2023年02期目次

  • Analysis of titer stability and inactivation kinetics of harvest solution of SARS-CoV-2

    GUO Bing-feng;HAN Bin;HAO Yi-nan;WANG Kui;YIN Ji-xiang;LI Yan;LI Nan;LING Xiang-ping;PAN Ruo-wen;Hualan Biological Vaccine Co.,Ltd.;

    Objective To investigate the titer stability of the harvest solution of severe acute respiratory syndrome coronavirus2(SARS-CoV-2)at 2 ～ 8 ℃ and the inactivation effect of β-propiolactone inactivator on the virus.Methods Three batches of SARS-CoV-2 harvest solution(batch numbers:202111001,202111002 and 202111003)were stored at 2 ～ 8 ℃ for 12 d and sampled every 3 d(0,3,6,9 and 12 d)for detection of the titers by Karber method;Three batches of virus harvest solution equilibrated overnight at 2 ～ 8 ℃ were inactivated by adding β-propiolactone at a volume fraction of 1∶4 000 and detected for the titers at different inactivation time points(0,0.5,1,1.5,2,3,4,8,16 and 24 h),of which samples inactivated for 8,16 and 24 h were taken for inactivation verification,and samples inactivated for 24 h were observed by transmission electron microscope.Results The titers of SARS-CoV-2 decreased with the prolongation of storage time at 2 ～8 ℃,which showed no obvious decrease during 0 ～ 3 d,while decreased from the initial 7.75,6 and 7.5 lgCCID_(50)/mL to5.75,4.625 and 6.25 lgCCID_(50)/mL on day 12,indicating that the virus activity showed a gradual decrease trend at 2 ～8 ℃；With the inactivation time,the virus titer decreased continuously and could not be detected after inactivation for 3 h.Transmission electron microscope observation showed that the inactivated virus particles were intact and the spike protein was evenly distributed.Conclusion The virulence of SARS-CoV-2 stored at 2 ～ 8 ℃ was unstable,so the subsequent inactivation and purification process should be carried out as soon as possible;The titer of virus could not be detected after3 h of inactivation,which provided a reference for the determination of the inactivation process.

    2023 02 v.36 [Abstract][OnlineView][Download 938K]

  • Effect of transient forebrain ischemia-reperfusion on the binding of brain-derived neurotrophic factor promoters to histone deacetylase 3 in hippocampus of rat and its mechanism

    ZHANG Qian;YAN De-ping;SHI Jin-chao;ZHONG Jin;ZHOU Yang;ZHAO Xin;ZHANG Yu;LI Jian-guo;Key Laboratory of Cellular Physiology,Ministry of Education,Department of Physiology,Shanxi Medical University;

    Objective To evaluate the effect of transient forebrain ischemia-reperfusion(I/R)on the binding of brainderived neurotrophic factor(BDNF)promoters to histone deacetylase 3(HDAC3)in the hippocampus of rat and investigate its mechanism.Methods The I/R model of SD rats(I/R group)was established by Pulsinelli four-vessel clamping method,and sham operation group(Sham group)was set at the same time,which were observed for the survival of neurons in the hippocampus of rats by Nissl staining,detected for the binding of BDNF promoters(Bdnf-p1,Bdnf-p2,Bdnf-p4 and Bdnf-p6)to HDAC3 by chromatin immunoprecipitation(ChIP)and determined for the expression of brain derived neurotrophic factor antisense(BDNF-AS)by qPCR.Results Compared with Sham group,the quantity of neurons in hippocampal CA1 region of rats decreased significantly in I/R group,while those in CA3 region and DG region showed no significant changes.The binding levels of Bdnf-p1 and Bdnf-p2 to HDAC3 in hippocampal CA1 region decreased significantly in I/R Group(t = 2.575 and 2.241 respectively,each P<0.05),while there was no significant difference in the binding levels of Bdnf-p4 and Bdnf-p6 to HDAC3(t = 1.033 and 0.348 respectively,each P>0.05);The binding levels of Bdnf-p1 and Bdnf-p2 to HDAC3 in CA3 region increased significantly(t = 12.600 and 3.191,P<0.001 and<0.05,respectively),while the binding level of Bdnf-p6 to HDAC3 decreased significantly(t = 4.029,P<0.05)and no significant difference was observed in the binding level of Bdnf-p4 to HDAC3(t = 0.175,P>0.05);In DG region,the binding level of each BDNF promoter to HDAC3 showed no significantly difference(t = 0.630 ～ 1.687,each P>0.05).Meanwhile,the expression level of BDNF-AS in hippocampal CA1 region of rats decreased significantly(t = 2.560,P<0.05),but increased significantly in hippocampal CA3 and DG regions(t = 3.543 and 3.637 respectively,each P<0.01)in I/R group.Conclusion I/R showed a significant effect on the binding level of BDNF promoter to HDAC3 in rat hippocampus,which may play a role by changing the expression level of BDNF-AS.

    2023 02 v.36 [Abstract][OnlineView][Download 871K]

  • Identification and transcriptional activity analysis of core regulatory region of human guanylate binding protein 5 gene promoter

    YE Ting;YANG Kang;WANG Tian-tian ;LIAO Yu-jiao ;DU Wen-qian ;HUANG Min ;JIANG Pei-wen;LI Min-hui ;YANG Ping;School of Basic Medicine,Chengdu Medical College;

    Objective To construct luciferase reporter plasmids of truncated fragments of different lengths of human guanylate binding protein 5(GBP5)gene promoter and analyze the transcriptional activity of each fragment to determine the core regulatory region.Methods GBP5promoter sequence was amplified by PCR,truncated into five fragments of different lengths and connected to pGL3-basic plasmid.The constructed recombinant plasmids pGL3-GBP5-11/21/31/41/51were transfected into 293FT cells and detected for luciferase activity.The binding sites of transcription factors in GBP5promoter region were predicted by JASPAR software,and Yin-Yang transcription factor 1(YY1)targeting the core regulatory region was selected and verified for the transcriptional regulatory activity.The CDS sequence of YY1 was amplified by PCR to construct the overexpression plasmid pIRES2-EGFP-YY1,which was then co-transfected to 293FT cells with plasmids pGL3-GBP5-21(-1 623 ～ +47 bp)and internal reference plasmid pRL-CMV,and detected for luciferase activity to analyze the regulation of transcription factor YY1 on GBP5 promoter activity.Results Colony PCR and double enzyme digestion identification proved that the plasmid of human GBP5 promoter reporter gene was correctly constructed;JASPAR software predicted that there were multiple transcription factor binding sites such as STAT1,YY1 and Foxp3 in GBP5promoter region.Double luciferase activity assay showed that pGL3-GBP5-21(-1 623 ～ +47 bp)showed the highest promoter activity,while the promoter activity of pGL3-GBP5-41(-520 ～ +47 bp)decreased significantly,suggesting that the core region of GBP5 promoter was located at upstream-1 623 ～-520 bp of 5 'UTR;Overexpression of YY1 significantly activated the GBP5 promoter activity and regulated the expression of GBP5.Conclusion The core regulatory region of human GBP5 promoter was located in upstream-1 623 ～-520 bp of the 5 'UTR,with a binding site of transcription factor YY1 existing in this region.Meanwhile,overexpression of YY1 significantly effected the activity of GBP5 promoter.

    2023 02 v.36 [Abstract][OnlineView][Download 898K]

  • Construction of interferon alpha/beta receptor subunit 1 gene knockout Caco-2 cell line based on CRISPR/Cas9 system

    LIU Xin-yi;AN ni ;ZHANG Qing ;WANG Hong ;KONG Xiang-yu ;WANG Ming-yue;PANG Li-li ;DUAN Zhao-jun;School of Public Health,Gansu University of Traditional Chinese Medicine;

    Objective To knockout interferon alpha/beta receptor subunit 1(IFNAR1) gene in human colorectal adenocarcinoma cells Caco-2 using clustered regularly interspaced short palinmic repeats(CRISPR)/CRISPR-associated protein 9(Cas9)system to construct IFNAR1 knockout Caco-2 cell line.Methods The single guide RNA(sgRNA)sequence was designed to specifically recognize the exon region of IFNAR1 gene using CRISPR/Cas9 technology,and the LentiCRISPRv2-IFNAR1-sgRNA recombinant plasmid was constructed.Caco-2 cells were infected with the plasmid packaged by lentivirus and screened by puromycin resistance.The obtained monoclonal cell lines were cultured by limited dilution method,which were verified for the effect of IFNAR1 gene knockout by target gene sequencing and Western blot,and detected for the mRNA levels of CXC chemokine ligand 10(CXCL10)and interferon-stimulatd gene 20(ISG20)in IFNAR1knockout cells by adding exogenous IFNβ.Results Sequencing results of plasmid LentiCRISPRv2-IFNAR1-sgRNA showed that the insertion sites were all located at the sticky end of BsmBⅠenzyme digestion.Two IFNAR1 knockout monoclonal cell lines were obtained.The sequencing results showed that Caco-2-IFNAR1-KO1 had 5 bp deletion in the sixth exon of IFNAR1,and Caco-2-IFNAR1-KO2 had 18 bp deletion and 1 bp insertion in the seventh exon.Compared with wild-type Caco-2 cells,Caco-2-IFNAR1-KO1 and Caco-2-IFNAR1-KO2 cells showed no expression of IFNAR1 protein.Compared with no IFNβ stimulation,the mRNA levels of CXCL10 gene(t = 0.566 and 1.268 respectively,P>0.05)and ISG20 gene(t =1.522 and 1.733 respectively,P>0.05)in Caco-2-IFNAR1-KO1 and Caco-2-IFNAR1-KO2 cells stimulated by 50 ng/mL IFNβ showed no significant increase.While compared with those of wild-type Caco-2 cells,the mRNA levels of CXCL10gene(t = 6.763 and 6.777 respectively,P<0.05)and ISG20 gene(t = 5.664 and 5.65 respectively,P<0.05)in Caco-2-IFNAR1-KO1 and Caco-2-IFNAR1-KO2 cells decreased significantly under the stimulation of 50 ng/mL exogenous IFNβ.Conclusion Caco-2 cell line with IFNAR1 knockout was successfully constructed by using CRISPR/Cas9 technology,and the downstream molecules activated by IFNAR(interferon alpha/beta receptor)in this cell line were obviously inhibited,which provided a powerful tool for further exploration of the innate immune response and replication packaging mechanism of Caco-2 cells after virus infection.

    2023 02 v.36 [Abstract][OnlineView][Download 968K]

  • Detection of virulence genes and analysis of antimicrobial resistance of E.coli from calves with diarrhea in Tongliao City,Inner Mongolia Autonomous Region,China

    YANG Si-qin;WANG Ping ;CHAGAN Hasi ;SHI Shun-li;College of Animal Science and Technology,Inner Mongolia Minzu University;

    Objective To detect the virulence gene of E.coli from calves with diarrhea in Tongliao City,Inner Mongolia Autonomous Region,China and analyze its antimicrobial resistance as well as the distribution of antimicrobial resistant genes.Methods The sensitivities of 82 E.coli isolates from the fecal samples of calves with diarrhea to thirteen kinds of antibiotics were determined by disk diffusion test.The carrying statuses of thirteen virulence genes and twelve antimicrobial resistant genes of the E.coli isolates were determined by PCR,based on which the phylogenetic background was investigated.Results Of the 82 pathogenic E.coli isolates,48.78%(40/82)、31.71%(26/82)、14.63%(12/82)and 4.88%(4/82)belonged to phylogenic groups A,B1,B2 and D respectively,indicating that the prominent one was group A.A total of 11 virulence genes were detected in 82 isolates.The detection rates of irp2,fyuA,eaeA and STb genes were 79.27%(65/82),63.41%(52/82),53.66%(44/82)and 50%(41/82)respectively,while those of other virulence genes were less than 50%,and no tsh or LT1 was detected.The 82 isolates were significantly resistant to 13 kinds of antibiotics,in which the resistant rates to tetracycline,doxycycline and amoxicillin were 100%(82/82),97.56%(80/82)and 90.24%(74/82)respectively.All the isolates were mutidrug resistant,most of which were resistant to eight kinds of antibiotics(16/82,19.51%).A total of twelve antimicrobial resistant genes were detected in the 82 isolates,in which the positive rates of genes resistant to β-lactams(bla_(TEM)),sulfonamide(sul1 and sul2),tetracycline(tetB and tetD)and aminoglycosides(aadB)were more than 70%.Conclusion The 82 pathogenic E.coli isolates mainly belonged to group A,with high detection rates of virulence gene and antimicrobial resistant gene as well as high and multiple drug resistance.The study provided a reference for the prevention and treatment of and clinical medication of E.coli-associated diseases in calves in Tongliao Region.

    2023 02 v.36 [Abstract][OnlineView][Download 848K]

  • Pharmacodynamics of human interferon α1b against severe acute respiratory syndrome coronavirus 2 Omicron strain in vitro

    LIU Lin-lin;LI Yu-wei ;ZOU Yong;ZHANG Xue-mei;LU Jia ;LIU Xiao-ke ;WANG Ze-yun ;LIU Yu-lin;LIU Jing-hui;Changchun Institute of Biological Products Co.,Ltd.;

    Objective To evaluate the pharmacodynamics of human interferon(IFN)α1b against severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)Omicron strain in vitro.Methods Total four drugs human IFNα1b bulk,human IFNα1b eye drops,human IFNα1b spray and Remdesivir were detected for cytotoxicity by CCK-8 assay.The inhibitory effect of human IFNα1b on SARS-CoV-2 Omicron strains(BA.5/BA.2/BA.1)was determined by qPCR.Results Human IFNα1b bulk of the maximum concentration(1 × 10~7IU/mL)and Remdesivir of the maximum concentration(150 μmol/L)did not achieve half cytotoxicity to Vero cells;The median cytotoxicity concentrations(CC_(50))of human IFNα1b eye drops and human IFNα1b sprays were 29 958 and 37 550 IU/mL,respectively,showing toxicity to Vero cells.The median effective concentrations(EC_(50))of human IFNα1b against virus strains BA.1,BA.2 and BA.5 after incubation for 2 h in advance were 9.30,13.38 and 12.33 IU/mL and those of Remdesivir were 0.314 7,0.291 0 and0.300 3 μmol/L.When incubation with virus simultaneously,the EC_(50)of human IFNα1b to BA.1,BA.2 and BA.5 were19.68,10.91 and 18.84 IU/mL and those of the control drug Remdesivir were 0.320 5,0.274 4 and 0.304 1 μmol/L,respectively.Conclusion At the cell level in vitro,human IFNα1b of very low activity showed a good inhibitory effect on SARS-CoV-2 Omicron strain,which was expected to be a clinical specific drug for the treatment of SARS-CoV-2 Omicron strain infection.

    2023 02 v.36 [Abstract][OnlineView][Download 854K]

  • Effect of silencing E6-associated protein on level of p53 protein in human papilloma virus negative cervical cancer cells

    XIE Yi-hang;GUO Yu-wei;SUN Bo-xuan;XIN Yang;YU Jia-min;ZHAO Chun-yan;Department of Clinical Biochemistry,College of Laboratory Medicine,Dalian Medical University;

    Objective To investigate the effect of silencing E6-associated protein(E6AP)on the level of p53 protein in human papilloma virus(HPV)negative cervical cancer cells(C33A cells).Methods The siRNA sequence silencing E6AP(siE6AP)and silencing control disordered siRNA sequence(siControl)were transfected into C33A cells with the mediation of LipofectamineTM2000 transfection reagent respectively.The silencing effect of siRNA on E6AP and the expression of p53and cleaved-caspase-3 proteins were detected by Western blot.Results The levels of E6AP protein in C33A cells of siE6AP group were significantly lower(t =-4.597,P<0.05),while the levels of p53 and cleaved-caspase-3 proteins were significantly higher than those of siControl group(t = 4.533 and 7.099 respectively,each P<0.05).Conclusion Silencing of E6AP significantly increased the expression of p53 protein in C33A cells,indicating that silencing of E6AP may restore the activity and function of p53 protein in C33A cells.

    2023 02 v.36 [Abstract][OnlineView][Download 811K]

  • Effect of caloric restriction on myocardial ischemia/reperfusion injury in mice and its mechanism

    WANG Wen-li;HE Zhong-mei;JI Ye-nan;SUN Si-yu;YANG Rui-rui;YAN Zi;CAO Ji-min;Department of Physiology,Key Laboratory for Cellular Physiology of Ministry of Education,Shanxi Medical University;

    Objective To investigate the effect of caloric restriction(CR)on myocardial ischemia/reperfusion injury(MI/RI)in mice and its mechanism.Methods C57 mice were randomly divided into normal diet group(AL group,free feeding)and CR group(diet decreased by 10% every 2 weeks)for 8 weeks and monitored for weight changes.Each group was divided into sham operation group and MI/RI group,total 4 groups,AL + Sham group,AL + I/R group,CR + Sham group and CR + I/R group).The left anterior descending coronary artery was ligated for 30 minutes and then reperfused for 24 hours in mice of MI/RI group and mice in Sham group were only threaded but not ligated.The mice were determined for myocardial ischemia and infarct size by Evans blue/TTC staining,observed for the pathology of myocardium by HE staining,determined for the activities of lactate dehydrogenase(LDH),superoxide dismutase(SOD)and the contents of creatine kinase-MB(CK-MB)and malondialdehvde(MDA)in myocardium by the corresponding kits,determined for serum levels of IL-1β and IL-18 by ELISA and detected for the expression of pyroptosis-associated proteins in myocardium by Western blot.Results After 8weeks,the weights of mice in CR group[(24.54 ± 0.41)g]were significantly lower than those in AL group[(31.46 ±0.25)g](t = 14.34,P<0.05).Compared with those in AL + I/R group,the area of myocardial ischemia in CR + I/R group showed no significant difference(t = 0.783 0,P>0.05),while the area of myocardial infarction decreased significantly(t = 7.250,P<0.01);The myocardial arrangement was relatively neat,and the degree of pathological changes was obviously reduced;LDH activity,CK-MB and MDA contents decreased significantly(t = 4.331,2.875 and 5.343 respectively,each P<0.05),while SOD activity increased significantly(t = 4.211,P<0.05);Serum levels of IL-1β and IL-18 decreased significantly(t = 3.375 and 4.266 respectively,each P<0.05);The expression levels of nod-like receptor protein 3(NLRP3),gasdermin D(GSDMD),apoptosis-associated speckle-like protein(ASC)and caspase-1 significantly decreased(t = 3.412,3.420,3.480 and 2.585 respectively,each P<0.05).Conclusion CR alleviated MI/RI in mice,and its mechanism was related to the inhibition of cardiac pyroptosis.

    2023 02 v.36 [Abstract][OnlineView][Download 892K]

  • Effects of polysorbates on stability of monoclonal antibody drugs

    ZHU Sheng-ying;CAO Jia-wei;XU Jin;CHEN Chen-hui;GUO Qing-cheng ;LI Jun ;ZHANG Da-peng;QIAN Wei-zhu;HOU Sheng;GUO Huai-zu;Shanghai Zhangjiang Biotechnology Co.,Ltd,State Key Laboratory of Antibody Medicine and Targeted Therapy;

    Objective To evaluate the effects of various polysorbates(PS)on the stability of different types of monoclonal antibody(mAb)drugs.Methods Three types of monoclonal antibodies mAbA(IgG1 proantibody drug),mAbB(IgG1 mAb)and mAbC(IgG1 mAb with Fc N297A mutation)were used as model proteins,and different kinds or contents of PS were added into the mAb formulations respectively to investigate the influencing factors.The effects of PS on the stability of mAb drugs were evaluated comprehensively by detecting the changes of quality attributes,such as protein aggregates and insoluble particles.Results PS20 and PS80 showed no significant difference in inhibiting the formation of aggregates and charge variants in the three mAbs(P>0.05),while the addition of PS80 in mAbB and PS20 in mAbC significantly inhibited the increase of insoluble particles respectively(P<0.05);The content of PS20 showed a significant effect on the detection indexes of charge variants and insoluble particles in mAbC(P<0.05).Conclusion Different types of mAbs have different sensitivities to various kinds and contents of PS.Therefore,when designing the formulation of mAbs,it is necessary to select appropriate kinds and contents of PS to further improve the stability of mAb drugs.

    2023 02 v.36 [Abstract][OnlineView][Download 907K]

  • Meta-analysis of safety of human purified Vero cell rabies vaccine after exposure

    WU Hao-fei;WANG Lei;GE Ling-rui;ZHANG Jing;YANG Wen-bin;XU Qi;MENG Sheng-li;Rabies Vaccine Research Department,Ab&B Bio-Tech Co.,Ltd.JS;

    Objective To evaluate the safety of human purified Vero cell rabies vaccine(PVRV)after exposure in China by Meta-analysis.Methods With rabies,vaccine and safety as key words,a systematic search was performed in PubMed,EMBASE,Cochrane and China National Knowledge Infrastructure(CNKI),supplemented by manual retrieval.A Meta-analysis was performed to analyze the incidence of adverse events of two immunization regimens Zagreb and Essen using Review Manager 5.4 software after literature screening and data extraction according to the inclusion and exclusion criteria.Results A total of 12 studies were included,of which 7 were prospective studies and 5 were retrospective studies.Most included in the studies showed a low risk of bias.The incidence of adverse events in Zagreb regimen was significantly higher than that in Essen regimen[relative risk(RR)= 1.01,95% CI = 0.90 ～ 1.14;I~2= 73.00%,P<0.05],but there was a high degree of heterogeneity.The incidence of fever,pain and induration in Zagreb regimen was significantly higher than that in Essen regimen(RR = 1.14,0.92 and 0.86,95% CI = 0.82 ～ 1.60,0.73 ～ 1.14 and 0.29 ～ 2.51;I~2= 81%,65% and 92%,respectively,P<0.01).Conclusion Two regimens of PVRV vaccination after exposure showed good safety.However,when adopting Zagreb regimen,attention should be paid to the physical conditions of children and the elderly with relatively poor immunity to avoid adverse events.

    2023 02 v.36 [Abstract][OnlineView][Download 1042K]

  • Genetic sequence characteristics of varicella-zoster virus epidemic strains in Changchun City from 2021 to 2022

    XIA Li-xin;SUN Yue-qiu;WANG Meng-han;JIANG Xue-ou;ZHOU Ying;GU Jian-yang;Changchun Keygen Biological Products Co.,Ltd.;

    Objective To identify the genotypes and analyze the molecular characteristics of varicella-zoster virus(VZV)endemic strains in Changchun from 2021 to 2022.Methods A total of 59 patients with varicella or herpes zoster treated in China Japan Union Hospital of Jilin University in Changchun from 2021 to 2022 were selected as the research objects,which were identified for the serum VZV-specific antibodies by fluorescent antibody to membrane antigen(FAMA)method.The viral DNA was extracted from herpetic fluid of the confirmed patients,and multiple open reading frames(ORFs)including ORF1,ORF12,ORF16,ORF17,ORF21,ORF22,ORF37,and ORF54 of VZV were amplified by PCR,which was genotyped according to the single nucleotide polymorphisms(SNPs) of the ORFs.Furthermore,multiple ORFs,ORF38,ORF54 and ORF62 were identified by using restriction fragment length polymorphism(RFLP)to distinguish clinical strains and vaccine strains.Results 59 serum samples were positive,indicating that all the patients were infected with VZV.Among the 59 clinical samples,4 samples were completely matched with Clade 2 genotype,4 samples showed 2 SNPs,including ORF1(SNP790)mutation of C→A and ORF12(SNP18 082)mutation of T→C,and 51 samples showed ORF12(SNP18 082)mutation of T→C.All 59 VZV clinical isolates were PstⅠ~+BglⅠ~+SmaⅠ~-,which was different from the vaccine strain PstⅠ~-BglⅠ~+SmaⅠ~+.Conclusion All the VZV endemic strains in Changchun from 2021 to 2022 were Clade 2 genotype,and no vaccine strain was found to be pathogenic.

    2023 02 v.36 [Abstract][OnlineView][Download 811K]

  • Development and verification of double antibody sandwich ELISA for quantitative detection of TrypLE

    BI Hua;LU Ning ;NIU Lin-ru ;WANG Yu-zhe ;ZHU Xiang ;LI Wen-hui ;YANG Ling ;ZHOU Yong;LIANG Ya-li;Recombinant Drug Room of the Institute of Bioassay,National Institutes for Food and Drug Control;

    Objective To develop and verify a double antibody sandwich ELISA method for quantitative detection of TrypLE.Methods The optimal concentration of capture antibody and detection antibody were determined by orthogonal experiments to develop TrypLE double antibody sandwich ELISA quantitative detection method,which was verified for linear range,specificity,limit of detection(LOD),limit of quantitation(LOQ),accuracy and reproducibility.A variety of biological products were detected by the developed method to verify the applicability.Results The TrypLE double antibody sandwich ELISA quantitative detection method was established by using 3 μg/mL capture antibody and 15 000 times dilution of detection antibody,with a linear range of 0.41 ～ 40.00 ng/mL,a LOD of 0.258 ng/mL,a LOQ of 0.5 ng/mL.The measurement deviation was less than 5% and the CV of reproducibility verification was less than 5% when detecting standards and samples.The recovery rates of different types of samples were within 80% ～ 120%.Conclusion The established TrypLE double antibody sandwich ELISA quantitative detection method accurately,effectively and quickly detected residual amount of TrypLE in various types of biological products with good specificity,accuracy and reproducibility.

    2023 02 v.36 [Abstract][OnlineView][Download 815K]

  • Verification of detection method for multiple pathogens in plasma of convalescent patients with Coronavirus Disease 2019

    WANG Yue;ZHANG Jin;YU Jian-hong;LIU Ying;LUO Yan;ZHANG Lin-lin;GUO Jia-ru;XIANG Yang;ZHANG Xue;ZHAO Chuan-bo;PENG Gan;CHEN E-xiang;HE Yan-lin ;LI Ce-sheng;YANG Xiao-ming;Sinopharm Wuhan Plasma-derived Biotherapies Co.,Ltd;

    Objective To systematically verify the detection method for multiple pathogens in plasma of convalescent patients(CPs)with Coronavirus Disease 2019(COVID-19).Methods According to the actual situation of plasma samples and the requirements of kit,the molecular biological detection method for multiple pathogens in plasma of CPs with COVID-19 was systematically verified for specificity,reproducibility,intermediate precision and limit of detection(LOD),and confirmed for applicability by detecting 50 plasma samples of CPs with COVID-19.Results The results of interference test and cross test showed that the detection of positive samples and negative samples were not affected;The RSDs of melting temperature values(Tm)of the positive control four pathogens by the same or different test personnels at different time under the same test conditions were 0.07%,0.14%,0.07%,0.14% and 0.06%,0.23%,0.23%,0.20%,and those of internal control(IC)and amplification control(AC)1 and 2 were 0.07%,0.01%,0.07%,0.14% and 0.11%,0.10%,0.15%,0.22%,respectively.Meanwhile,the RSDs of reproducibility and intermediate precision were less than 15% and20% respectively,which met the requirements;The minimum LOD of 22 pathogens were determined;No pathogen was detected in 50 plasma samples of CPs with COVID-19.Conclusion The method for detecting pathogens in plasma of CPs with COVID-19 was specific,stable,reliable and reproducible,which was suitable for the detection of pathogens in plasma of CPs with COVID-19.

    2023 02 v.36 [Abstract][OnlineView][Download 898K]

  • Optimization of glycosylation type of recombinant human growth hormone-Fc fusion protein expressed in CHO cells

    ZHANG Kai-ning;LIU Han ;ZHU Qiu-mei ;FU Rui-li ;LIU Yu-lin ;SUN Rui-xin ;LIU Jing-hui ;ZHAO Jing-nan ;HAN Xue-rong;School of Life Science and Technology,Changchun University of Science and Technology;

    Objective To optimize the expression of recombinant human growth hormone-Fc(rhGH-Fc)fusion protein in CHO cells in order to obtain better glycosylation ratio and lower content of highmannose.Methods CHO cells expressing rhGH-Fc were cultured in a 7 L bioreactor.The glycosylation modifications of rhGH-Fc were adjusted by improving the composition of feeding media(using three commercial media:Gly-1:EX-CELL Glycosylation Adjust,Gly-2:SHEFF-CHO PLUS PG ACF and Gly-3:EfficientFeed C + AGT Supplement & GlycanTune C + Total Feed),and the glycosylation type and proportion of the target proteins were analyzed by mass spectrometry.Results The G0F(main glycosylation types:G0,G1 and G2;F:fucose)of Gly-1,Gly-2 and Gly-3 were 32.89%,58.66% and 33.28%,the G1F were 31.39%,18.03%and 34.90%,and the G2F were 31.39%,18.03% and 34.90%,respectively.Gly-1 and Gly-3 made the target protein contain less G0F while more G2F;Gly-3 feeding scheme-showed less high mannose modification than the other two schemes.Conclusion Gly-1 medium changed the glycosylation modification from G0F to G1F and G2F,while Gly-2 medium changed that from G2F and G1F to G0F.However,Gly-3 medium changed the glycosylation modification from G0F to G1F and G2F,and the contentof high mannose was less than 5%,which may have a better effect on modifying glycosylation type and proportion of the target protein.

    2023 02 v.36 [Abstract][OnlineView][Download 1016K]

  • Development and verification of plaque method for detection of tick-borne encephalitis virus titer

    CHEN Na-na;CHEN Shi-yang ;WANG Ying;DU Xiang-yu;MIAO Hui;GONG Xiao-tang;LIU Shuang-jun;LI Jing-liang;Department Ⅲ of Vaccine,Changchun Institute of Biological Products,Co.,Ltd.;

    Objective To develop and verify a plaque method for detection of infectious titer of tick-borne encephalitis virus(TBEV)strain(PHKT strain for short)adapted to primary hamster kidney(PHK)cells.Methods PHK cells were infected with TBEV,a primary mouse brain adaption strain,and passed consecutively for 12 passages.The titer of PHKT was detected by plaque method(Monolayer BHK-21 cells were infected with PHKT of various passages at different dilution ratios,and the plaque number was calculated by neutral red staining)and challenge titration in mouse brain(Mice were challenged with PHKT of various passages at different dilution ratios through brain cavity,0.03 mL for each,observed continuously for 14 days,and calculated for the median lethal dose(LD50)by Reed-Muench method)respectively,and the correlation between the results of two methods was analyzed.The developed plaque method for the detection of TBEV titer was verified for specificity,repeatability and intermediate precision.Results The plaque titer of PHKT virus was up to8.9 lgPFU/mL;The correlation between the results of plaque method and mouse brain challenge titration method was good(r = 0.92);The specificity of plaque method for detecting infectious titer of PHKT virus was good,and the coefficients of variation(CVs)of repeatability and intermediate precision were both less than 5%.Conclusion A plaque method for detecting infectious titer of PHKT virus was developed,which may be used as an alternative method for challenge titration in mouse brain.

    2023 02 v.36 [Abstract][OnlineView][Download 822K]

  • Development and verification of high performance liquid chromatography for determination of residual 4-dimethylaminopyridine content in pneumococcal polysaccharide-protein conjugate vaccine

    LI Shuang-hua;ZHAO Ping;TIAN Hong-min;LI Hong;DONG Wei;CHEN Yu;BAO Lu-wei;WU Ke;Hubei Provincial Engineering Laboratory of Novel Vaccine & Recombinant Protein,Wuhan Bravovax Co.,Ltd.;

    Objective To develop and verify a high performance liquid chromatography(HPLC) for determination of residual 4-dimethylamino-pyridine(DMAP)content in pneumococcal polysaccharide-protein conjugate vaccine.Methods A HPLC method for determination of residual DMAP content in pneumococcal polysaccharide-CRM197 protein conjugate vaccine was developed by optimization of type of chromatographic column and composition of mobile phase,verified for specificity,accuracy,repeatability and reproducibility,and determined for limit of detection(LOD),limit of quantitation(LOQ)and linear range.The residual DMAP contents in pneumococcal polysaccharide-CRM197 protein conjugate vaccine of 13 serotypes and the intermediates of 1-cyano-4-dimethylamino pyridinium tetrafluoroborate(CDAP)-activated pneumococcal polysaccharide were determined by the developed method.Results The condition for HPLC was optimized as follows:Sepax HP-C18 chromatographic column(5 μm,4.6 mm × 250 mm)was adopted,using the mixture of 5 mmol/L sodium heptanesulfonate containing 20 mmol/L potassium dihydrogen phosphate(the pH value was adjusted to 3.0 with phosphoric acid)and acetonitrile at a ratio of 87∶13(v/v)as mobile phase at a detection wavelength of 280 nm,a flow rate of 1 mL/min,a sample load of 10 μL and a column temperature of 35 ℃.The mechanism of samples and solvent for pre-treatment showed no interference to the determination result,indicating good specificity of the developed method.The LOD and LOQ were 3.6 and 14.4 ng/mL respectively,while the linear range was 0.05 ～ 10 μg/mL with a R2value of0.999 9.The spike recovery rate was 83% ～ 105%,while the RSDs in repeatability and reproducibility tests were 0.28% ～0.72% and 7.38% respectively.The residual DMAP contents in pneumococcal polysaccharide-CRM197 protein conjugate vaccine of 13 serotypes were 0.135 ～ 1.635 μg/mL.DMAP was detected in the first step of activation of pneumococcal polysaccharide with CDAP,while no CDAP was detected.Conclusion The developed HPLC method is simple,specific,accurate,repeatable and reproducible,which is effective for quality control of pneumococcal polysac-charide-protein conjugate vaccine.

    2023 02 v.36 [Abstract][OnlineView][Download 839K]

  • Research progress of broadly neutralizing antibodies against human immunodeficiency virus

    ZHANG Hui;DENG Ting-ting ;LI Shao-wei;GU Ying;State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics,School of Public Health,Xiamen University;

    Acquired immune deficiency syndrome(AIDS)is a global public health issue,which has a major impact on human life and health.As the main pathogen of AIDS,human immunodeficiency virus type 1(HIV-1)has high variability and latent characteristics,there are no available vaccines and drugs to prevent and cure AIDS.The broadly neutralizing antibodies(bNAbs)against HIV-1 can induce effective immune response to HIV-1 infection in vivo,and neutralize different types of strains at the same time,which have great application potential in preventing HIV-1 infection and controlling the process of AIDS.In this paper,the production and acquisition,classification and characteristics as well as application of HIV-1bNAbs are reviewed so as to provide a reference for the subsequent research on HIV-1 vaccine and development of antibody drugs.

    2023 02 v.36 [Abstract][OnlineView][Download 836K]

  • Progress of proteomics in research of colon cancer

    WU Ao;ZHANG Li-jun;Shanghai Public Health Clinical Center;

    The diagnosis and treatment of colon cancer has always been a difficult problem in the medical field.In recent years,there have been more and more researches on differential proteins in colon cancer.It has become a new trend to find differential proteins in cancer tissues by proteomic technology and study their functions and roles in cancer,which was so as to used in cancer diagnosis and treatment.We have found two differential proteins ATP-binding cassette sub-family G member 2(ABCG2)and protein disulfide-isomerase A2(PDIA2)in colon cancer by proteomic technology before,and this article reviews the expression and function of these two proteins in colon cancer.

    2023 02 v.36 [Abstract][OnlineView][Download 870K]

  • Development and application progress of standards for antibody against SARS-CoV-2

    ZHANG Hui;MAO Qun-ying;LIANG Zheng-lun;XU-Miao;National Institutes for Food and Drug Control;

    Coronavirus Disease 2019(COVID-19)caused by severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)seriously threatens human health and public health safety.Vaccination plays an important role in effectively controlling the epidemic situation.More than 150 SARS-CoV-2 vaccines have entered the clinical trial stage and 38 have been approved for emergency use or marketing.Neutralizing antibody level is the main index for evaluation of the immunogenicity of vaccines,but there has been no standardized detection method for SARS-CoV-2 neutralizing antibody till now,which makes it difficult to compare the neutralizing antibody levels among different laboratories and different products horizontally,and seriously restricts the development and evaluation of vaccines and antibody therapeutic drugs.With the rapid sale of the first generation of standards for SARS-CoV-2 antibody and the emergence of variants of concern(VOC)of SARS-CoV-2,WHO and China carried out the development of the second generation of standards simultaneously in 2022.This paper reviews the development and application progress of the standards for SARS-CoV-2 antibody in WHO and China.

    2023 02 v.36 [Abstract][OnlineView][Download 796K]

  • Present situation and prospect of development and vaccination of acellular pertussis vaccine

    CUI Le-le;CHEN Zhi-yun;HE Qiu-shui;Department of Medical Microbiology,School of Basic Medical Sciences,Capital Medical University;

    Whooping cough(pertussis)is a highly contagious respiratory disease,which mainly affects infants and young children.Vaccination is an effective measure to prevent pertussis infection.Due to the fewer side effects,acellular pertussis vaccines(aPVs)have replaced whole-cell pertussis vaccine(wPVs)in many countries.Despite vaccination coverage is high,the short immunity induced by aPVs is considered to be significant reason for the re-emergence of pertussis.For improving pertussis vaccine,genetically detoxified vaccine and live attenuated vaccine have showed obvious clinical results and other strategies including using novel adjuvants in aPVs,increasing antigen or packaging aPVs with nanoparticles also have good prospects.This paper reviews the antigen composition and protective effects of aPVs,vaccination programs in different countries,potential candidate components of pertussis vaccine and new strategies for prevention of pertussis.

    2023 02 v.36 [Abstract][OnlineView][Download 783K]

  • Research progress on regulation of protein tyrosine kinase 6 by micro RNA in malignant tumors

    GAO Na;CHU Hui-yuan;CHEN Che;Gansu University of Traditional Chinese Medicine;

    Protein tyrosine kinase 6(PTK6)is a non-receptor tyrosine kinase,which plays a key role in the genesis and progression of various tumors and shows a dual role.MicroRNAs(miRNAs)are small non-coding RNAs that regulate the expression of post-transcriptional genes.In recent years,more and more studies have shown that the regulation of PTK6 by miRNA plays an important role in tumor.This paper illustrates the effect of miRNA on PTK6 expression in various cancers so as to provide theoretical basis for cancer treatment.

    2023 02 v.36 [Abstract][OnlineView][Download 797K]

  • Pathogenic mechanism of Acinetobacter baumannii and research status of corresponding vaccines

    MA Yuan;SONG Lei;LUO Zhao-qing;Department of Respiratory Medicine and Center of Pathogen Biology and Infectious Diseases,The First Hospital of Jilin University;

    As a Gram-negative bacillus,Acinetobacter baumannii is one of the main opportunistic pathogens causing nosocomial infection,and mainly infects various human organs such as lungs,which finally causes bacteremia,pneumonia and other diseases,and seriously threatens lives of patients.People with weak immunity are the main susceptible people.The strain has strong stress resistance and multiple antibiotic resistance,making its infection a difficult problem in clinical treatment.The outer membrane complex(OMC),outer membrane vesicles(OMVs),secretory system and lipopolysaccharide of A.baumannii play an important role in the process of infection of host cells.At present,the vaccines developed against these pathogenic components can play a certain role in preventing and treating A.baumannii infection,and have a good application prospect.In this paper,the pathogenesis of A.baumannii and the current situation of vaccine development are reviewed,so as to provide a reference for the development of novel prevention and treatment methods of it in clinic.

    2023 02 v.36 [Abstract][OnlineView][Download 816K]


@2012 Editorial Department of "Chinese Journal of Biologicals"