2025年02期目次

  • Construction and immune effect evaluation of recombinant M13 phage vaccine targeting outer membrane protein P6 of nontypeable Haemophilus influenzae

    WANG Cai;CHANG Yueli;HU Nan;LI Weifeng;ZHAO Zihong;AN Jing;ZHANG Yutuo;Institute of Pathogenic Biology and Immunology, Hebei North University;

    Objective To construct a recombinant M13 phage vaccine targeting the outer membrane protein P6 of nontypeable Haemophilus influenzae(NTHi) and evaluate its immunogenicity in order to provide new ideas for further development of NTHi vaccines. Methods The NTHi P6 gene was fused with the vector pMECS Phagemid by gene recombination technique.After packaging and purification, the obtained recombinant P6-M13 phage was prepared into recombinant P6-M13 phage vaccine. The expression of P6-M13 PⅢ fusion protein in the vaccine was detected by Western blot, the vaccine titer was determined by double-layer agar plate method, and the recombinant P6-M13 phage morphology was observed under transmission electron microscope. Ninety 5-week-old BALB/c female mice were randomly divided into PBS group, M13 phage group and recombinant P6-M13 phage vaccine group, 30 for each, and intraperitoneally injected with PBS(500 μL/mouse), M13 phage[1 × 10~(12)pfu/(500 μL·mouse)]and recombinant P6-M13 phage vaccine[1 × 10~(12)pfu/(500 μL·mouse)]on the 0, 14th and28th day, separately. Two weeks after the last immunization, the levels of specific IgG in serum and IFNγ, IL-2, IL-4, IL-5and IL-17A in spleen lymphocyte culture supernatant were detected by ELISA, and the proliferation of spleen lymphocytes was analyzed by CCK-8. Three weeks after the last immunization, the mice were challenged with 1. 5 × 108cfu/mL NTHi through the nasal cavity. After one week of challenge, the pathological changes of nasal mucosa and lung tissue were observed by HE staining. Four weeks after the last immunization, the heart, liver, spleen, lung and kidney of mice were weighed, the organ coefficients were calculated, and histopathological sections were prepared for pathological observation.Results The recombinant P6-M13 phage could correctly express P6-M13 PⅢ fusion protein with a titer of 5. 5 × 10~(14) pfu/mL,and the recombinant P6-M13 phage with regular morphology was observed under microscope. Compared with M13 phage group and PBS group, the level of serum specific antibody IgG in mice of recombinant P6-M13 phage vaccine group was significantly higher(F = 71. 489, P < 0. 05); the levels of IFNγ, IL-2, IL-4 and IL-5 secreted by mouse spleen cells decreased significantly(F = 8. 315, 16. 986, 39. 204 and 6. 291, respectively, each P < 0. 05), while there was no significant difference in IL-17 level among the three groups(F = 0. 863, P > 0. 05); the spleen cell stimulation index increased significantly(F =22. 952, P < 0. 05). After challenge, the nasal mucosa and lung tissue structures of mice in PBS group and M13 phage group were seriously damaged, and inflammatory cells increased, while in the recombinant P6-M13 phage vaccine group, the structures of nasal mucosa and lung tissue were normal with few inflammatory cells. There was no significant difference in the organ coefficients of heart, liver, spleen, lung and kidney of mice in each group(F = 1. 012, 1. 642, 0. 300, 2. 079, and 0. 405, respectively,each P > 0. 05), and no pathological changes were found in the general color morphology and pathological sections of the main organs. Conclusion The constructed recombinant P6-M13 phage vaccine targeting NTHi outer membrane protein P6 can induce effective humoral and cellular immunity in mice with certain immune protection ability and good safety.

    2025 02 v.38 [Abstract][OnlineView][Download 1279K]

  • Isolation, purification and identification of a novel varicella-zoster virus Oka-7S strain

    SUN Yueqiu;LIU Hongtao;JIANG Xueou;LI Meng;WEI Bo;TAO Jinyu;WANG Menghan;ZHOU Hongda;ZHANG Fukun;Research Department of Changchun Keygen Biological Products Co.Ltd.;

    Objective To isolate, purify and identify the genetically modified varicella-zoster virus(VZV) Oka-7S strain, so as to lay a foundation for the establishment of a seed bank for the novel live attenuated varicella vaccine Oka-7S strain.Methods The Oka-7S strain was purified twice on 2BS human diploid cells using plaque purification method, and multiple plaque strains were selected for amplification and passage. Typical CPE strains were selected according to the cytopathic effect(CPE) and virus titer. The virus titer, gene sequence and protein of the isolated virus strains were detected, and the quality testing was performed in accordance with the methods of Chinese Pharmacopoeia(Vol Ⅲ, 2020 edition).Results Two candidate strains of Oka-7S were successfully screened, and the identification test showed that the candidate strains were VZV, of which the virus titers remained above 6. 0 lgPFU/mL after serial passages. Western blot revealed that the main antigen gE protein was expressed normally, while no ORF7 protein was detected. Meanwhile, during the passage of Oka-7S strain, the main gene sequences ORF62 and ORF68 encoding gE did not mutate. The results of bacterial testing, mycoplasma testing, and exogenous viral factor testing were all negative.Conclusion Two new VZV Oka-7S strains have been successfully screened, which can be used as the candidate vaccine strains for novel varicella vaccines.

    2025 02 v.38 [Abstract][OnlineView][Download 904K]

  • Genetic detoxification of pertussis toxin S1 subunit

    LIU Hongbo;ZHU Dewu;CAI Mengyao;MA Lin;WANG Fen;HU Yuan;CHEN Wen;FU Lie;DUAN Kai;Wuhan Institute of Biological Products Co., Ltd., National Engineering Technology Research Center of Combined Vaccines,Vaccine Technology Innovation Center of Hubei Province, State Key Laboratory of Novel Vaccines for Emerging Infectious Diseases;

    Objective To edit the gene of pertussis toxin(PT)-S1 subunit in order to complete the genetic detoxification of Bordetella pertussis CS strain.Methods Electroporation competent cells of Bordetella pertussis CS strain were prepared and electro-transfected with the linear recombinant DNA fragments carrying Kana resistance genes, which was then exchanged with the PT-S1 subunit through recombination of the homologous arms on both sides. The recombinant strains were screened with Kana antibiotics, and the recombinant sequence was determined by gene sequencing. The PT protein was obtained from the culture supernatant of the recombinant strains for ELISA, Western blot and in vitro toxicity analysis.Results S1-R9K/E129G double mutant strain FE3 and S1-R9K single mutant strain FE16 were obtained by genome and mRNA sequencing.The growth rate of FE3 and FE16 decreased slightly, but the maximum bacterial concentration increased. The content of PT protein in shake flask culture supernatant of strain FE3 and FE16 was 796. 8 and 185. 9 ng/mL, respectively, and the PT proteins could be recognized by S1 subunit monoclonal antibody 1B7. The in vitro toxicity of PT proteins from strain FE3 and FE16 decreased to 0. 001 6% and 0. 008 1%, respectively, compared with that of PT standard.Conclusion Gene editing can be performed on Bordetella pertussis CS strain using electroporation of linear DNA fragments and Kana antibiotic screening, and result in genetically detoxified pertussis strains with PT protein of significantly reduced toxicity, which lays a foundation for further research on pertussis vaccines.

    2025 02 v.38 [Abstract][OnlineView][Download 1058K]

  • Effect of bovine lactoferrin on proliferation and apoptosis of lung cancer A549 cells and its mechanism

    SU Jinxian;SHU Xingfu;CHEN Yao;ZHAO Yu;ZHANG Bowen;ZHANG Haixia;Key Bio-engineering and Technology Laboratory, Biomedical Research Center of the Northwest Minzu University;

    Objective To investigate the effect of bovine lactoferrin(bLF) on the proliferation and apoptosis of lung cancer A549 cells and its possible mechanism, so as to provide an experimental basis for the study of anti-lung cancer effect of bLF.Methods A549 cells were treated with different concentrations of bLF. The effect of bLF on cell morphology was observed under microscope, the effect on cell activity was detected by CCK-8, the effects on cell proliferation and migration were verified by clone formation and scratch assay, and the expression of apoptosis-related genes and proteins was determined by qRTPCR and Western blot.Results The bLF had a significant inhibitory effect on the growth of A549 cells in a concentrationand time-dependent manner. Compared with the 0 mg/mL bLF group, the colony formation ability of A549 cells treated with0. 5 and 1. 0 mg/mL bLF significantly decreased(t = 6. 016 and 30. 24, P = 0. 003 8 and 0. 000, respectively), and the in vitro migration ability was gradually weakened. In addition, bLF induced the activation of apoptosis signaling pathway in A549cells. Compared with the control group, the expression levels of pro-apoptotic proteins Bax(t = 6. 454 and 11. 11, P = 0. 023 2and 0. 008, respectively) and P53(t = 6. 962 and 49. 46, P = 0. 002 2 and 0. 000 4, respectively) significantly increased and the expression level of anti-apoptotic protein Bcl-2(t = 4. 333 and 10. 44, P = 0. 049 4 and 0. 009 1, respectively) significantly decreased in the 0. 5 and 1. 0 mg/mL bLF groups. The bLF at a high concentration of 1. 0 mg/mL bLF group could cleave pro-caspase-3(t = 5. 759, P = 0. 028 9) signaling and activate caspase-3.Conclusion This study showed that bLF can significantlyinhibittheproliferationofA549lungcancercellsandpromotetheapoptosis,thusexertingananti-cancereffect.

    2025 02 v.38 [Abstract][OnlineView][Download 1030K]

  • Epidemiological characteristics of hand-foot-mouth disease in China: A retrospective analysis

    MA Zhijing;WU Jinjuan;WU Hailan;LIU Ning;JIN Yuqin;LEI Zehua;LIANG Yu;LI Qiming;National Vaccine and Serum Institute;

    Objective To understand the epidemiological characteristics of hand-foot-mouth disease(HFMD) in China, and provide evidence for the prevention and control of HFMD and vaccine clinical research design.Methods Using PubMed,China National Knowledge Infrastructure(CNKI) and Wanfang databases as retrieval sources, the Chinese and English articles on epidemiological characteristics of HFMD in China published from 2012 to 2021 were systematically retrieved, and relevant information was extracted. Chi-square test was used for the statistical analysis of the enterovirus detection rate, type composition, the distribution of gender, age, year, season, region and symptoms.Results A total of 327 articles were included in this study, of which 295 were included in the overall detection rate of HFMD enterovirus. The total number of people involved was 4 786 389, the number of enterovirus nucleic acid positive cases was 709 086, and the laboratory-diagnosed cases accounted for about 14. 81% of the total number. The four types with the highest detection rates were Coxsackievirus A6(CA6), enterovirus 71(EV71), CA16 and CA10. A total of 327 articles with 61 serotypes were included in the type proportion, and the four types more than 1% were EV71, CA16, CA6 and CA10, and other types such as CA4, CA2 and CA5accounted for a lower proportion. Among the laboratory-diagnosed cases, there were more men than women, with a sex ratio of about 1. 72∶1; children under 5 years of age were at high risk of HFMD, with 1 year of age being the most and 0 year of age being the least; the composition ratio of enteroviruses varied with the year and season, the proportion of EV71 showed an overall downward trend from 2008 to 2019, and the peak season for HFMD was from April to June. The distribution of HFMD exhibited geographical differences, and the top 5 provinces were: Guangdong, Jiangsu, Hebei, Hunan and Zhejiang; HFMD was mainly common type/mild type, and the proportion of EV71 in severe cases and deaths was significantly higher than that of other types.Conclusion The type composition of HFMD enteroviruses in China was mainly EV71, CA16, CA6 and CA10,while other types such as CA4, CA2 and CA5 accounted for a relatively low proportion. Type distribution varied in sex, age,year, season, region and symptoms.

    2025 02 v.38 [Abstract][OnlineView][Download 1062K]

  • Analysis of vaccine-associated immune thrombocytopenia reports based on the US Vaccine Adverse Event Reporting System

    WANG Junfang;CHENG Juan;First Clinical Medical College of Lanzhou University;

    Objective To investigate the clinical characteristics of vaccine-associated immune thrombocytopenia(VA-ITP)based on the US Vaccine Adverse Event Reporting System(VAERS), and to provide a reference for vaccine safety monitoring, vaccination prevention, and clinical diagnosis and treatment.Methods Data from VAERS on all reported cases of immune thrombocytopenia(ITP) after vaccination since the establishment of the database in 1990 to the data lock point of 7th November 2023 were analyzed retrospectively for information on such as sex, age, vaccine type, vaccine dose, and occurrence time of ITP.Results Among the 877 cases of VA-ITP, there were 868 cases of effective statistical vaccine types, involving 43 vaccine types. The top 10 vaccine types were COVID-19 vaccine(434 cases, 50%), MMR vaccine(82 cases, 9. 4%),HEPA vaccine(48 cases, 5. 5%), FLU3 vaccine(41 cases, 4. 7%), FLU4 vaccine(32 cases, 3. 7%), HPV4 vaccine(30 cases,3. 5%), DTAP vaccine(29 cases, 3. 3%), MMRV vaccine(32 cases, 3. 7%), VARZOS vaccine(17 cases, 2. 0%) and HPV9vaccine(13 cases, 1. 5%). Among the 877 reported cases, 843 cases with definite sex were counted, including 374 males and469 females, with a male-to-female ratio of 1∶1. 25. The 377 cases of patients were under 18 years of age, accounting for about 44. 7% of the total, and there was significant difference in the composition ratio of sexes among patients with VA-ITP at different ages(χ~2= 10. 029, P < 0. 05). The incidence of ITP within 8 days after vaccination, with a total of 336 cases, was39.9%, and there was no significant difference in the occurrence time composition ratio of ITP between different sexes(χ~2=3. 296, P > 0. 05). The 54. 9% of ITP occurred after the first dose of vaccine, and there was no significant difference in the composition ratio of different vaccination doses between male and female recipients(χ~2= 5. 323, P > 0. 05), but there was significant difference in the composition ratio of vaccination doses among different age groups(χ~2= 30. 591, P < 0. 01).Conclusion By analyzing the summary data from VAERS database, the adverse reaction of VA-ITP was fully recognized.After clinical vaccination, attention should be focused on the platelet status of minors and the elderly, as well as those who were vaccinated within 8 days, and the continuous vaccine safety monitoring should be carried out, which is helpful to reduce the missed diagnosis of vaccine adverse events(AE) and improve the efficiency of clinical diagnosis and treatment.

    2025 02 v.38 [Abstract][OnlineView][Download 864K]

  • Establishment and verification of a TaqMan one-step real-time fluorescence quantitative PCR method for Coxsackievirus B4

    GUO Wei;CHEN Junwei;LIU Yuhan;ZHANG Ming;FENG Changzeng;MA Shaohui;Institute of Medical Biology, Chinese Academy of Medical Sciences & Peking Union Medical College, Key Laboratory of Vaccine Research and Development for Major Infectious Diseases in Yunnan Province;

    Objective To establish and verify a one-step real-time fluorescence quantitative PCR(RT-qPCR) method for the detection of Coxsackievirus B4(CVB4) with TaqMan probe, in order to provide a reliable method for rapid quantitative detection of CVB4. Methods According to the more conserved segment of VP1 sequence of CVB4, primers and probes were designed. The positive RNA standard was obtained by in vitro transcription and the standard curve was obtained. The TaqMan probe one-step RT-qPCR method for CVB4 was established and verified for the sensitivity, linear range, reproducibility and specificity. Sixteen clinical samples were detected by using the established method. Results The sensitivity of the method was 10~3 copies/μL. In the concentration range of 10~3-10~(10) copies/μL, the positive RNA standard showed a good linear y x R~2CV relationship with relative fluo rescence unit(RFU),and the line a requation was:=-3.742+48.651,= 0. 998. The of reproducibility verification was less than 2%. The method had no cross-reactivity with other serotypes of coxsackievirus B(CVB2, CVB3, CVB5, CVB6), enterovirus 71(EV71), Coxsackievirus A8, A10 and A12(CVA8, CVA10, CVA12), as well as echovirus 11(E11). Twelve of the 16 clinical samples were positive for CVB4. Conclusion The TaqMan one-step RT-qPCR method developed in this experiment has good sensitivity, specificity and reproducibility, and can be used for the detection of CVB4 clinical samples.

    2025 02 v.38 [Abstract][OnlineView][Download 907K]

  • Development and verification of a high-performance liquid chromatography method for determination of polysorbate 80 content in oil-in-water emulsions and its comparison with other methods

    JIA Lanxin;NIAN Xuanxuan;MA Ning;ZHENG Jiahao;NIE Xilin;ZHANG Zhegang;ZHANG Jiayou;Second Laboratory of Viral Vaccine Research, Wuhan Institute of Biological Products Co., Ltd.;

    Objective To develop and verify a high-performance liquid chromatography(HPLC) method for the determination of polysorbate 80 in oil-in-water emulsions, and to compare it with the modified cobalt ammonium thiocyanate colorimetric method for the determination of polysorbate 80 in oil-in-water emulsions. Methods Eclipse Plus C8 column, TSKgel G2000SW_(XL) column and ChromCore SAA column were used to detect polysorbate 80 standard or test sample to determine the optimum chromatographic column, and the elution gradient of mobile phase B(50%, 75% and 90% as the elution gradient of mobile phase B in the second stage to detect polysorbate 80 standard solution; 90% and 75% as the elution gradient of mobile phase B in the second stage to detect test sample solution) and the flow rate(0. 8, 1. 0, 1. 2 mL/min) were optimized.The specificity, linear range, repeatability, accuracy and durability of the method were verified, and the linear range, repeatability and accuracy were compared with those of the modified cobalt ammonium thiocyanate colorimetric method. Results The conditions of HPLC method were as follows: ChromCore SAA chromatographic column was adopted at a column temperature of 25 ℃, the mobile phase A was aqueous solution containing 0. 1%(v/v) acetic acid, and the mobile phase B was isopropanol containing 0. 1%(v/v) acetic acid, with an injection volume of 10 μL and gradient elution with the optimum elution gradient of 75% for mobile phase B in the second stage at a flow rate of 1. 2 mL/min. Polysorbate 80 chromatographic peak was observed at 3. 1 min in both polysorbate 80 standard solution and test solution, and other chromatographic peaks were observed at 6 min in test solution. In the concentration range of 300-1 000 μg/mL, polysorbate 80 standard solution exhibited good linear relationship with the peak area, and the regression equation was: y = 1. 44 x + 4. 91, r = 0. 999 8. The RSD of polysorbate 80 content in six test solutions was 1. 13%. The spike recovery rates of low, medium and high content samples were 96. 51%-104. 82%. The RSD of polysorbate 80 content was 2. 38% after the test solution was placed at room temperature for 0, 2, 4, 6 and 8 hours. The linear relationship of the two methods was both good. The repeatability and accuracy of HPLC method were better than those of modified cobalt ammonium thiocyanate colorimetric method. Conclusion The developed HPLC method is superior to the modified cobalt ammonium thiocyanate colorimetric method with good specificity, precision, accuracy and durability, which provides a reliable method for the determination of polysorbate 80 in biological products containing various surfactants.

    2025 02 v.38 [Abstract][OnlineView][Download 971K]

  • Evaluation of removal efficiency of human interferon α2b chromatography process for impurity proteins using liquid chromatography-high resolution mass spectrometry

    GUO Ying;LONG Zhen ;HUANG Min ;YU Lei;LI Xiang;PEI Dening;ZHOU Yong;National Institutes for Food and Drug Control;

    Objective To evaluate the removal efficiency of chromatography process for impurity proteins[IFN-related proteins and host cell proteins (HCPs)]in the purification process of human interferon α2b (h IFNα2b) by liquid chromatogra-phyhigh resolution mass spectrometry (LC-HRMS),in order to provide experimental basis for improving the production process of h IFNα2b.Methods The whole protein levels of IFN-related proteins (including deamidated IFN,oxidized IFN and acetylated IFN) and HCPs during the chromatography process of h IFNα2b were analyzed by LC-HRMS,and the h IFNα2b post-translational modification sites and HCPs were detected on the polypeptide levels.Results HCPs could be removed by hydrophobic interaction chromatography (HIC) M column,HCPs,deamidated IFN and acetylated IFN could be removed by anion exchange chromatography (AEC),and oxidized IFN could be removed by HIC (B column).The major acetylation sites (K34,K121,K131 and K134),major oxidation sites (M16,M21,M59 and M148),as well as Q5 and Q21 of major deamidation sites (N45,N156,Q5,Q21,Q46 and Q124) of h IFNα2b were located on the surface of the protein,which were susceptible to environmental conditions and subjected to acetylation and oxidation.Conclusion In the purification process of h IFNα2b,chromatography purification can effectively remove IFN-related proteins and HCPs,which also indicated that LC-HRMS technology can be used for the research of IFN purification process.

    2025 02 v.38 [Abstract][OnlineView][Download 1080K]

  • Verification of virus inactivation/removal efficacy in pilot-scale procedure of recombinant human growth hormone-Fc

    GAO Qihang;LIU Yulin;YU Jian;ZHAO Lei;SUN Zhongtao;ZHANG Kaining;ZHAO Jingnan;LIU Han;Changchun Institute of Biological Products Co., Ltd.;

    Objective To verify the virus inactivation/removal efficacy in pilot-scale procedure of recombinant human growth hormone-Fc(rhGH-Fc) fusion protein in order to ensure the product quality and patient safety.Methods Four viruses, pseudorabies virus(PRV), xenotropic murine leukemia virus(X-MuLV), minute virus of mice(MVM), and reovirus-3(Reo-3),were used as indicator viruses. Under the condition of scaling down, the inactivation ability of solvent and detergent(S/D)method on PRV and X-MuLV, the removal ability of X-MuLV and MVM by affinity chromatography, and the removal ability of MVM and Reo-3 by nanofilm filtration, were investigated.Results The inactivation ability of PRV and X-MuLV by S/D method was ≥ 4. 95 and > 5. 15 Log, respectively. The removal ability of X-MuLV and MVM by affinity chromatography was ≥ 4. 36 and 1. 68 Log, respectively. The removal capacity of MVM and Reo-3 by nanofilm filtration was > 5. 36 and >5. 40 Log, respectively.Conclusion The established rhGH-Fc fusion protein inactivation/removal process can effectively inactivate/remove the unknown and emerging viruses.

    2025 02 v.38 [Abstract][OnlineView][Download 858K]

  • Quantitative study of genomic DNA in HEK293 cell line by digital PCR

    BI Hua;WEN Fenfen ;TAO Lei;WEI Linghui ;YANG Jingqing;LU Ning ;QIN Xi;LIANG Chenggang;National Institutes for Food and Drug Control;

    Objective To perform quantirative study of HEK293 cell genomic DNA by microfluidic chip-based digital PCR technology, and to provide new ideas for more accurate gene quantification and genomic DNA related detection.Methods Firstly, the HEK293 cell DNA was obtained by Spin Column, identified and analyzed for the purity by agarose gel electrophoresis. Then, the genomic DNA of HEK293 cells was quantified by traditional ultraviolet spectrophotometry, Qubit fluorescence quantification and microfluidic chip-based digital PCR technology and statistically analyzed.Results The concentration of genomic DNA in HEK293 cells was detected as 100. 08 ng/μL by ultraviolet spectrophotometry, 93. 98 ng/μL by Qubit fluorescence quantification and the copy number was determined as 29 722. 81 copies/μL by digital PCR. Roughly calculated based on human single-copy genome of about 3. 3 pg, the converted copy numbers of spectrophotometry and Qubit fluorescence quantification were about 30 327 and 28 479 copies/μL, respectively. The deviation of the digital PCR detection value from the spectrophotometry detection value was only 2%, and the deviation from the Qubit detection value was only4%.Conclusion In this study, HEK293 genomic DNA was detected and compared by digital PCR, spectrophotometry and Qubit fluorescence, which provides a new detection method and idea for DNA quantification, and also provides a certain reference for the quantification of other nucleic acids.

    2025 02 v.38 [Abstract][OnlineView][Download 943K]

  • Screening and application of a verification method for influenza virus inactivation in MDCK cells

    LV Yuan;LI Chenfeng;YANG Yue;SUI Lina;LI Shuang;WU Yehong;WANG Qingshuang ;ZHANG Xuemei;Changchun Institute of Biological Products, Co., Ltd.;

    Objective To screen a verification method for influenza virus inactivation in MDCK cells, and apply it to the sample detection before and after influenza virus inactivation, so as to provide a reliable method for the quality detection of influenza vaccines. Methods Four types of influenza viruses adapted to MDCK cells, B/Yamagata(BY), B/Victoria(BV),H1N1 and H3N2, were diluted to 10, 1, 0. 1, 0. 01, 0. 001, and 0. 000 1 lgCCID_(50)/mL separately, which were then inoculated into MDCK cells and cultured by changing the medium(discarding after adsorption for 1. 5 h and adding virus culture medium, passaging every 3 d for a total of 3 generations), rehydration method(adding virus culture medium directly after adsorption for 1. 5 h, passaging every 3 d for a total of 3 generations), and method in European Pharmacopoeia(passaging every 4 d for a total of 3 generations). The cytopathic effect(CPE) was observed every day, the virus solutions of different passages were harvested, and the hemagglutinin(HA) titers were detected to determine the optimum inactivation verification method. Finally, the optimum inactivation verification method was used to detect samples before and after inactivation of influenza virus. Results Influenza virus could be detected at the titer of 0. 1-1 lgCCID_(50)/mL by both changing medium method and rehydration method, while influenza virus could be detected in the titer range of 1-10 lgCCID_(50)/mL by test protocol in European Pharmacopoeia, and the titer of HA detected by rehydration method was higher than those by the other two methods. Therefore, rehydration method was determined as the optimum inactivation verification method. The samples detected by rehydration method were all positive before influenza virus inactivation and negative after virus inactivation.Conclusion Rehydration method is the optimum method to verify the influenza virus inactivation in MDCK cells, which can be used to detect the quality of influenza vaccines.

    2025 02 v.38 [Abstract][OnlineView][Download 917K]

  • Establishment, verification and application of detection method for size distribution of residual DNA fragments in vaccine products based on MultiNA microchip electrophoresis technology

    YUAN Yue;MA Jing;CHEN Jie;CHEN Kerong;SU Chunyan;SHU Congyan;LI Yan;WANG Shuqiao;YANG Lei;Sichuan Institute for Drug Control (Sichuan Testing Center of Medical Devices), NMPA Key Laboratory for Quality Control and Evaluation of Vaccines and Biological Products, SCMPA Key Laboratory for Quality Monitoring and Risk Assessment of Biological Products;

    Objective To establish a method for determination of residual DNA fragments based on Multi NA microchip electrophoresis system,thus analyzing the size distribution of DNA fragments in a rapid way.Methods Based on Multi NA microchip electrophoresis system,a method for determination of the size distribution of residual DNA fragments was established,and the specificity,detection range and precision of the method were verified.Furthermore,the method was applied for the detection of the size of residual DNA fragments in the bulks and finished products of Sabin strain inactivated poliomyelitis vaccine (IPV).Results This method showed good specificity,with the detection range of 0-10 000 bp and the CVs of precision detection less than 20%.The results showed that the sizes of residual DNA fragments in Sabin strain IPV bulks were about 400 and 500 bp,accompanied with no detection in finished products.Conclusion A method based on Multi NA microchip electrophoresis technology for determination of residual DNA fragments was established,which might provide a reference for determination of the size distribution of residual DNA in biological products.

    2025 02 v.38 [Abstract][OnlineView][Download 887K]

  • Evaluation of application of enzyme immunoassay workstation in detection of antibodies against Streptococcus pneumoniae

    ZHOU Shanshan;BAI Shuang;CHEN Weixin;LV Min;WANG Jian;LAN Wenwen;LI Jing;ZHAO Dan;WU Jiang;Institute for Immunization and Prevention, Beijing Center for Disease Prevention and Control;

    Objective To apply automated enzyme immunoassay workstation instead of manual operation to facilitate detection of Streptococcus pneumoniae(S.pneumoniae) IgG antibodies, so as to improve the detection efficiency.Methods Procedures of the assay were set up on the automated workstation according to the World Health Organization(WHO) Pneumococcal Capsular Polysaccharide Types-pecific IgG Antibody Detection Manual Pn PS ELISA. The number of working station panels was maximized to preset all reagent consumables and more samples to be tested, thereby improving the detection flux and reducing the number of manual interventions. The serum PnG was repeatedly detected to verify the test precision. The WHO S.pneumoniae international calibration serum panel 12/278 was detected to verify the accuracy of the test. To compare the consistency of workstations and manual operation, 16 same serum samples were tested by manual operation and on the workstation meanwhile. Detections of four calibration serum samples were performed on the workstation by double-(WHO standard method) and single-column methods, and the consistency of the results was compared.Results With the layout of 21 plate positions, 24 serum samples could be examined in one program run, requiring only 2 manual interventions. The CV of 32 detection results of serum PnG was lower than 15%. The assay results of 75% of the calibration serum panel in 14 out of 23 serotypes exhibited a percent error of 40% or less compared to the assigned values, and the total accuracy was 80%.The correlation coefficient(Pc) of agreement between workstation and manual test results was 0. 996 7, and the correlation coefficient of agreement between workstation single-and double-column test results was 0. 999 8, both of which showed high agreement.Conclusion The automated enzyme immunoassay workstation has shown high precision, accuracy and large detection throughput, which is labor-saving, and can be applied to the detection of S.pneumoniae antibodies. The results of single-and double-column assay provide experimental basis for single-column detection in place of double-column detection.

    2025 02 v.38 [Abstract][OnlineView][Download 841K]

  • Research progress on rodent models infected by SARS-CoV-2 variant strains

    WANG Ruyu;SUN Kaili;LU Jia;Biosafety Level Ⅲ Laboratory, Wuhan Institute of Biological Products Co., Ltd.;

    The infection caused by SARS-CoV-2 is a very widespread epidemic disease. Rodent models have played an important role in studying the pathogenesis and immune response of SARS-CoV-2 infection, as well as in developing drugs or vaccines for treatment and prevention. However, the accumulation of SARS-CoV-2 mutations and their rapid spread in the population have led to the emergence of mutant strains that evade host immune responses. The current SARS-CoV-2 vaccine may not be able to effectively control the spread of mutant strains, so the research and development of vaccines and drugs for the prevention and treatment of mutant strains will continue. Rodents are crucial to the study of virus etiology, transmission and pathogenesis, and have played an important role in the early development of related inactivated vaccines and antibodies.This review mainly summarizes the research progress of rodent models in SARS-CoV-2 Alpha, Beta, Gamma, Delta, Omicron mutants.

    2025 02 v.38 [Abstract][OnlineView][Download 842K]

  • Research progress on pathogenesis mechanism of psoriasis and treatment of IL-17 antagonists

    ZHAO Zeyuan;ZHOU Baisong;LIU Yulin;LIU Jinghui;The Second Laboratory, Changchun Institute of Biological Products Co., Ltd.;

    Psoriasis is a chronic non-communicable skin disease characterized by red scaly patches on the scalp, knees,elbows, and other areas. It is often accompanied by various diseases such as metabolic arthritis, cardiovascular disease, and depression, which bring a heavy burden to individuals and society. This paper mainly discusses the pathogenesis of psoriasis,the establishment of animal models of psoriasis, the pharmacodynamics evaluation index of psoriasis drugs, and the research progress of current drugs related to interleukin(IL)-17 antagonists, aiming to provide certain references for the development of IL-17-related drugs and the treatment of psoriasis.

    2025 02 v.38 [Abstract][OnlineView][Download 1005K]

  • Research and application progress of methods for determination of hemagglutinin content in influenza vaccine

    LIN Yongjian;WU Yehong;Vaccine Research Department, Changchun Institute of Biological Products Co., Ltd.;

    Hemagglutinin(HA) is a glycoprotein on the surface of influenza virus, which can induce protective immune responses in a variety of hosts. It is the main antigen of influenza vaccines, and its content is an important indicator of influenza vaccine efficacy. The common detection method for HA content in influenza vaccines is single-radial immunodiffusion(SRID), which requires standard reference reagents and needs long preparation time. However, in the early stage of the pandemic influenza outbreak, it was difficult to obtain international reference materials in time, and it was hard to use SRID method to detect HA content, which seriously hindered the efficiency of vaccine research and development. Therefore, there is an urgent need to develop rapid HA content assay methods with good accuracy, sensitivity, and reproducibility to replace SRID method, improve the speed of vaccine development, and cope with influenza epidemic outbreaks. In this paper, the research and application progress of HA content detection methods for influenza vaccines in recent years is summarized, with a view to providing ideas for the development of detection methods for HA content and other antigens.

    2025 02 v.38 [Abstract][OnlineView][Download 916K]

  • Research progress on recombinant polymeric protein vaccines

    HUANG Luxia;LI Zhihua;WANG Youchun;LI Qianqian;Institute of Medical Biology, Chinese Academy of Medical Science & Peking Union Medical College;

    Infectious diseases are the second leading cause of death globally, and vaccination is the most effective prevention strategy. Ideal immunogenicity is a prerequisite for vaccine efficacy. Therefore, ensuring the natural conformation of antigen proteins to improve their immunogenicity is the focus for the research of recombinant protein vaccines, among which constructing a polymeric protein vaccine is an effective method to enhance vaccine immunogenicity. This paper reviews the current research of recombinant protein vaccines, the methods to construct protein polymers, and the application of polymer proteins in the development of viral vaccines, aiming to provide references for the development of recombinant polymeric protein vaccines.

    2025 02 v.38 [Abstract][OnlineView][Download 944K]

  • Advances in metabolic mechanism of innate immune response against SARS-CoV-2

    HAO Bingbing;CHEN Lei ;LIU Zhixiao ;HAN Chaofeng;School of Basic Medical Sciences, Naval Military Medical University;

    COVID-19 caused by SARS-CoV-2 infection poses a great threat to the lives of people around the world and has become a major global public health challenge. The innate immune system is the body's first line of defense against viral invasion and plays an important role in the development of COVID-19. As the main immune cells in the innate immune system, monocyte macrophages have pattern recognition receptors on their surface that recognize SARS-CoV-2 singlestranded RNA and damage associated molecular patterns(DAMPs) and induce antiviral innate immune responses. However,SARS-CoV-2 has a strong immune escape ability and can rapidly expand and replicate in host cells and induce host cell death, which further induces an inflammatory response and exacerbates COVID-19. Studies have shown that macrophage infiltration-induced hyperinflammatory responses(i.e., cytokine storms) are an important lethal mechanism of COVID-19,and that during SARS-CoV-2 infection, the over-activation of glycolysis is an important metabolic mechanism for viral immune escape and inflammatory response. This paper reviews the related research progress of glucose metabolism in the innate immune response against SARS-CoV-2.

    2025 02 v.38 [Abstract][OnlineView][Download 837K]

  • Research progress on effects of compound probiotics on animal immune mechanism

    GONG Zifeng;YE Guisheng;College of Agriculture and Animal Husbandry, Qinghai University;

    In recent years, with the abuse of antibiotics, the development of animal husbandry industry suffered a serious impact. Therefore, research and development of a new type of natural, safe and residue-free green feed additives has become a research focus. Probiotics originate from nature and have outstanding advantages in improving intestinal flora, antagonizing pathogenic bacteria and improving immune function, which has attracted the attention of scholars in related fields. This paper reviewed the effects of compound probiotics on animal immune mechanism, so as to provide a reference for studying the application of microecological agents in animal husbandry.

    2025 02 v.38 [Abstract][OnlineView][Download 824K]

  • CMC considerations of manufacturing site changes for marketed therapeutic recombinant protein drugs from regulatory review

    SAI Wenbo;MA Xiaojuan ;QIU Xiao;WEI Wei;Center for Drug Evaluation, National Medical Products Administration;

    With the continuous improvement of China's drug regulatory laws and regulations, Life-cycle regulatory management has become a key means and an inevitable development trend to ensure the safety of public drug use. The management of post-approval changes to biological products is gradually shifting towards the risk-based model from the item-based model.In recent years, there has been a significant increase in supplementary applications for manufacturing site changes of marketed therapeutic recombinant protein drugs. However, the risks associated with manufacturing site changes vary under different circumstances. Therefore, the research that needs to be carried out is also different. This paper provides an in-depth discussion on three key aspects: risk assessment and grading, general requirements and special considerations for pharmaceutical research, and common application issues. It outlines general requirements for manufacturing site change, aiming to offer valuable insights for industry needs. Through the collaborative efforts between regulatory authorities and the industry, patient rights and interests can be effectively protected, and high-quality drugs can be made available to the public.

    2025 02 v.38 [Abstract][OnlineView][Download 804K]
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@2012 Editorial Department of "Chinese Journal of Biologicals"