2023年08期目次

  • Evaluation of immune effect of ESAT6-Fc DNA vaccine against Mycobacterium tuberculosis

    YAO Si;YANG Jieqiong;YANG Yuxin ;LI Hui;WANG Tiansong;ZHANG Wei;MA Guorong;WAN Qiaofeng;Department of Pathogen Biology and Immunology,School of Basic Medicine,Ningxia Medical University;

    Objective To evaluate the immune effect of ESAT6-Fc DNA vaccine against Mycobacterium tuberculosis(M.tb),so as to lay a foundation of the next protective test of the vaccine against M.tb infection.Methods The female C57BL/6J mice were randomly divided into two groups with 5 mice in each group,which were injected with the recombinant plasmid pcD-ESAT6-Fc containing HSV2gD signal peptide gene,Rv3875 gene and mouse Fc gene(1 116 bp nucleotides)as well as PBS intramuscularly through thigh,respectively,with the dosage of 50 μg/leg,100 μg in total. After the first immunization,immunization was boosted once a week for three times continuously. Blood samples were collected 14,28,42 d after the first immunization,and the titers of IgG,IgG1 and IgG2a in serum were detected by indirect ELISA;Serum and spleen samples were collected 42 d after the first immunization,spleen cells were isolated and detected for the ratio of CD4~+ and CD8~+T cells secreting IFNγ by flow cytometry,and the contents of IFNγ and IL-2 in serum were determined by ELISA.Results IgG,IgG1 and IgG2a specific antibodies were detected in the serum of mice immunized with the recombinant plasmid 14 d after the first immunization,and the titers continued to increase;42 d after the first immunization,The ratio of CD3~+CD4~+IFNγ~+ and CD3~+CD8~+IFNγ~+ cells and the contents of IFNγ and IL-2 in the recombinant plasmid immunized group were significantly higher than those in the PBS group(t = 12. 43,13. 35,7. 04 and 6. 65,respectively,each P <0. 001).Conclusion The recombinant plasmid pcD-ESAT6-Fc can effectively induce the humoral and cellular immune responses in mice,which lays an experimental foundation for the further development of ESAT6-Fc DNA vaccine.

    2023 08 v.36 [Abstract][OnlineView][Download 835K]

  • Effect of non-structural protein amino acid site-directed mutation of dengue virus Ban18HK20 strain on virus proliferation and virulence

    LI Ming;FANG Enyue;LIU Xiaohui;LI Yuhua;National Institutes for Food and Drug Control;

    Objective To investigate the effect of non-structural protein amino acid mutations(NS1-G53D,NS3-E250V)on viral proliferation and virulence of Dengue virus(DENV)type 4 Ban18HK20 strain.Methods The mutant sites in Ban18-HK20 strain were determined by sequence alignment with an attenuated strain of DENV(PDK53). Point mutation plasmids pSPTM-Ban18HK20(G53D),pSPTM-Ban18HK20(E250V)and pSPTM-Ban18HK20(G53D+E250V)were constructed by using homologous recombination technique,and identified by enzymatic cleavage and sequencing. Viral RNA was obtained by in vitro transcription and then electrotransfected to Vero cells to rescue the point mutant virus. The biological characteristics of point mutant virus were identified by plaque assay,indirect immunofluorescence assay,virus genome sequencing,growth kinetics test,CCK-8 test and mouse neurotoxicity test.Results Enzyme digestion and sequencing confirmed that the point mutation plasmids were constructed correctly. The results of immunofluorescence assay and virus sequencing showed that the virus was rescued successfully. The point mutation virus showed smaller plaque than the wildtype virus. The virus titers of Ban18HK20(G53D),Ban18HK20(E250V)and Ban18HK20(G53D+E250V)reached the peak on the 4th day,which were 7. 67,7. 84 and 7. 78 LgPFU/mL,respectively. The titer of the wild-type virus reached the peak value 7. 68 LgPFU/mL on the 5th day. The proliferation activity of BHK21 cells infected with Ban18HK20(G53D)was significantly higher than those infected with the wild-type virus(P = 0. 003 6)and the proliferative activity of C6/36 cells infected with Ban18HK20(G53D)and Ban18HK20(G53D+E250V)was significantly higher than those infected with the wild-type virus(P < 0. 000 1). Both the point mutation virus and wild-type virus had no neurotoxicity to 4-week-old BALB/c mice. The neurotoxicity of point mutation viruses Ban18HK20(E250V)(LD_(50)=8. 51 PFU)and Ban18HK20(G53D+E250V)(LD_(50)= 0. 69 PFU)on 3-day-old BALB/c neonatal mice was lower than that of the wildtype virus(LD_(50)= 0. 69 PFU). The survival rate(60%)of nude mice injected with point mutation virus Ban18HK20(G53D +E250V)was higher than those injected with the wild-type virus(0)after intracerebral injection of the same amount of virus. The point mutation virus was genetically stable,and no recurrent mutation occurred in Vero cells after passage to the10th generation.Conclusion The non-structural protein NS1-G53D amino acid mutation weakens the cytotoxicity of Ban18HK20 strain to C6/36 and BHK21 cells,and the two-point combined mutation could weaken the neurotoxicity of Ban18HK20 strain in nude mice,which laid a foundation of the study of DENV virulence sites and the development of the vaccine.

    2023 08 v.36 [Abstract][OnlineView][Download 933K]

  • Expression and identification of recombinant human interferon λ1 in HEK-293F cells

    YAN Jiali;LIU Mingyang;LIU Jianwei;LI Suzhen;LIU Sen;WANG Jian;The Fourth Laboratory,National Vaccine and Serum Institute;

    Objective To express recombinant human interferon λ1(rhIFNλ1)by transient transfection in HEK-293F cells and identify it. Methods Two signal peptides[T cell receptor(TCR)and nature signal peptide(NSP)],three vectors[pcDNA3.4,ubiquitous chromatin opening element(UCOE)and PFR]and the target gene rhIFNλ1 were used to construct recombinant plasmids of six signal peptide-vector combinations. Using HEK-293F as host cells,the recombinant plasmids were transfected transiently to express rhIFNλ1 on a shake flask scale. The recombinant plasmid UCOE-Q46-λ with low glycosylation rhIFNλ1 was constructed by using NSP and vector UCOE,and transfected transiently into HEK-293F cells. The expressed product was purified by cation exchange chromatography(HiTrap SP FF),blue gel chromatography(HiTrap Blue HP)and gel filtration chromatography(Sephacryl S-100 HR),which was then analyzed by Western blot and reversed-phase HPLC,and determined for its molecular mass and N-terminal amino acid sequence by mass spectrometry. Results Six recombinant plasmids were constructed correctly as identified by double enzyme digestion and sequencing. With the extension of transfection time,the expression levels of the six expressed products increased gradually,and reached the highest level of10 ～ 20 mg/L at the 6th day of transfection. Double enzyme digestion and sequencing identification proved that the recombinant plasmid UCOE-Q46-λ with low glycosylation rhIFNλ1 was constructed correctly. The recombinant plasmid UCOE-Q46-λwas transfected into HEK-293F cells for 6 d. The purified product of cell culture supernatant showed a relative molecular mass of about 27 800 and a purity of 97. 372%,which showed specific binding to mouse anti-human IL-29/IFNλ1 monoclonal antibody;Two peaks were detected by reversed-phase HPLC,and the peak was showed at 15. 6 min and 20. 0 min respectively;The mass spectrometry molecular mass was about 24 000;The N-terminal five amino acids were G-P-V-P-T.Conclusion The rhIFNλ1 expressed by HEK-293F cells has high purity,which lays a foundation of further study of the protein.

    2023 08 v.36 [Abstract][OnlineView][Download 857K]

  • Prokaryotic expression,purification and activity of CamA protein with methylase activity from Clostridiodies difficile

    DUAN Cidong;CHEN Yu;SONG Xiaojun;LIN Shan;JIN Dazhi;School of Laboratory Medicine and Bioengineering,Hangzhou Medical College,Key Laboratory of Biomarkers and In Vitro Diagnosis Translation of Zhejiang Province;

    Objective To express CamA(Clostridiodies difficile adenine methltransferase A,CamA)protein with methylase activity in prokaryotic expression system.Methods The gene sequence of CamA protein of the standard strain of C.difficile1870 was amplified by PCR,cloned into plasmid pGEX-4T-1-MBP,transformed into E.coli HB101 and induced by 1 mmol/L IPTG to express the recombinant target protein. After purification with Amylose Resin,CamA protein was digested by tobacco etch virus(TEV)protease,and verified for its methylase activity using MTase-Glo~(TM)Methyltransferase Assay in vitro.Results PCR sequencing showed that the cloned CamA gene sequence was correct,in which no base mutation occurred. The recombinant expression plasmid pGEX-4T-1-MBP-CamA was digested by EcoRⅠand BamHⅠ,which showed that 1 731 bp of CamA gene was connected to the vector plasmid. The relative molecular mass of the recombinant protein was about 108 800(with 1 MBP tag),and the purity was over 90% after purified with Amylose Resin. Under the condition of20 μmol/L DNA and 20 μmol/L SAM at room temperature for 30 min,0. 5 μg CamA produced SAH at a concentration of about 101. 60 nmol/L and the enzyme activity was(0. 339 ± 0. 027)U/mg.Conclusion The CamA protein of C.difficile has been successfully expressed and purified with methylase activity,which lays a foundation of further study on the function of CamA and its role in the occurrence,development and treatment of C.difficile infection.

    2023 08 v.36 [Abstract][OnlineView][Download 846K]

  • Prokaryotic expression and activity detection of insulin-degrading enzyme mutant T142A

    XIAO Shu;HUANG Chen;MA Shutao;ZHANG Kai;LI Yimeng;LI Zhen;LU Changrui;CHEN Ting;College of Chemistry,Chemical Engineering and Biotechnology,Donghua University;

    Objective To express insulin-degrading enzyme(IDE)mutant T142A in prokaryotic cells and detect its activity.Methods According to the results of multi-sequence alignment and IDE substrate co-crystal structure,an active residue in β6-strand structure of IDE were predicted.The recombinant plasmid ppSUMO-T142A,with the site mutation of threonine 142 to alanine,was constructed by point mutation technique and expressed by E.coli prokaryotic expression system.After purification by nickel ion column affinity chromatography,ion exchange chromatography and gel filtration chromatography,the mutant T142A was obtained and determined for the activity by fluorescence method.Results IDE amino acid sequence is highly conserved among 16 species.T142 directly participates in substrate binding,interacts with substrate cleavage sites,and is close to important structures such as catalytic active sites and door-subdomains.The mutation of recombinant plasmid ppSUMO-T142A was proved to be correct by sequencing.The expressed fusion protein His-SUMO-T142A was mainly existed in soluble form in the supernatant at a concentration of 18 mg/mL,with a relative molecular mass of about 131 000;After three steps of purification,the purity of mutant T142A reached 86%.The maximum reaction rate(V_(max))of T142A catalytic degradation of fluorescent substrate V was 501.06 min~(-1) and the Michaelis constant(K_m) was 9.01μmol/L.Compared with wild-type IDE(V_(max) was 2 814.32 min~(-1),K_m was 11.93μmol/L),the activity of T142A decreased significantly.Conclusion The activity of IDE mutant T142A expressed in this study greatly decreases,while T142 is an important residue for IDE to play its enzymatic function,which provides an experimental basis for the development of new IDE activity regulatory molecules.

    2023 08 v.36 [Abstract][OnlineView][Download 936K]

  • Effect of cold stimulation on phenotype of alveolar macrophages in mice

    LI Qianru;ZHOU Bowen ;LI Xinyue;LU Jingjing;XU Bin;YANG Huanmin;College of Animal Science and Veterinary Medicine Basic Veterinary Science,Heilongjiang Bayi Agricultural University;

    Objective To investigate the effect of cold stimulation on the phenotype of alveolar macrophages(MH-S cells) in mice. Methods MH-S cells were cultured at 37 ℃ for 24 h,and cold stimulated at 36,34 and 32 ℃ for 0,0. 5,1,3,6,9 and 12 h respectively. The mRNA transcription levels of interleukin-1β(IL-1β) and interleukin-10(IL-10) genes in MH-S cells were detected by qRT-PCR. MH-S cells were cultured at 37 ℃ for 24 h,and cold stimulated at 34 ℃ for 0. 5 h,which were detected for the mRNA transcription levels of tumor necrosis factor-α(TNF-α),inducible nitric oxide synthase(iNOS)and Arginase1(Arg1)genes by qRT-PCR(MH-S cells with 0 h cold stimulation as control),detected for the expression of iNOS and Arg1 by immunofluorescence assay(MH-S cells cultured at 37 ℃ for 0. 5 h as negative control)and detected for the expression levels of iNOS,TNF-α and nuclear factor-kappa B(NF-κB)by Western blot(MH-S cells cultured at 37 ℃ for 0. 5 h as negative control). Results The mRNA transcription levels of IL-1β and IL-10 genes in MH-S cells were the highest when the cells were cultured at 34 ℃ for 0. 5 h,therefore,the cold stimulation model of MH-S cells was established under this condition. Compared with the cells cultured for 0 h,the mRNA transcription levels of iNOS,TNF-α and Arg1genes in MH-S cells cultured at 34 ℃ for 0. 5 h increased significantly(t = 3. 733,12. 190 and 6. 793,respectively,each P < 0. 05). Compared with the negative control group,the fluorescence expression intensity of iNOS and Arg1 in MH-S cells in the stimulation group increased,especially iNOS,the expression levels of iNOS and TNF-α proteins increased with no significant difference(t = 0. 675 and 1. 514,respectively,each P > 0. 05),and the expression level of NF-κB increased significantly(t = 3. 092,P < 0. 05). Conclusion Cold stimulation at 34 ℃ for 0. 5 h can increase the expression of inflammatory factors such as IL-1β,IL-10,TNF-α,iNOS,Agr1 and NF-κB in MH-S cells,activate NF-κB signaling pathway in MH-S cells,induce the expression of inflammatory proteins and promote cell activation.

    2023 08 v.36 [Abstract][OnlineView][Download 851K]

  • Isolation,identification and virulence gene distribution and pathogenicity of three Bacillus cereus strains

    FAN Peichao;LU Yafei;LIU Chunyu;MA Dehui;XUE Jiangdong;Laboratory of Preventive Veterinary Medicine,College of Animal Science and Technology,Inner Mongolia University for Nationalities;

    Objective To isolate and identify the pathogenic bacteria in Tongliao area,Inner Mongolia Autonomous Region,China in 2019 and determine the virulence gene distribution and pathogenicity.Methods Three Gram positive isolates,FL1,FL2 and FL3,from the clinical specimens in Tongliao area were subjected to biochemical test,drug sensitivity test,16S rDNA sequencing,virulence gene test and pathogenicity test.Results Bio-chemical test proved the three isolates as Bacillus cereus. The strains FL1 and FL2 from bovine origin were resistant to penicillin and carbamazillin,while the strain FL3 from horse origin to Cefradine. Strains FL1 and FL2 showed the closest relationship to the isolate(KR063181. 1)from Hunan,China,while strain FL3 to the isolate(HM104658. 1)from Shandong,China. However,strain FL3 showed relatively far relationship to strains FL1 and FL2. All the distributions of eight virulence genes of B.cereus were observed in the three strains,while the distributions of four virulence genes of B.anthracoides were different. All the three isolates showed pathogenicity.Conclusion All the three isolates were pathogenic B.cereus,which showed a certain drug resistance. However,there were differences in virulence gene distri-bution between bovine and equine B.cereus.

    2023 08 v.36 [Abstract][OnlineView][Download 918K]

  • Preparation of colloidal gold immunochromatographic test strip for rapid detection of Pseudomonas aeruginosa

    ZHANG Shikai;ZHU Huihuang;LI Jiaji;WANG Yi;HU Zheng;School of Food and Biological Engineering,Hubei University of Technology;

    Objective To develop a colloidal gold immunochromatographic test strip for rapid and accurate detection of Pseudomonas aeruginosa(P.aeruginosa,Pa).Methods After bioinformatics analysis of Pa outer membrane protein OprF,the gene sequence with abundant antigenic determinants and high intraspecific homology was chemically synthesized,and then connected to pET-28a(+)vector to construct the expression vector pET-28a-OprF,which was transformed into E.coli BL21(DE3)and induced by IPTG. The recombinant OprF protein was purified by Ni Sepharose~(TM)6 Fast Flow and used to immunize two female BALB/c mice for 3～4 times by multi-point subcutaneous injection in the back at the first immunization and intraperitoneal injection at subsequent immunizations. The monoclonal antibodies were screened by animal cell fusion technique,and the colloidal gold immunochromatographic test strip for rapid detection of Pa was prepared by using monoclonal antibody and double antibody sandwich immunochromatography technique. The specificity,sensitivity and stability of the test strip were evaluated.Results Two monoclonal antibodies,Pa-1# and Pa-2#,were obtained with the titer of 1∶409 600,and both of them recognized OprF specifically. The prepared colloidal gold immunochromatographic test strip showed a sensitivity of 1. 0×10~6CFU/mL and had no cross reaction with 9 common respiratory pathogens with a good stability.Conclusion The prepared colloidal gold immunochromatographic test strip can detect Pa rapidly within 15 min,with high specificity and good stability.

    2023 08 v.36 [Abstract][OnlineView][Download 891K]

  • Effect of tyrosine kinase inhibitor BGJ398 on proliferation,apoptosis and migration of human hepatocellular cancer Huh-7 cells and its mechanism

    LIU Chang;YU Anqi ;CHI Fenqing ;GONG Tao ;LIANG Wenting ;YU Baofeng;Department of Biochemistry and Molecular Biology,Changzhi Medical College;

    Objective To evaluate the effect of tyrosine kinase inhibitor BGJ398 on the proliferation,apoptosis and migration of human hepatocellular cancer Huh-7 cells and explore the mechanism.Methods The effects of 10 tyrosine kinase inhibitors on the survival of Huh-7 cells were detected by MTT assay,and the sensitivity of Huh-7 cells to BGJ398 was analyzed by single-target kinetic equation and biphasic kinetic equation respectively.Huh-7 cells were added with 10,30 and 90 nmol/L BGJ398 respectively,and the control group(without drugs)was set.The effects of BGJ398 on the apoptosis and cell cycle of Huh-7 cells were detected by flow cytometry after culturing at 37℃for 24 h,the effect on the migration ability was detected by wound healing assay and the effect on the expression of multiple pathway-related proteins was detected by Western blot.Results All of 10 tyrosine kinase inhibitors inhibited the proliferation of Huh-7 cells,among which Huh-7 cells were most sensitive to BGJ398 and the IC_(50)was(0.020±0.013)μmol/L;The response of Huh-7 cells to BGJ398 was composed of two phases with F_1 accounted for 92.8%(K_(d1)was 36 nmol/L)and F_2 accounted for 7.2%(K_(d2)>1 000μmol/L).Compared with the control group,the apoptosis rate and the percentage of Huh-7 cells in G1 phase increased significantly in 30 and 90 nmol/L BGJ398 groups(t=-6.407～-4.459,each P<0.05),while the percentage of Huh-7 cells in S phase decreased significantly in 10,30 and 90 nmol/L BGJ398 groups(t=2.982,7.859 and 12.425,respectively,each P<0.05);After 24 and 48 h of scratching,the scratch area of 30 and 90 nmol/L BGJ398groups decreased significantly(t=5.376～18.197,each P<0.05);The expression levels of phosphorylated fibroblast growth factor receptor(FGFR)and phosphorylated extracellular signal-regulated kinase 1/2(Erk1/2)protein decreased significantly in 30 and 90 nmol/L BGJ398 groups(t=4.015～6.729,each P<0.01).Conclusion BGJ398 can inhibit the proliferation and migration of human hepatocellular cancer Huh-7 cells,induce apoptosis and cell cycle arrest,which might be achieved by inhibiting FGFR phosphorylation and MAPK signaling pathway.BGJ398 is expected to be a potential agent for the treatment of hepatocellular cancer.

    2023 08 v.36 [Abstract][OnlineView][Download 939K]

  • Safety of human papilloma virus vaccine based on Vaccine Adverse Event Reporting System from 2006 to 2021

    LIU Yan;WANG Jietao ;YANG Beifang;WANG Zhao;DENG Peng;SHEN Heng;LUO Jinjun ;ZHAN Faxian;Institute for Communicable Disease Control and Prevention,Hubei Provincial Center for Disease Control and Prevention;

    Objective To investigate the characteristics of distribution of adverse event(AE)associated with human papillomavirus(HPV)vaccine by analysis of data on AE collected from the Vaccine Adverse Event Reporting System(VAERS).Methods The data on AE reported in VAERS from January 1st,2006 to December 31st,2021 were analyzed and compared by using Pearson Chi-square test and Mann-Whitney U test.Results A total of 53 571 cases of AE were included in the study,in which the ratio of male to female was 0. 25∶1,and the median age of vaccinees was 15 years. A portion of 36. 1%of AE occurred after the first dose,while 90. 7% occurred within 3 d after vaccination. Both the gender ratios(χ~2=72. 570,P < 0. 001) and the median ages(Z = 4. 255,P < 0. 001)of vaccinees in non-serious and serious adverse event(SAE)showed significant difference. In terms of classification of SAE,hospitalization,prolonged hospitalization and disability were more common in females than in males,of which the percentages decreased with the increasing age. Among the AE,syncope was the most common clinical symptom. In the SAE,the highest proportion of deaths was caused by HPV2 vaccine,which was 19. 0%. The proportion of prolonged hospitalization caused by HPV4 vaccine was higher than that by HPV9vaccine. In general,HPV4 vaccine was more prone to cause SAE than HPV9 vaccine(χ~2=183. 267,P < 0. 001).Conclusion In all the AE,the largest proportion occurred in the age group of 9 ～ 17 years,followed by those in the groups of 18 ～ 26 and 27 ～ 45 years. Most of the AE occurred after the first dose. The clinical symptoms of AE caused by three vaccines were different. The analysis of distribution characteristics of AE may provide a reference for the study on clinical safety of HPV vaccine and optimization of vaccination.

    2023 08 v.36 [Abstract][OnlineView][Download 811K]

  • Epidemiological characteristics of measles in Jilin Province from 2013 to 2022

    CHENG Tao;FU Simei;WANG Shuang;SHAN Yuanchun;LI Chunmei;CAO Fengrui;WU Yi;LI Yongqiang;LI Xiang;Jilin Provincial Center for Disease Control and Prevention;

    Objective To analyze the epidemiological characteristics of measles in Jilin Province from 2013 to 2020.Methods Through the measles surveillance system of China Disease Prevention and Control Information System,a national health security information project,the measles incidence data of Jilin Province from 2013 to 2022 were collected,the measles incidence and incidence characteristics were analyzed by descriptive epidemiological method,while the virus was isolated and the genotype was identified at the same time.Results From 2013 to 2022,3 202 measles cases were reported in Jilin Province,and the reported incidence rates were 0. 57/100 000,9. 88/100 000,0. 71/100 000,0. 32/100 000,0. 12/100 000,0. 13/100 000,0. 13/100 000,0. 05/100 000,0. 05/100 000 and 0. 06/100 000,respectively.By age group,people aged 0 ～ 1,2 ～ 6,7 ～ 18,19 ～ 29,30 ～ 49 and over 50 years accounted for 42. 19%,10. 31%,6. 28%,14. 52%,24. 83% and 1. 87% of all the reported incidence cases,respectively,while among cases aged less than8 months,8 ～ 12 months,2 ～ 18 years and over 19 years,98. 49%,71. 34%,33. 33% and 39. 77% had no vaccination history,respectively. From 2013 to 2018,a total of 44 measles virus strains were obtained in the measles laboratory of Jilin Province,except for one vaccine strain A genotype,the rest were all H1a genotype,and no measles virus strain was obtained since 2019.Conclusion From 2013 to 2022,the reported incidence of measles in Jilin Province showed a downward trend year by year,while there is still a need to maintain the coverage of two doses of vaccination of measles containing vaccine. At the same time,adult measles vaccination should be strengthened,and the sensitivity of surveillance system should be improved to prevent measles outbreaks.

    2023 08 v.36 [Abstract][OnlineView][Download 790K]

  • Screening and optimization of the formulation of human recombinant neutrophil inhibitory factor and hirulog hybrid for injection

    LI Na;WANG Ke;ZHANG Peibiao;LIU Zhong;ZHANG Guimin;Shandong Engineering Laboratory of Protein Drugs,Shandong Newtimes Pharmaceutical CO.,Ltd.;

    Objective To screen and optimize the formulation and technology of human recombinant neutrophil inhibitory factor and hirulog hybrid(TNHH)for injection,and investigate its stability.Methods Based on the results of the single factor experiment,with the pH range,mannitol dosage and povidone K30 dosage as independent variables,and the content of high molecular protein as response value,the response surface design(CCF)test was used to analyze the effects of the respective variables and their interaction on the content of high molecular protein in TNHH for injection to screen out the optimal formulation. In order to facilitate the operation,the optimal formulation was adjusted to prepare three batches of samples in pilot scale,which were placed at 40 ℃,75% relative humidity(RH)for 2,4 weeks and 2 ～ 8 ℃ for 3,6 months,respectively. The samples were taken and the appearance,pH,purity of reversed phase-high performance liquid chromatography(RP-HPLC)and purity of size exclusion chromatography-high performance liquid chromatography(SECHPLC)were detected to verify the stability of this formulation and process.Results The optimal formulation was pH 4. 982 6,mannitol 7. 986 4% and povidone K30 1. 902 7%,which was finally adjusted to pH 5. 0,mannitol 8. 0% and povidone K302. 0%. The TNHH preparation for injection prepared by the optimized prescription and process were stable in quality and met the clinical medication requirements.Conclusion The optimum formulation of TNHH preparation for injection is reasonable in the process and suitable for industrial production.

    2023 08 v.36 [Abstract][OnlineView][Download 813K]

  • Optimization,verification and application of ELISA method for quantitative detection of varicella-zoster virus IgG antibody

    ZHOU Zhijun;CHEN Kejin;LIN Lianzhen;LI Juan;WANG Min;CHEN Limei;CHENG Shuang ;LUO Minhua ;ZENG Shuangying;LI Taojing ;HU Yong;LI Cesheng;Sinopharm Wuhan Plasma-derived Biotherapies Co.,Ltd.;

    Objective To optimize and verify the ELISA method for quantitative detection of varicella-zoster virus(VZV)IgG antibody potency,and use it for the screening of plasma with high potency VZV-IgG in healthy donors.Methods The VZV-IgG indirect ELISA kit from Institut VirionSerion GmbH was selected,the first international standard for varicellazoster immunoglobulin(NIBSC code:W1044)was diluted to 2 IU/mL as the standard,and 4-parameter fitting curve was used to develop the quantitative ELISA method. The method was determined for the optimal linear range and verified for the precision and accuracy. VZV-IgG antibody potency of 1 962 human plasma samples and some batches of human immunoglobulin preparations from 10 plasma stations under Sinopharm Wuhan Plasma-derived Biotherapies Co.,Ltd.(SWPB)were detected by the developed method.Results The linear range of the standard curve was 16. 25 ～ 2 000 mIU/mL,the CV values of precision in intra-and inter-assays were 1. 3% ～ 10. 6% and 4. 270% ～ 7. 636%,and the accuracy in intra-and inter-assays were 92. 30% ～ 111. 02% and 98. 40% ～ 104. 88%,respectively;Sample-adding experiment showed that the measured value of the added sample was 95. 79% ～ 111. 03% of the theoretical value. The positive rate of 1 962 human plasma samples was 94. 29%,and the samples with potency greater than 3 000 mIU/mL accounted for 1. 02%. The potency of VZV-IgG antibody in different kinds of human immunoglobulin preparations was lower,while higher than that of intravenous human immunoglobulin(pH 4).Conclusion The optimized VZV-IgG quantitative detection method can be used for the screening of VZV-IgG in healthy people. The positive rate of VZV-IgG antibody in naturally infected healthy plasma donors is high,while the potency is low,thus,vaccine immunization is required to obtain qualified plasma with high potency.

    2023 08 v.36 [Abstract][OnlineView][Download 811K]

  • Development and verification of loop-mediated isothermal amplification for detection of 11 common mycoplasmas

    KE Xiaomei;WU Yuman;KONG Weisheng ;CHEN Zhiyan ;LIU Shuai;WU Huazuo;ZHU Xiayan;ZHANG Jian;Department of Bioengineering,Zhuhai Campus of Zunyi Medical University;

    Objective To develop and verify a loop-mediated isothermal amplification(LAMP)method for simultaneous detection of multiple mycoplasma contamination.Methods The conserved sequences of 16S rRNA genes of 13 mycoplasma species were aligned by the multiple sequence alignment tool of SnapGene software.Two internal primers FIP and BIP,two external primers F3 and B3 and loop primer LOOP(LF/LB)were designed according to the conserved sequences.The LAMP system was developed as follows:Bst DNA polymerase large segment(8 U/μL),10×ThermoPoly reaction buffer[20 mmol/L Tris-HCl,10 mmol/L KCl,10 mmol/L(H_4)_2SO_4,2 mmol/L MgSO_4 and 0.1%Triton X-100,pH 8.8],MgSO_4(100 mmol/L),dNTP Mix,FIP/BIP Primers,F3/B3 Primers,LOOP Primers,nuclease-free water and template DNA(13 plasmids carrying mycoplasma 16S rRNA).The products were identified by 1%agarose gel electrophoresis.The feasibility,specificity and repeatability of the method were verified,and the detection limit was determined.Results A total of 11 mycoplasma sequences were detected simultaneously by using two sets of LAMP primers screened.There was no nonspecific amplification in other nonspecific template detection,and the primer group had good specificity.The developed method showed good reproducibility and good sensitivity with the minimum detection limit of 30.1 copies/μL,and no false positive or false negative amplification.Conclusion LAMP technology can be used to detect a variety of mycoplasma contamination in cell culture,with good specificity and short time consumption,which can be used as one of the technical platforms for rapid detection of mycoplasma contamination in cell culture.

    2023 08 v.36 [Abstract][OnlineView][Download 969K]

  • Evaluation of uncertainty in determination of cetrimonium bromide residue in polysaccharide vaccines by ultra performance liquid chromatography-mass spectrometry

    LI Jingtao;WU Wenqian ;WANG Yuejiao;CHEN Liang;BAO Xiaohua;Jilin Province Drug Inspection Research Institute;

    Objective To determine the cetrimonium bromide(CTAB)residue in polysaccharide vaccines using ultra performance liquid chromatography-mass spectrometry(UPLC/MS-MS),and analyze and evaluate the uncertainty of the determination results.Methods By establishing a mathematical model,the sources and values of uncertainty introduced in the measurement process were analyzed,the uncertainty components of each influencing factor were calculated,and the standard uncertainty and expanded uncertainty were synthesized to form an uncertainty report.Results At 95% confidence interval,the expanded uncertainty was 0. 002 8 mg/kg. The determination result of CTAB residue in polysaccharide vaccine was reported as(1. 000 6 ± 0. 002 8)mg/kg(k = 2,confidence interval p = 95%).Conclusion The main factors affecting the accuracy of determination results are the preparation of standard solution and the introduction of recovery rate,which should be focused on and controlled in the experiment process to make the detection results more reliable.

    2023 08 v.36 [Abstract][OnlineView][Download 867K]

  • Development and verification of chemical chromogenic method for determination of residual cetyltrimethyl ammonium bromide content in ACYW135 meningococcal polysaccharide stock solution

    YANG Baifeng;WU Lijie;LIU Ting;ZHANG Na;LI Shihui;Bacterial Vaccine Department of Research,Beijing Institute of Biological Products Co.,Ltd.;

    Objective To develop and verify a chemical choromogenic method for the determination of residual cetyltrimethylammonium bromide(CTAB)content in ACYW135 meningococcal polysaccharide stock solution. Methods A chemical chromogenic method was developed for the determination of residual CTAB content by using titan yellow as chromogenic reagent,and verified for the linear range,intermediate precision and accuracy. ACYW135 meningococcal polysaccharide stock solution were determined for residual CTAB content by the developed method. Results The CTAB reference at concentrations of 4. 0～10. 0 μg/mL showed good linear relationship to A_(500),with a R~2 value of more than 0. 990. The recovery rates of CTAB standard at concentrations of 5. 0,7. 0,and 9. 0 μg/mL were all within 95% ～ 110% in six repeated tests,with the CV values of determination results of less than 10%. All the residual CTAB contents in four batches of meningococcal polysaccharide stock solutions were less than 8. 0 μg/mL. Conclusion The chemical chromogenic method showed good linearity,intermediate precision and accuracy,which might be used for the determination of residual CTAB content in ACYW135 meningococcal polysaccharide stock solution.

    2023 08 v.36 [Abstract][OnlineView][Download 806K]

  • Role of microRNAs in tumor progression by regulating macrophage phenotypes and functions

    PAN Fengyan;LIU Qianqian;LIU Fang;CHEN Che;Gansu University of Chinese Medicine;

    MicroRNAs(miRNAs) are a class of small non-coding RNAs that regulate the pathogenesis of many diseases,including cancer. As the main permeable immune cells in the tumor microenvironment(TME),macrophages play an important role in tumor initiation,angiogenesis,metastasis,drug resistance and so on. The altered expression of miRNAs may regulate the function of macrophages and thus affect the progression of cancer. This paper reviews the mechanism of miRNAs influencing tumor progression by regulating the phenotype and function of macrophages,and provides a new idea for tumor treatment.

    2023 08 v.36 [Abstract][OnlineView][Download 788K]

  • Application progress of high performance liquid chromatography in detection of hemagglutinin content in influenza vaccine

    TONG Guangwei;WU Yehong;Vaccine Laboratory of Changchun Institute of Biological Products Co.,Ltd.;

    At present,the most commonly used method for detecting hemagglutinin(HA)content in influenza vaccines is still single-radial immunodiffusion(SRID). However,the preparation of standards required by this method takes a long time,usually 2 ～ 3 months. Therefore,how to quantitatively analyze HA accurately has always been a difficult problem in the detection of HA content in the situation that reference products can not be obtained at the early stage of the pandemic influenza. High performance liquid chromatography(HPLC)has its own characteristics of rapidity,high sensitivity,good repeatability and high accuracy,which can rapidly determine HA content by using different separation principles and has been widely used in the detection of HA content in influenza vaccine. This paper reviewed the research progress of the application of HPLC in the determination of HA content in influenza vaccine.

    2023 08 v.36 [Abstract][OnlineView][Download 785K]

  • Research progress on regulation of Staphylococcus aureus Ebh protein by ArlRS two-component system

    GUAN Xiaowen;LIU Meng ;WEI Lianhua;Gansu University of Chinese Medicine;

    Staphylococcus aureusis a major cause of hospital and community acquired infection,which can express many kinds of cell wall-related and extracellular virulence factors,causing a variety of infectious diseases. Ebh protein is involved in the adhesion,aggregation and biofilm formation of Staphylococcus aureus,and thus closely related to its strong pathogenicity. The pathogenesis of Staphylococcus aureus is controlled collaboratively by different two-component signal transduction systems and transcriptional regulation factors,among which ArlRS is the key two-component system(TCS)necessary for the adhesion,biofilm formation and virulence. At the same time,ArlRS regulates Ebh protein through global regulation factor MgrA,thus affecting the role of Staphylococcus aureus in human infectious diseases. In this paper,the regulatory mechanism was reviewed.

    2023 08 v.36 [Abstract][OnlineView][Download 860K]

  • Progress in research on detection techniques for peste des petits ruminants virus

    HE Yunjiang;JIA Weijuan;CHI Shanshan;WANG Xueli;College of Animal Science and Technology,Inner Mongolia Minzu University;

    Peste des petits ruminants(PPR)is an acute and highly contagious disease caused by peste des petits ruminants virus(PPRV),which mainly infects goats and sheep with high morbidity and mortality. Because of its serious pathological damage and wide spread,PPR has caused huge economic losses to the aquaculture industry and international trade in various countries,so it is particularly important to establish a rapid and accurate detection method for the prevention and control of the disease. This paper reviews the progress in research on biological detection techniques for PPRV detection,such as routine RT-PCR,routine multiplex PCR,real-time fluorence quantitative PCR(RT-qPCR),pyrosequencing,nested PCR(nPCR),loop-mediated isothermal amplification(LAMP)and recombinase polymerase amplification(RPA),so as to provide bases and ideas for scientific detection and identification of PPRV.

    2023 08 v.36 [Abstract][OnlineView][Download 827K]


@2012 Editorial Department of "Chinese Journal of Biologicals"