2022年04期目次

  • Immune responses induced with different doses of inactivated COVID-19 vaccine at different time intervals

    CANG Tian-le;LI Ke-lei;JIANG Cong-li ;HU Wei;LIU Si-yuan;WANG Hai-xin;WANG Mei-rong;LIU Jian-kai;LIU Jian-dong;Beijing Minhai Biotechnology Co.Ltd,Beijing Engineering Technology Research Center for New Conjugate Vaccine;

    Objective To compare the immune responses induced with inactivated COVID-19 vaccine by different immunization schedules in mice in order to provide an experimental basis for formulation of subsequent immune strategies.Methods BALB/c mice were immunized with three doses of inactivated COVID-19 vaccine,1 μg per mouse,each at an interval of 14 d,or with two doses at an interval of 28 d. Serum samples were collected 14 d after each dose and determined for immune response against the prototype strain of SARS-CoV-2. Results The neutralizing antibody titer against the prototype strain of SARS-CoV-2 induced by two doses of inactivated COVID-19 vaccine at an interval of 14 d was significantly lower than that by three doses(P < 0. 000 1). The GMT of neutralizing antibody 14 d after immunization with two doses of vaccine at an interval of 28 d was significantly higher than that at an interval of 14 d(P < 0. 05).However,the neutralizing antibody titer against prototype strain in immune sera was significantly higher than those against Beta and Delta variants of SARS-CoV-2(each P < 0. 05). Conclusion The immunization with three doses of inactivated COVID-19 vaccine greatly improved the induced neutralizing antibody immune response. The immune response induced by the vaccine at an interval of 28 d was superior to that at an interval of 14 d. The mouse sera immunized with inactivated COVID-19 vaccine may also neutralize the SARS-CoV-2 variants.

    2022 04 v.35 [Abstract][OnlineView][Download 303K]

  • Role of human bone marrow mesenchymal stem cell culture supernatant in repairing of oxidative stress injury of SH-SY5Y cells and relevant mechanism

    PU Wen-xing;MI Xu-guang ;ZHOU Yang;WANG Wen-tao;JING Meng ;MENG Fan-kai;School of Clinical Medicine,Changchun University of Chinese Medicine;

    Objective To investigate the role of human bone marrow mesenchymal stem cell culture supernatant(hBMSCs-Sup)in repairing of oxidative stress injury of SH-SY5Y cells as well as the relevant mechanism. Methods SHSY5Y cells were divided into control,MPP+,MPP+ + hBMSCs-Sup and MPP+ + hBMSCs-Supexo-groups,and cultured in complete culture media containing an equal volume of phosphate buffer,1-methyl-4-phenylpyridine ion(MPP+)at various concentrations(0. 25,0. 5,1,2 and 4 mmol/L),0. 25 mmol/L MPP+ + hBMSCs-Sup or 0. 25 mmol/L MPP+ +hBMSCs-Sup without exosomes(hBMSCs-Supexo-)respectively for various hours(24,48 and 72 h). The cell viability was determined by MTT assay,while the intracellular reactive oxygen species(ROS) content and apoptosis rate by flow cytometry,and the expression of related proteins by Western blot. Results The viability of SH-SY5Y cells gradually decreased with the increasing MPP+concentration and the prolongation of treatment time. Compared with those in control group,the survival rate of cells in MPP+ group decreased significantly(P < 0. 001),while the ROS content and apoptosis rate increased significantly(P < 0. 001),the expression level of apoptosis-associated protein Bax increased significantly(P < 0. 01),and the expression levels of propagation-associated protein P-Akt and anti-apoptosis-associated protein Bcl-2decreased significantly(P < 0. 01). However,compared with those in MPP + group,the cell survival rate in MPP + +hBMSCs-Sup group increased significantly(P < 0. 001),while the ROS content(P < 0. 001)and apoptosis rate(P < 0. 01)decreased significantly,the Bax expression level decreased significantly(P < 0. 05),and the expression levels of P-Akt and Bcl-2 proteins increased significantly(P < 0. 05). The cell survival rate and ROS content in MPP + + hBMSCs-Supexogroup showed no significant difference with those in MPP+group(P > 0. 05). Conclusion The hBMSCs-Sup decreased the intracellular ROS content and activated the AKT pathway through its exosome components,while restored the survival rate down-regulated by MPP+and the apoptosis rate increased by MPP+of SH-SY5Y cells.

    2022 04 v.35 [Abstract][OnlineView][Download 368K]

  • Effect of miR-455-5p targeting RECK gene on A549 cell migration and microvascular angiogenesis

    LIU Cui-hua;ZHAO Fang-xin;SUN Peng;HONG Mei;WU Jian-qiang;ZHANG Xuan;College of Basic Medicine,Inner Mongolia Medical University;

    Objective To investigate the effect of miR-455-5p targeting RECK gene on migration and microvascular angiogenesis of non-small-cell lung carcinoma(NSCLC) A549 cells. Methods The data on expressions of mi R-455-5p and RECK genes in lung cancer in TCGA database were analyzed by UALCAN online,and the targeting relationship between miR-455-5p and RECK genes was predicted by miRanda. The target was validated by double luciferase reporter gene assay. A549 cells were transfected with miR-455-5p mimic,inhibitor and negative control respectively,and determined for the relative expressions of miR-455-5p and RECK mRNAs by qRT-PCR,and for the expressions of RECK,MMP2 and MMP9 by Western blot. The migration ability of A549 cells was determined by TranswellTMassay. The expression of vascular endothelial growth factor(VEGF)-A in culture supernatant of A549 cells was determined by ELISA.Human umbilical vein endothelial cells(HUVECs)were treated with the culture supernatant of A549 cells and determined for microvascular angiogenesis ability by in vitro microtube formation assay. Results The expression level of miR-455-5p was significantly higher,while that of RECK was significantly lower in lung cancer tissue than in normal tissues. The miR-455-5p had a binding domain in the 3′-UTR region of RECK gene which was the direct target. The miR-455-5p down-regulated the expression of target gene RECK while up-regulated those of MMP2 and MMP9, promoted the migration of A549 cells and the secretion of VEGF-A significantly,and enhanced the ability of inducing HUVEC to differentiate into microtubules. Conclusion The miR-455-5p promoted the A549 cell migration and microvascular angiogenesis by up-regulating the expressions of MMPs and VEGF-A via directly targeting RECK.

    2022 04 v.35 [Abstract][OnlineView][Download 396K]

  • Prokaryotic expression and polyclonal antibody preparation of Escherichia coli heat-labile enterotoxin B subunit

    GONG Zhi-yuan;ZHANG Meng-yao;JIN Hong-li;HUANG Pei;ZHANG Hai-li;LI Yuan-yuan;WANG Hua-lei;College of Veterinary Medicine,Key Laboratory of Zoonosis Research,Ministry of Education,Jilin University;

    Objective To express the heat-labile enterotoxin B(LTB)subunit of Escherichia coli in prokaryotic cells and prepare its polyclonal antibody. Methods LTB gene was amplified by PCR and inserted into prokaryotic expression vector pET30a(+). The constructed recombinant plasmid pET30a(+)-LTB-8 × His was transformed to E. coli BL21(DE3) and induced by IPTG. The condition for expression of the target protein was optimized. The target protein was purified by His nickel ion affinity chromatography,with which white rabbits were immunized to prepare polyclonal antibody against LTB. Results Both restriction analysis and sequencing proved that recombinant plasmid pET30a(+)-LTB-8 × His was constructed correctly. The expressed recombinant LTB protein,with a relative molecular mass of about13 000,existed in a form of inclusion body. The optimal condition for induction was treatment with 0. 4 mmol/L IPTG at30 ℃ for 4 h. The concentration of purified protein was 3. 7 mg/mL. The prepared polyclonal antibody could be used for Western blot and IFA of LTB. Conclusion Recombinant LTB protein was successfully expressed by using E. coli expression system,and polyclonal antibody with good reactogenicity and specificity was prepared,which laid a foundation of diagnosis of enterotoxigenic Escherichia coli(ETEC) diseases and study on the properties of LTB mucosal immune adjuvant.

    2022 04 v.35 [Abstract][OnlineView][Download 298K]

  • Isolation,identification and screening of lactobacillus from Yao chicken feces

    JIN Zheng-yu;DENG Yong-jun;YANG Ze-min;LI Shuang;YANG Ying;College of Animal Science,Guizhou University;

    Objective To screen high-quality host-derived lactobacillus from Yao chicken feces. Methods The feces from healthy adult Yao chicken were collected and inoculated onto MRS broth medium for enrichment culture,and 5%volume of the bacterial liquid was treated with PBS(pH 3)medium for 1 h. The bacterial sediment after centrifugation was treated with PBS containing 0. 3% bile salt for 1 h,then diluted doubly and spread onto MRS agar plate. The dominant strains were screened,observed for the colony morphology and subjected to microscopy with Gram staining and 16S r RNA gene sequencing,of which the growth curve,acid production performance,acid resistance,bile salt resistance and bacteriostatic ability were analyzed. Results The isolated strain was Enterococcus haynii named as GZYY2021,of which the growth reached a stable stage 8 h,and the pH value was nearly 4. 43 24 h after culture. The strain showed high antibacterial activity against Salmonella pullorum,of which the diameter of inhibition zone was about 18. 46 mm.However,the strain was resistant to low pH value and bile salt environment. Conclusion The strain isolated provided an usable microbial resources for the subsequent research and application of microecological preparations for Yao chichen and other poultries.

    2022 04 v.35 [Abstract][OnlineView][Download 309K]

  • Prokaryotic expression and purification of RcDof protein associated with castor dwarfing

    ZHANG Shuai;WANG Shuang ;LI Yue ;LI Guo-rui ;HUANG Feng-lan ;CHEN Yong-sheng;Agricultural College,Inner Mongolia University for Nationalities;

    Objective To express RcDof protein associated with castor dwarfing in prokaryotic cells and purify the expressed product. Methods The signal peptide and structural domain of RcDof gene were predicted by using SignalP 4. 0server and Iprscan software respectively. RcDof(44-101)gene was amplified from plasmid pMD-18T-RcDof by PCR and cloned into prokaryotic expression vector pETMBP-XE. The constructed recombinant plasmid pETMBP-XE-RcDof(44-101) were induced with IPTG,and the expressed recombinant protein was purified by affinity,cation exchange and molecular sieve chromatography. Results There was no signal peptide cleavage sequence in RcDof gene,while a zinc finger conserved domain between bases 44 and 101. Both restriction analysis and sequencing proved that recombinant plasmid pETMBP-XE-RcDof(44-101) was constructed correctly. The expressed recombinant protein,with a relative molecular mass of about 43 000,existed in supernatant in a soluble form. The purified recombinant protein reached a high purity,of which the concentration was 10 mg/mL. Conclusion Soluble RcDof with high purity was obtained,which provided an experimental basis for analyzing the crystal structure of RcDof protein and exploring the molecular mechanism of the protein in castor.

    2022 04 v.35 [Abstract][OnlineView][Download 331K]

  • Genetic characteristics of epidemic strain of varicella-zoster virus in Changchun City,Jilin Province,China in winter of 2020—2021

    FU Xing-ye;LI Qing;LV Pin;JIANG Xue-ou;MENG Di;ZHANG Fu-kun;Changchun Keygene Biological Products Co.,Ltd.;

    Objective To analyze the genetic characteristics of epidemic strain of varicella-zoster virus(VZV)in Changchun City,Jilin Province,China in the winter of 2020 — 2021. Methods Vesicle fluid samples were collected from 48patients with varicella or zoster in Changchun in 2020 — 2021,from which the VZV DNA was extracted and used as a template for amplification of specific ORF22,ORF38,ORF54 and ORF62 fragments. The PCR products were sequenced,and the results were compared with those of single nucleotide polymorphism(SNP)sites of ORF22 of representative strains of various genotypes. The restriction sites of ORF38-Pst Ⅰ,ORF54-Bgl Ⅰ and ORF62-Sma Ⅰ were searched from the sequencing results by using SnapGene software,and compared with those of Oka strain(PstⅠ-BglⅠ+SmaⅠ+). Results All the bases of ORF22-SNP sites of 48 samples were consistent with those of the pOka strain,which were of genotype Clade2. Forty-seven of the samples were PstⅠ-BglⅠ+SmaⅠ+,while the remained one was PstⅠ-BglⅠ+SmaⅠ+,all of which were wild virus. Conclusion The epidemic strain of VZV in Changchun in the winter of 2020 — 2021 was highly conserved wild strain of genotype Clade2.

    2022 04 v.35 [Abstract][OnlineView][Download 389K]

  • Comparison of two methods for biological activity assay of Bevacizumab

    DENG Chun-ping;CHEN Hang;WANG Ying-hua;CAO Di;YU Jin-quan;LIU Cui-hua;Bio-Thera Solutions,Ltd;

    Objective To compare the performances of two methods for biological activity assay of Bevacizumab.Methods Two methods for biological activity assay of Bevacizumab,human umbilical vein endothelial cell(HUVEC)proliferation inhibition assay and luciferase reporter gene assay(RGA),were validated to compare their detection performances. Fifty-three batches of Bevacizumab(including original patented drug and its biosimilar)were determined simultaneously by the two methods,and the stability indicating properties of the two methods was compared. Results RGA showed a shorter experiment period,wider detection range and higher precision,of which the R2 value of curve,signal/noise ratio and linear range were superior to those of HUVEC proliferation inhibition assay. No significant difference was observed between the biological activities of Bevacizumab determined by RGA and HUVEC proliferation inhibition assay(P > 0. 05). RGA was similar to or slightly better than HUVEC proliferation inhibition assay in reflecting the biological activity change of Bevacizumab except strong photostress. Conclusion The performance of RGA was superior to that of HUVEC proliferation inhibition assay. The method effectively reflected the biological activity change of Bevacizumab,thus may be used as an alternative to classic HUVEC proliferation inhibition assay for product release and stability test of the McAb.

    2022 04 v.35 [Abstract][OnlineView][Download 429K]

  • Preparation of national reference for serotype 19A pneumococcal polysaccharide

    CHEN Qiong;WANG Shan-shan;LV Xue-yan ;ZHOU Xue-yi ;HAN Fei ;ZHANG Ting ;YE Qiang;National Institutes for Food and Drug Control;

    Objective To prepare the national reference for serotype 19A pneumococcal polysaccharide. Methods According to the requirements in Chinese Pharmacopoeia(VolumeⅢ,2020 edition),the quality of serotype 19A pneumococcal polysaccharide candidate reference,including the contents of foreign matters such as protein and nucleic acid,the groups such as aminohexose and methyl pentose,as well as molecular size distribution,were analyzed. The pneumococcal polysaccharide candidate reference was collaboratively by rate nephelometry in three laboratories using the reference from manufacturer as standard. Fifty containers of candidate were randomly selected and investigated for the uniformity. The candidate was subjected to accelerant thermal stability test after storage at 37,25 or 2 ～ 8 ℃ for a certain time durations respectively by rate nephelometry by the National Institutes for Food and Drug Control(NIFDC).The effect of repeat freezing-thawing for 3 times on serotype 19A pneumococcal polysaccharide content of the candidate reference was observed. Results All the quality indexes of candidate reference met the requirements in Chinese Pharmacopoeia(Volume Ⅲ,2020 edition). 1H NMR confirmed the candidate as serotype 19A pneumococcal polysaccharide. The collaborative calibration results showed that the mean polysaccharide content determined by three laboratories was 565 μg/mL,of which the RSDs were less than 5%. The RSD of polysaccharide content of 50 containers of candidate was 3. 16%. The candidate showed high stability after storage at 2 ～ 8 ℃ for 8 weeks,at 25 ℃ for 14 d,at37 ℃ for 3 d,and after repeat freezing-thawing for 3 times. Conclusion The national reference for serotype 19A pneumococcal polysaccharide was successfully prepared,of which the polysaccharide content was determined by collaborative calibration. The reference may be used for determination of polysaccharide content in pneumococcal polysaccha-ride vaccine.

    2022 04 v.35 [Abstract][OnlineView][Download 180K]

  • Antibody levels against mumps in healthy population in Changning District,Shanghai

    PANG Hong;SHI Wei;LIU Xiao-xiang;ZHU Xiao-hua;ZHENG Min;Center for Disease Control and Prevention of Changning District,Shanghai City;

    Objective To analyze the IgG antibody level against mumps in populations of various age groups in Changning District,Shanghai City in 2017 and observe the changes of mumps antibody level after inclusion of live attenuated measles,mumps and rubella combined vaccine(MMR) into the EPI. Methods A total of 2 662 serum samples were collected from the populations at various ages in Changning District,Shanghai City in 2017,and determined for IgG antibody levels against mumps by ELISA,of which the results were compared with those of investigation on serum IgG level against mumps in healthy population in 2009. Results The overall age-adjusted positive rate of IgG antibody against mumps was 90. 09% in 2017. The IgG antibody positive rate(99. 13%)and GMC(632. 06 U/mL)were highest in the children at ages of 5 ～ 9 years then decreased gradually with the increasing age. Compared with those in 2009,both the positive rates and GMCs in the age groups of 1 ～ 4,5 ～ 9 and 10 ～ 14 years in 2017 increased significantly(P < 0. 05). Conclusion The IgG antibody positive rate against mumps was high in Changning District,Shanghai City in 2017,and both the positive rate and GMC of antibody of vaccinated population increased significantly. Two-dose schedule of MMR vaccine maintained high positive rate and GMC of antibody against mumps.

    2022 04 v.35 [Abstract][OnlineView][Download 179K]

  • Development and application of a double antibody sandwich ELISA for neuraminidase content of influenza vaccine

    DENG Tao;LV Chuan-shuo ;LIU Jing;MA Ning;ZHOU Rong;ZHANG Guo-mei;LE Yang;ZHANG Jia-you;YANG Xiao-ming;Second Laboratory of Viral Vaccine Research,Wuhan Institute of Biological Products Co.,Ltd;

    Objective To develop and verify a double antibody sandwich ELISA method for quantitative determination of neuraminidase(NA)content in influenza vaccine. Methods The conserved sequence of NA was synthesized by peptide synthesis,with which Japanese white rabbits and guinea pigs were immunized s. c. in several sites for 5 times. Serum samples were collected 2 weeks after the last immunization and purified by protein G/A chromatography to prepare a universal antibody. A double antibody sandwich ELISA method was developed using the universal antibody from guinea pigs as coating antibody and that from rabbits,labeled with horseradish peroxidase(HRP),as enzyme labeled antibody.The working concentration(32,16,8,4,2 and 1 μg/mL)of coating antibody and dilution(1 ∶ 50 ～ 1 ∶ 6 400)of enzyme labeled antibody were optimized. The developed method was verified for linear range,accuracy,reproducibility,intermediate precision and durability. The NA contents in monovalent bulks of influenza virus split vaccine of types H1N1,H3N2,BV and BY were determined by the developed method, and the result was compared with that by fluorescent substrate assay. Results The titers of universal antibodies from rabbit and guinea pig were 128 000 and64 000,while the purities were 95% and 96%,and the protein concentrations were 2 339 and 1 780 μg/mL,respectively. The optimal working concentration of coating antibody was 16 μg/mL,while the optimal dilution of enzyme labeled antibody was 1 ∶ 400. The concentration of reference of NA(type H3N2)at a range of 20 ～ 640 ng/mL showed a good linear relationship to the A450value,and all the R2 values were more than 0. 99. The average recoveries of NA(type H3N2)reference at concentrations of 500,200 and 50 ng/mL were 102. 15%,100. 89% and 100. 70% respectively. The CVs of six repeat test results were less than 10%,while those of three repeat tests by either of two workers were less than 15%. The recovery rates of samples when the antigen and enzyme labeled antibody were incubated for various time durations were 85. 41% ～ 103. 81%. The NA contents of monovalent bulks of types H1N1,H3N2,BV and BY were 8. 06,13. 20,6. 93 and 6. 18 μg/mL respectively,which was positively related to the NA activities determined by fluorescent substrate assay(29 833,36 800,28 907 and 25 871 U/L)(R2 = 0. 979 2). Conclusion A double antibody sandwich ELISA method for quantitative determination of NA content was successfully developed,which showed good accuracy,reproducibility,precision and durability and might be used for control tests on influenza vaccine and quality control of NA content in the production process.

    2022 04 v.35 [Abstract][OnlineView][Download 311K]

  • Comparison and evaluation of various methods for determination of hepatitis B surface antibody

    CHEN Shi-yi;ZHAO Yu-ping;ZHAO Ting;LI Jing;SHI Hong-yuan;LI Ying;YANG Jing-si;Institute of Medical Biology,Chinese Academy of Medical Sciences & Peking Union Medical College;

    Objective To determine hepatiits B surface antibody(HBsAb)by electrochemiluminescence immunoassay(ECLIA)and enzyme-linked immunosorbent assay(ELISA)and compare the application effectiveness. Methods A total of 955 serum samples were collected from the subjects immunized with hepatitis B(HepB)vaccine on time according to the requirements in National Immunization Program in Liuzhou City,Guangxi Zhuang Autonomous Region,China,and determined for HBsAb by ECLIA and ELISA respectively. The accuracy,correctness and reportable range(linearity evaluation) of the two methods were verified before determination,and the differences between the results were compared. Results The verification results of precision,correctness and reportable range of the two methods were within the acceptable limits. The qualitative determination results of serum samples by the two methods were in a strong agreement(with a Kappa coefficient of 0. 787 8),while the quantitative determination results were in poor agreement(P < 0. 001). Conclusion ECLIA and ELISA showed no significant difference in the qualitative determination while showed significant difference in quantitative determination of HBsAb. The low sensitivity of ELISA for low-level antibody may result in false-negative result,while ECLIA is more sensitive,resulting in a lower false-negative rate and a more reliable result.

    2022 04 v.35 [Abstract][OnlineView][Download 240K]

  • Development and verification of a reverse phase high performance liquid chromatography for determination of purity of glucagon-like peptide-1 receptor agonist fusion protein

    GAO Xue-feng;WANG Wan-ru;ZHENG Wei-feng;YANG Jun;ZHANG Yan-li;MA Ya-ru;CHEN Yong;Department 4 of Research,Lanzhou Institute of Biological Products Co.,Ltd.,Center for Gansu Provincial Vaccine Engineering Research;

    Objective To develop and verify a reverse phase high performance liquid chromatography(RP-HPLC)for determination of purity of glucagon-like peptide-1(GLP-1)receptor agonist fusion protein. Methods Agilent ZORBAX 300 ExtendC18(4. 6 mm × 150 mm)analytic column and ZORBAX 300Extend-C18(4. 6 mm × 12. 5 mm)protective column were adopted. A 0. 1% trifluoroacetic acid aqueous solution was used as mobile phase A,while a 0. 1% trifluoroacetic acid acetonitrile solution as mobile phase B. The sample load was 100 μL,while the flow rate was 0. 50 mL/min. The chromatographic column was equilibrated with phase A until the baseline was stable,onto which the samples were loaded and eluted according to the following procedure:0 ～ 5 min:0 ～ 50% B;5 ～ 15 min: 50% ～ 60% B;15 ～ 20 min:60% ～100% B;20 ～ 30 min: 100% B;8 min:100% A. The detection wavelength was 280 nm,while the column temperature was 40 ℃. The developed method was verified for system suitability,specificity,sensitivity,reproducibility,intermediate precisio n and duration,by which the purities of six bathes of GLP-1 receptor agonist fusion protein were determined.Results The number of theoretical plates was 26 891,while the symmetry factor was 0. 64. No interference peaks of blank control(diluent),negative control(antithrombin-Ⅲ,AT-Ⅲ)or ciliary neurotrophic factor(CNTF)were observed on the chromatogram,and the resolution of GLP-1 receptor agonist fusion protein with the negative control was more than 1. 5.The sensitivity of the developed method was 4. 8 μg/mL. The relative standard deviation(RSD) of area percentage in verification for reproducibility was 0. 35%,while that of retention time was 0. 07%. However,in verification for intermediate precision,the RSD of area percentage was 0. 75%,while that of the retention time was 0. 07%. The RSD of area percentage determined at various system temperatures was 3. 94%,while that of the retention time was 0. 10%. All the purities of six batches of GLP-1 receptor agonist fusion protein were not less than 95. 0%. Conclusion A RP-HPLC method for determination of purity of GLP-1 receptor agonist fusion protein was successfully developed,which showed good system suitability,sensitivity,precision and durability,and might be used for quality control of the protein.

    2022 04 v.35 [Abstract][OnlineView][Download 211K]

  • Development and verification of capillary isoelectric focusing method for determination of isoelectric point of recombinant Candida uricase

    LIU Jia-xin;ZHANG Ting-ting;YANG Li-hua;TANG Qi-feng;JIN Zheng;Shenyang Sunshine Pharmaceuticals CO.,Ltd.;

    Objective To develop and verify a capillary isoelectrie focusing(cIEF)method for determination of isoelectric point(pI)of recombinant Candida uricase. Methods The cIEF method was developed by optimization of key parameters such as the proportion of stabilizer and focusing time,and verified for specificity,precision,accuracy and durability.Results The optimal ratio of arginine(ARG)to iminodiacetic acid(IDA)in stabilizer was 60 μL ∶ 6 μL,while the optimal focusing time was 15 min. No interference peak of blank matrix was observed in the position of main peak of recombinant C. uricase. The relative standard deviations(RSDs) of test results of reproducibility,intermediate precision and interbatch precision were 0. 81% ～ 0. 87%,0. 91% ～ 1. 5% and 0% ～ 0. 79% respectively. The changes of lot numbers of 3-10 amphoteric electrolyte carriers,cIEF gel,capillary column showed no interference to the test results. Conclusion The developed cIEF method showed good specificity, precision, accuracy and durability, which was suitable for determination of pI of recombinant C. uricase.

    2022 04 v.35 [Abstract][OnlineView][Download 279K]

  • Development and verification of a method for rapid detection of activity of BCG vaccine

    HUANG Yue;WU Yu-jie;ZHU Ke-jun;JIANG Qiu-hong;ZHANG Jian;LU Rong-jiang;WEI Fen;TAO Li-feng;PU Jiang;Anhui Zhifei Longcom Biopharmaceutical Co.,Ltd;

    Objective To develop and verify a method for rapid detection of Bacille Calmette-Guérin (BCG) vaccine.Methods The standard for ATP was diluted to concentrations of 6.25×10~(-10)～2×10~(-8)mol/L and determined by bioluminescence assay to obtain the relative light unit (RLU),based on which a standard curve of ATP concentration against RLU was plotted.The ATP in nine batches of low dose (0.25,0.375 and 0.5 mg/m L)and six batches of high dose (60 mg/mL) BCG were extracted by centrifugation,background and ATPase methods separately,of which the contents were determined by bioluminescence assay.The correlation between the ATP content and the corresponding viable count was evaluated,and the reproducibility and intermediate precision of the developed method were verified.Results The ATP at concentrations of 6.25×10~(-10)～2×10~(-8)mol/L showed good linear relationship to the RLU,of which the regression equation was y=1 680.4 x-574.46 (R~2=0.998 7).The ATP contents in high and low dos BCG extracted by centrifugation showed no significant relationship (P>0.05),while those in high dose BCG extracted by background and ATPase methods showed significant relationship (P<0.05),to the corresponding viable counts.However,the ATP content in low dose BCG extracted by the latter two methods showed no significant relationship to the corresponding viable countes(P>0.05).The RSDs in verification for reproducibility and intermediate precision were less than 8%.Conclusion The method for rapid determination of activity of high dose BCG was successfully developed,which showed good reproducibility and intermediate precision.

    2022 04 v.35 [Abstract][OnlineView][Download 200K]

  • Development and application of ion chromatography for determination of lactose content

    ZHUO Xiao-qin;CHU Bing-jie;JIANG Shan;QIANG Peng-xia;FU Yuan-xin;FENG Xiao;LUO Li-hong;FENG De-jie;The Quality Control Department,Lanzhou Institute of Biological Products Co.,Ltd.,Center for Gansu Provincial Vaccine Engineering Research;

    Objective To develop an ion chromatography method for determination of lactose content in raw and subsidiary materials and polysaccharide vaccines. Methods The lactose as raw or subsidiary material was dissolved with purified water and filtered for loading. The polysaccharide vaccines were dissolved with purified water,centrifuged and filtered,and the filtrate was collected for loading. All samples were separated by CarboPac PA20 column(3 mm × 150 mm).Calibration curve was established according to external standard method. The developed method was verified for systemic suitability,specificity,accuracy and precision,determined for detection limit,quantification limit and linear range,by which the content of lactose in raw and subsidiary materials and polysaccharide vaccines were determined. Results The developed method showed good systemic suitability and specificity. The recoveries in accuracy test were 93. 9% ～ 100. 2%,while both the relative standard deviations(RSDs)in reproducibility and intermediate precision tests were less than 1. 0%.The detection limit and quantification limit were 0. 001 and 0. 05 mg/L respectively. The lactose contents at a range of2 ～ 10 mg/L showed good linear relationship to the peak area,while the correlation coefficients(r2) of standard curve were more than 0. 999. The method also showed good durability. The RSDs of determination results in the practical application were 0. 2% ～ 0. 8%. Conclusion The method was simple,reliable,accurate,precise and sensitive,which might be used as a routine method for the determination of lactose content in polysaccharide vaccines.

    2022 04 v.35 [Abstract][OnlineView][Download 229K]

  • Scale-up of procedure for interleukin-12 expression by using WAVE25 bioreactor

    QIAO Lei;ZHENG Bin-bin;YANG Pan-pan;CHEN Wen-cheng;YAN Le-le;WANG Jian-gang;WU Ming-yuan;Kanglitai Biomedical(Qingdao)Co.LTD;

    Objective To scale up the procedure for interleukin-12(IL-12) expression and provide a reference for subsequent large-scale industrial production. Methods The procedure for IL-12 expression was scaled-up by using BIOSTAT B 10 L and GE WAVE25 50 L bioreactors respectively. Based on the growth and metabolic parameters such as cell living rate,density,pH,glucose,lactic acid,ammonium ions and glutamine,the intermediate control in course of scale-up was monitored. Primary control tests were performed on the expressed product based on physical and chemical testing indexes such as SDS-PAGE,yield of product,isoelectric point and peptide map. Results The change curves of intermediate process control parameters and the quality indexes of expressed products before and after scale-up were in agreement,all of which were within the range of quality standard. Conclusion The procedure may be steadily scaled-up from BIOSTAT B 10 L to GE WAVE25 50 L bioreactors,which provides a reference for subsequent large-scale industrial production.

    2022 04 v.35 [Abstract][OnlineView][Download 293K]

  • Progress in research on detection methods for SARS-CoV-2

    HUANG Hui-li;WU Jia-jing;LIANG Chun-nan;National Institutes for Food and Drug Control;

    The Coronavirus Disease 2019(COVID-19)outbreak has spread globally in 2020,which shows strong communication ability,causes a wide range of infection,and is difficult to prevent and control. The International Committee on Taxonomy of Viruses(ICTV) officially named the novel coronavirus causing this outbreak as severe acute respiratory syndrome coronavirus 2(SARS-CoV-2),which is mainly transmitted by respiratory droplet and close contact of humans. There is an asymptomatic incubation period of several days after infection in general,followed by the symptoms such as weakness,dry cough and fever. However,partial asymptomatic carriers enhanced the difficulty in diagnosis and are un-favorable to the control of outbreak. Rapid and accurate diagnosis is critical to the prevention and control of COVID-19. This paper reviews the progress in research on the diagnostic method for COVID-19,including the tests for nucleic acid,antibody and antigen.

    2022 04 v.35 [Abstract][OnlineView][Download 204K]

  • Progress in research on monoclonal antibodies against rabies virus

    CAI Mei-na;XU Si-hong;Peking Union Medical Collage;

    Rabies is a severe infectious disease caused by rabies virus(RV),of which the mortality is nearly 100%once the clinical symptoms appear. In general,anti-rabies immunoglobulin(RIG) is used for post-exposure prophylaxis(PEP) of rabies. However,the RIG which is mainly derived from human or horse immune sera may have some disadvantages such as high cost,supply shortage,safety and immunogenicity. Since protective neutralizing antibodies are mainly specific to the glycoprotein(G) of RV,WHO recommends that the monoclonal antibodies(MAbs) with at least two non-over-lapping epitopes may be used instead of RIG. This review focused on the progress in research on structure characteristics and neutralizing epitopes of RV glycoprotein as well as the candidate MAbs with broad neu-tralizing activity that may be used instead of RIG,which will provide a reference for the study on combination of MAbs in cocktail therapy.

    2022 04 v.35 [Abstract][OnlineView][Download 258K]

  • Strategies for optimization of recombinant expression of antimicrobial peptides

    ZONG Xi-cui;ZHANG Yi ;FANG Peng-hua ;CHEN Yu-qing;Medical Department Experimental Training Center,Hanlin College,Nanjing University of Chinese Medicine;

    Antimicrobial peptides are polypeptides with low relative molecular mass,high thermal stability,unique action mechanism and wide bactericidal range,which are encoded by specified genes,and play important roles in stimulating the healing of wound as well as hormone and immune regulations. Gene engineering expression technology is the most economical,scientific and effective way to obtain a large number of antimicrobial peptides. However,antimicrobial peptides are restricted in large-scale production due to their low expression,unstable expressed products and toxic interaction with the host. In this paper,the strategies for optimization of recombinant expression of antimicrobial peptides were briefly reviewed in terms of fusion label selection,preferred codon selection,optimal strain selection,gene coexpression,fusion protein purification method and gene expression design,in order to provide a reference for the application and large-scale production of antimicrobial peptides.

    2022 04 v.35 [Abstract][OnlineView][Download 310K]

  • Progress in research on adeno-associated virus vectors

    CHEN Ping;ZHANG Li-juan;LI Na;Jiaming Biotechnology Company Limited;

    Adeno-associated virus(AVV)is one of the widely used viral vectors. As a tool for gene therapy,AAV vector has high biological safety,high stability,low immunogenicity,wide host range and high specificity,which may be used for long-term expression of foreign genes. Therefore,it is favored by researchers in gene therapy. This article reviews the discovery,biological characteristics,design of recombinant AAV(r AAV) vector,application of AAV vector in clinical gene therapy and possible adverse reactions so as to provide a reference for further research.

    2022 04 v.35 [Abstract][OnlineView][Download 277K]

  • Progress in research on role of human endogenous retroviruses in pathogenesis of multiple sclerosis

    YU Kun;WU Shi-ping;GONG Pan-pan;WANG Man-xia;Department of Neurology,The Second Hospital of Lanzhou University;

    Multiple sclerosis(MS)is an autoimmune demyelinating disease of the central nervous system,which is characterized by infiltration of immune cells and subsequent neuroinflammatory response,demyelination reaction and neuronal injury. However,the pathogenesis of MS is still unclear. As a large category of RNA virus,human endogenous retroviruses(HERV)may encode reverse transcriptase and integrate the viral genome into the host genome. HERV and its expressed product are closely related to the pathogenesis of MS. This article reviews the relationship of HERV to MS,the role of HERV in pathogenesis of MS as well as the treatment of MS.

    2022 04 v.35 [Abstract][OnlineView][Download 167K]
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@2012 Editorial Department of "Chinese Journal of Biologicals"