitation(LOQ).The serum sample S10 and reference serum were measured by the same method for 6 d,3 times of repeated test a day,and the CV was calculated;The inhibition rate of serum to polysaccharide was calculated by using the competitive inhibition ELISA with reference serum.The reference serum was detected by the method provided by the enterprise,the linear range was determined,the curve equation was obtained,and the correlation coefficient(R~2) was calculated.The verified method was used to detect 1 842 human serum samples before and after immunization with flexneri and sonnei dysentery bivalent vaccine.Results The recovery rates of Shigella flexneri 2a and Shigella sonnei polysaccharide antibody IgG in 10 serum samples were 73.21%～222.68% and 83.67%～123.56%,respectively;CV of precision verification between and within tests were all less than 30%;The inhibition rates of reference serum on 100 μg/mL Shigella flexneri 2a and Shigella sonnei polysaccharide were 85.95% and 88.42%,respectively;The linear ranges of Shigella flexneri 2a and Shigella sonnei polysaccharide antibody IgG were 0.05～0.125 and 0.025～0.125 EU/mL,with the R~2 not less than 0.99,and the LOQ were 0.050 and 0.025 EU/mL,respectively.The content of antibody IgG in serum after immunization with flexneri and sonnei dysentery bivalent vaccine was significantly higher than that before immunization.Conclusion The ELISA quantitative detection method provided by the enterprise had good accuracy,precision and specificity,and might be used for the detection of clinical serum of dysentery vaccine.

    2023 06 v.36 [Abstract][OnlineView][Download 881K]

  • Correlation analysis between three ELISA methods and micro-cell neutralization assay for detection of SARS-CoV-2 antibody titer

    JIANG Guorun;ZHAO Heng;LIAO Yun;FAN Shengtao;LI Dandan;ZHANG Ying;YU Li;WANG Lichun;LI Qihan;XU Xingli;Institute of Medical Biology,Chinese Academy of Medicine Sciences & Peking Union Medical College,Yunnan Key Laboratory of Vaccine Research and Development for Severe Infectious Diseases;

    Objective To analyze the correlation of different methods for the detection of antibody titer after immunization with severe acute respiratory symptom corona virus 2(SARS-CoV-2) vaccine,and provide a methodological basis for the specific antibody detection.Methods The seroconversion rate of IgG antibody and neutralizing antibody titer of SARS-CoV-2 inactivated vaccine clinical trial serum samples were detected by micro-cell neutralization assay and three ELISA methods(using S protein,N protein and inactivated whole virus particles as antigens respectively),and the correlation among the methods was analyzed.Results The seroconversion rates detected by neutralizing antibody,S antibody,N antibody and whole virus antibody were 84.3%,91.3%,65.2% and 46.6%,and the geometric mean titers were 16.6,945.9,72.7 and 18.8,respectively.The correlation coefficients(r) between the results of three ELISA methods(using S protein,N protein and inactivated whole virus particles as antigens) and micro-cell neutralization assay were 0.494,0.371 and 0.181respectively.Conclusion Detection of SARS-CoV-2 S antibody level reflected the activation of the humoral immune response characterized by elevated antibody level to a great extent.

    2023 06 v.36 [Abstract][OnlineView][Download 857K]

  • Application of reverse genetics in development of coronavirus vaccine

    WANG Mengjun;SHEN Shuo;Viral Vaccine Research Laboratory,Wuhan Institute of Biological Products Co.,Ltd.;

    In recent years,more and more coronaviruses(CoV)have crossed the species barriers and spread from animals to human,causing many serious public health events and a blow to the global economy. Vaccination is the most effective measure to prevent coronavirus pandemic. A safe,effective and time-save vaccine platform is a critical requirement of vaccine research and development. With the increasing maturity of reverse genetics technology,it also plays an increasingly important role in the development of CoV vaccines. This paper reviewed several reverse genetics techniques used in CoV research and their possible directions in CoV vaccine development.

    2023 06 v.36 [Abstract][OnlineView][Download 899K]

  • Advances in research and application of animal cell culture matrix fibracel carriers

    MA Chunying;WANG Meihao ;MA Zhongren ;WANG Jiamin;Life Science and Engineering College of Northwest Minzu University;

    Fibracel carriers based on polyester fiber have the advantages of good acid and alkali resistance,heat resistance,good biocompatibility,non-biodegradation,and can promote cell adhesion and growth. With no animal-derived ingredients and high biological safety,it is one of the preferred carriers for cell culture matrixes,which has been widely used in the development and production of cell matrix biological products with the development of biotechnology in recent years. This paper reviewed the structure and cell culture characteristics of fibracel carriers as well as the applications in vaccine production,cell therapy and tissue engineering,so as to provide a theoretical basis for the further development and application of fibracel carrier technology.

    2023 06 v.36 [Abstract][OnlineView][Download 1023K]

  • Research progress on role of apolipoprotein B mRNA-editing catalytic polypeptide 3B in occurrence and development of hematological tumors

    WANG Li;YANG Ying;College of Life Science,Tianjin University;

    The incidence of hematological tumors is increasing year by year. Childhood leukemia is a malignant tumor with the highest incidence in children. A variety of genetic mutations are closely related to the occurrence and development of hematological tumors. Apolipoprotein B mRNA-editing catalytic polypeptide 3B(APOBEC3B),a member of the cytosine deaminase family,can induce mutations in target DNA or RNA,which is one of the causes of tumor genome damage.APOBEC3B is highly expressed in many hematological tumors. This paper mainly reviewed the research progress on the role of APOBEC3B in hematological tumors,so as to provide potential targets for targeted therapy and accurate personalized therapy of hematological tumors.

    2023 06 v.36 [Abstract][OnlineView][Download 854K]

  • Research progress on non-clinical safety evaluation of SARS-CoV-2 vaccine

    XIAO Lichun;DU Juan;ZHANG Fangmei;ZHU Yingnan;Center for Drug Safety Evaluation and Research of ZZU;

    The Coronavirus Disease 2019(COVID-19) pandemic is having a dramatic impact on human health,lives,and the global economy. The development of a safe and efficacious vaccine is the most effective intervention to protect the population from the disease and limit the spread of the virus. Based on the current guidelines and research progress of severe acute respiratory symptom coronavirus 2(SARS-CoV-2) vaccines in various countries,this review summarized the research progress on non-clinical safety evaluation of SARS-CoV-2 vaccines by referring to the guidelines and relevant literatures over the world,in order to provide a reference for non-clinical research of SARS-CoV-2 vaccines.

    2023 06 v.36 [Abstract][OnlineView][Download 947K]

  • Research progress on mechanism and protein modification of Beclin1 in autophagy

    ZHANG Xuewen;WU Nianping;ZHOU Cefan;TANG Jingfeng;School of Food and Biological Engineering,Hubei University of Technology;

    Autophagy is a process of self-phagocytosis of lysosomal degrading substances in cells. Under normal conditions,cells can use autophagy to remove their own garbage to provide energy,while in a state of stress,autophagy can also be activated,resulting in cell damage and death,which can seriously lead to cancer. B-cell lymphoma-2-interacting myosin-like coiled-coil protein(Beclin1) is a central node in the autophagy pathway and interacts with a variety of proteins to regulate the formation and maturation of autophagosomes. Kinase mediates the protein modification of Beclin1 during autophagy,which will affect its activity and interaction with other autophagy-related proteins. Beclin1 is closely related to the occurrence and development of tumors. Therefore,this paper focused on the research progress on the mechanism and protein modification of Beclin1 in autophagy,in view to providing a reference for the research of targeted drugs of tumor autophagy.

    2023 06 v.36 [Abstract][OnlineView][Download 974K]

  • Progress in research on hydrogel and medium permeability

    SUN Gengtian;REN Jinna;QIAN Ming;Department of Oral Restoration,School of Stomatology,Jilin University;

    As a new research field,gel has been paid more attention and widely used for studies on tissue engineering,drug delivery and biosensor. Hydrogel is the carrier of cells,while the cell survival and death are keys to the construction of tissues and organs. However,the cell viability and biological behavior are limited by the exchange of hydrogel and nutrients in medium. This review summarizes the types of hydrogel,exchange mode of hydrogel and nutrients in medium and the relevant influencing factors,which will provide a reference for the development and research of tissue bioengineering.

    2023 06 v.36 [Abstract][OnlineView][Download 828K]

  • Research progress of bacterial flagellin as a vaccine adjuvant

    XIANG Tingting;MA Shumin;WO Enkang;School of Laboratory Medicine and Bioengineering,Hangzhou Medical College;

    Vaccines play an important role in the prevention and control of infectious diseases. Bacterial flagellin can activate Toll like receptor 5(TLR5) and NOD like receptor C4(NLRC4) in host cells,and has immune adjuvant effect. As a new immune adjuvant,flagellin can not only share with antigen protein,but also fuse with antigen protein,which can significantly improve the immune effect. At the same time,it has achieved good application effect in mucosal immunity and antitumor immunity,becoming a hot spot in the research and development of vaccine adjuvants. This review mainly discussed the research progress on application of bacterial flagellin as vaccine adjuvant,so as to provide new ideas for the development of flagellin adjuvant vaccine.

    2023 06 v.36 [Abstract][OnlineView][Download 844K]
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@2012 Editorial Department of "Chinese Journal of Biologicals"