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Analysis on integrity of target nucleic acid in recombinant adeno-associated virus

Chinese Journal of Biologicals.2024;37(6):641-645. doi: 10.13200/j.cnki.cjb.004235

Objective To explore a method for detecting the integrity of target nucleic acid in recombinant adeno-associated virus(rAAV),and establish a preliminary analysis algorithm.Methods The free DNA fragment of rAAV was digested,and the virus genome was extracted.Five pairs of overlapping primers were designed,using the orthogonal array method,which were detected by digital PCR respectively,with conventional conditions:20 μL reaction system with EveGreen and4 μL template,and digital PCR conditions:95 ℃ 5 min;95 ℃ 15 s,55 ℃ 30 s,72 ℃ 90 s for 45 cycles.The enhancement condition of ultra-long nucleic acid fragment was 25 μL reaction system with Mix B-2 000 bp and 2 μL template,and the quantitative analysis was performed by using the software attached to the instrument.Using the least square method,the number of full-length fragments was fitted and analyzed,and then the integrity distribution of target nucleic acid was interpreted.Results The fragments with the distance between primers of no more than 1 200 nt were amplified effectively,and a series of effective copies of fragments with a length of about 1 000 nt were obtained by systematic analysis.The copy number of common fragments was fitted and analyzed by the least square method.It was estimated that the full-length fragments in the sample were no more than 1 234 copies/μL,and there was a signficant difference(P < 0.05)between this value and the maximum measured value of 1 443 copies/μL,with the difference of approximately 16.9%.Conclusion A preliminary detection method for the integrity of target nucleic acid in rAAV has been developed,and a certain amount of incomplete target nucleic acids were analyzed in the test sample,laying a foundation for further in-depth research.

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Establishment and validation of a method for determination of interesting protein expression level in cells infected with recombinant adeno-associated virus

PEI Dening

Chinese Journal of Biologicals.2024;37(6):646-650+655 . doi:10.13200/j.cnki.cjb.004237

Objective To establish and validate a method for the determination of the interesting protein expression level of recombinant adeno-associated virus(rAAV)infected cells,so as to monitor the product quality in different stages of rAAV9production process.Methods After incubation of serial diluted rAAV samples with infection enhancer Envirus-AAV,the human malignant glioblastoma cells(U87-MG)pretreated with hydroxyurea(HU)were infected.Using rAAV9 reference as the standard,the expression level of glutaryl-CoA dehydrogenase(GCDH)was detected by ELISA,and the specificity,accuracy,precision,linear range,limit of quantitation(LOQ)and durability of the method were verified.Eight batches of rAAV9 samples were detected by the established method.Results The A_(450)-A_(630) value of the sample buffer was 0.3,which was slightly lower than the lowest dilution point(1 ng/mL)of the four-parameter standard curve for protein quantification.The average recoveries of samples with 150%,100% and 50% theoretical relative titer levels were in the range of 100.0%-107.3%.The RSDs of the target protein expression level of the samples with three theoretical relative titer levels detected by the same experimenter three times and different experimenters were all less than 25%.There was a good linear relationship between rAAV9 samples and the target protein expression levels in the range of 50%-150% theoretical relative titer levels,and the linear regression equation was y = 1.077 x-0.022,R~2= 0.984.The LOQ of the method was 0.59,namely 6.0×10~(12) vg/mL.After U87-MG cells were incubated with HU for different time(18,21,24 h),and the culture supernatant was stored under different conditions(room temperature for 0.5 h,below-60 ℃ for 12 h,below-60 ℃ for 24 h).The RSDs of target protein expression levels were all less than 25%.The target protein expression levels of 1-8 batches of rAAV9 samples were 111%,121%,72%,65%,86%,75%,102% and 91%,respectively.Conclusion The established method for the determination of the target protein expression level after rAAV infection has good specificity,accuracy,precision and durability,and can be used for the quality control of products in different stages of rAAV9 production.

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Effect of production site change on quality of live attenuated varicella vaccine

WANG Xiaoqin

Chinese Journal of Biologicals.2024;37(6):651-655 . doi:10.13200/j.cnki.cjb.004234

Objective To evaluate the effect of production site change of vaccine production on the quality of live attenuated varicella vaccine at the molecular level.Methods Using next-generation sequencing(NGS),the varicella-zoster virus(VZV)Oka strain virus seed(VW-before,SYVW-after)and vaccine bulk(P-before,YZP-after)produced before and after prodution site change were subjected to DNA extraction,purification,library construction and sequencing,and the quality control of the sequencing results were performed.Taking gene sequence of Dumas strain registered in GenBank(NC＿001348)as the position reference genome,the whole gene sequences of the four samples before and after prodution site change were compared with the Oka strain virus to obtain the mutation sites,base changes and variant allele frequency(VAF),and the consistency of the samples was evaluated.Results All the sequencing depth of the four samples was more than1 500 ×,and the GC content of the virus seed and vaccine bulk was about 46%,indicating that the sequencing quality was high.The mutation sites and base composition of the gene sequences of virus seed and vaccine bulk were consistent before and after prodution site change,and the VAF was close.Pairwise comparison of correlation coefficients was highly correlated,and the correlation coefficients were significantly different(each R~2≥ 0.990,each P < 0.001).Conclusion NGS test showed that the changes in vaccine production process have no effect on the quality of live attenuated varicella vaccine.

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Effect of different strains of mice immunized with rabies vaccine on detection of titer

ZHANG Yunchang

Chinese Journal of Biologicals.2024;37(6):656-659+665 . doi:10.13200/j.cnki.cjb.004247

Objective To study the effect of different strains of mice(KM and ICR)immunized with rabies vaccine on thedetection of titer.Methods The rabies vaccine and the national standard for the efficacy verification of rabies vaccine forhuman use(referred to as national standard)were diluted with PBS at the ratios of 1∶25,1∶125,1∶625 and 1∶3 125,and KM and ICR mice with half male and half female were immunized intraperitoneally respectively.Sixteen mice of each strain wereimmunized with 0.5 m L/mouse at each dilution.The immunization was strengthened once every one week at the same dose androute.The mice in each group were weighed 0,7 and 14 d after the initial immunization.After 14 d of the initial immunization,the mice were subjected to intracranial attack with rabies virus(RABV)CVS2(5-100 LD_(50)),0.03 m L/mouse.The numberof mice with death and typical rabies brain symptoms 5 d after the attack was counted.According to the national standard ED_(50),the relative efficacy was calculated by Reed-Muench method.Results The body mass of the two strains of miceshowed an increasing trend during the immunization stage,and the body mass of KM mice increased faster than that of ICRmice.The lgED_(50) values of the national standard in KM mice were all within the expected range of 2.10-2.75,while thevalues in ICR mice were higher than the range.The titers of rabies vaccine in KM mice were all significantly lower than thosein ICR mice(t = 2.887-6.619,each P < 0.05).Conclusion Mouse strains can significantly affect the results of rabies vaccine titer determination,and different standards should be adopted for different strains of mice to ensure the accuracy of vaccine detection results.

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Immunogenicity study of varicella-zoster virus Oka-7S strain

ZHANG Shujie

Chinese Journal of Biologicals.2024;37(6):660-665 . doi: 10.13200/j.cnki.cjb.004236

Objective To compare the immunogenicity of varicella-zoster virus(VZV)Oka-7S strain and VZV Oka strain.Methods Female BALB/c mice were subcutaneously immunized with VZV Oka-7S and Oka strains at high,medium and low doses(1 000,250 and 62.5 PFU/dose)for total two doses at an interval of 14 d,separately,with 12 mice in each group.The VZV-specific humoral immunity was evaluated based on fluorescent antibody to membrane antigen assay(FAMA),and the VZV-specific cellular immunity was evaluated by enzyme-linked immunospot assay(ELISPOT).Results Both VZV Oka-7S and VZV Oka strains induced specific humoral and cellular immune responses,and showed considerable immune effects;The level of anti-VZV specific IgG in serum of mice in Oka-7S-2.2 group increased significantly with the increase of immunization times and immunization doses.Conclusion The VZV Oka-7S strain had comparative immunogenicity with VZV Oka strain.

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Mechanism of neural tube defects caused by ethionine in mice

WANG Wenzhuo

Chinese Journal of Biologicals.2024;37(6):666-671. doi:10.13200/j.cnki.cjb.004043

Objective To prepare a neural tube defects(NTDs)model in mice with the structural analogue of methionineethionine,and investigate the effects of NTDs on the proliferation,apoptosis and migration of nerve cells.Methods Mouse NTDs embryo model was established.The C57BL/6J mice were injected intraperitoneally with 500 mg/kg ethionine after gestation to 7.5 d(E7.5),while the mice in control group were injected with the same dose of normal saline.Mouse embryos were taken at E11.5 and observed under stereomicroscope;The levels of S-adenosylhomocysteine(SAH)and Sadenosylmethionine(SAM)in plasma of pregnant mice were detected by ELISA;Western blot and RT-PCR were used to detect the expression of migration proteins E-cadherin and N-cadherin and the mRNA transcription in embryonic brain tissue.The expression of proliferation protein PCNA and apoptosis protein cleaved-Caspase3,and Wnt/β-catenin signaling pathway marker proteins β-catenin,TCF4 and C-myc were detected by Western blot.Results After the intervention of ethionine,the SAM content in pregnant mice decreased,the SAH content increased,and the SAM/SAH ratio decreased.Compared with the control group,the E-cadherin and cleaved-Caspase 3 were highly expressed and the apoptosis increased in NTDs group;The expression of N-cadherin and PCNA decreased,and the cell proliferation and migration decreased;The low expression of Wnt/β-catenin signaling pathway marker proteins β-catenin,TCF4 and C-myc indicated that the pathway was inhibited.Conclusion Ethionine may cause NTDs by promoting cell apoptosis via inhibiting Wnt/β-catenin signaling pathway and the proliferation and migration of nerve cells.

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Construction,expression,purification and quality identification of kanamycin resistant strain of recombinant human interleukin-1 receptor antagonist

YIN Tingting

Chinese Journal of Biologicals.2024;37(6):672-678+686 . doi: 10.13200/j.cnki.cjb.004242

Objective To construct a recombinant human interleukin-1 receptor antagonist(rhIL-1Ra)strain of kanamycin resistance,express,purify and identify rhIL-1Ra protein in order to reduce the risks of β-lactam antibiotics.Methods The ampicillin-resistant rhIL-1Ra(A-rhIL-1Ra)plasmid and PET-28a plasmid were used as templates to prepare the linearized vector and kanamycin gene fragment by PCR.The kanamycin resistant rhIL-1Ra plasmid(K-rhIL-1Ra)was constructed by homologous recombination,which was transformed into E.coli BL21(DE3)to construct the recombinant engineered bacteria after correct sequencing.The engineered bacteria were induced by IPTG and then purified by CM Bestarose Fast Flow and DEAE Bestarose Fast Flow column chromatography.The purified products were collected,and then detected for the purity by 15% SDS-PAGE,size-exclusion high-performance liquid chromatography(SEC-HPLC),and reversed-phase high-performance liquid chromatography(RP-HPLC),determined for the relative molecular mass by tandem mass spectrometry,identified for the specificity by Western blot,measured for the biological activity by reporter gene assay,and compared for the related impurities by liquid chromatography-mass spectrometry(LC-MS)analysis.Results Colony PCR and sequencing results showed that K-rhIL-1Ra plasmid was constructed correctly.The expressed K-rhIL-1Ra protein had a relative molecular mass of about 17 000,mainly existing in soluble form,and the expression amount accounted for more than 30% of the total bacterial proteins.The purity of K-rhIL-1Ra protein purified by two steps was 97% and 99%,the monomer content was 99.33%and 100%,and the chromatographic purity was 91.86% and 96.96%,respectively.The mass spectral molecular mass was consistent with that of the standard(A-rhIL-1Ra protein),and the protein reacted specifically relative with mouse antihuman IL-1Ra monoclonal antibody.The biological activity of the purified K-rhIL-1Ra protein was 1.29 × 105U/mg,and the chemical modification types of related proteins were the same as those of the standard(A-rhIL-1Ra protein).Conclusion The K-rhIL-1Ra strain was successfully constructed,and the expressed and purified protein was in line with the characteristics and quality standards of rhIL-1Ra,which lays a foundation for the study of rhIL-1Ra strain change and comparability

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Screening and evaluation of antigens for detection of antibodies against Mycobacterium avium subspecies paratuberculosis in naturally infected young sheep

SHEN Kefei

Chinese Journal of Biologicals.2024;37(6):679-686 . doi: 10.13200/j.cnki.cjb.004238

Objective To analyze the antibody responses of 10 serum reactive antigens(MAP1138c,MAP2121c,MAP0150c,MAP0862,MAP0209c,MAP2120c,MAP0038,MAP3420c,MAP 2154c and MAP2751)of Mycobacterium avium subspecies paratuberculosis(MAP)in naturally infected young sheep and evaluate their diagnostic value.Methods Serum samples and anal swab samples were collected from 6-month-old sheep without obvious PTB symptoms in the flocks with paratuberculosis(PTB)history.The serum samples were tested by PTB antibody detection ELISA kit,and anal swab samples were detected by the fluorescent quantitative PCR based on MAP F57 element.The sheep were grouped according to the test results.PCR was used to detect 10 MAP antigen genes in positive anal swabs.The antigen genes were cloned into pET-28a and induced to be expressed in E.coli BL21(DE3)strain by IPTG.The recombinant antigens were purified by Ni-Sepharose,and then coated on the ELISA plate for testing the collected serum samples to analyze the antibody reaction of the selected antigens in naturally infected sheep.The detection rates of serum antibodies against different antigens were analyzed to evaluate the diagnostic value of the antigens.Results The 72 sheep sampled were divided into three groups:anal swab positive-antibody positive(n = 34),anal swab positive-antibody negative(n = 23),and anal swab negative-antibody negative(n = 15).All 10 antigen genes were detected from positive anal swabs,and sequences of each gene were highly consistent.Through ELISA detection,MAP1138c,MAP2121c,MAP0150c and MAP0862 produced antibody reactions in infe-cted sheep.Antibodies against MAP1138c,MAP2121c,MAP0150c and MAP0862 were detected in 30,34,24 and 31 of the 57infected sheep,respectively.In the anal swab positive-antibody positive group,the detection rate of anti-MAP1138c antibody was the highest(76.47%).In the anal swab positive-antibody negative group,the detection rates of antibodies against MAP2121 and MAP0862(52.17% and 47.83%)were higher than those of the other two proteins.In the detection of 72serum samples,the overlap of ELISA coated with MAP2121c and MAP0862 exceeded 91%.Conclusion MAP1138c,MAP-2121c and MAP0862 may be dominant biomarkers to induce MAP antibody response in naturally infected young sheep.MAP2121c and MAP0862 can make up for the deficiency of sensitivity of commercial ELISA kits in early diagnosis of PTB.

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In vitro activity comparison between anti-human T lymphocyte porcine immunoglobulin prepared by Cohn ethanol fractionation and ammonium sulphate precipitation and same type of rabbit-derived products

YANG Fan

Chinese Journal of Biologicals.2024;37(6):687-695+702. doi: 10.13200/j.cnki.cjb.004243

Objective To compare the in vitro activity of anti-human T lymphocyte porcine immunoglobulin(P-ATG)prepared by Cohn ethanol fractionation and ammonium sulphate precipitation,commercial rabbit anti-human thymocyte immunoglobulin(trade name:Thymoglobuline),and anti-human T lymphocyte rabbit immunoglobulin(trade name:Grafalon)so as to evaluate the properties of P-ATG prepared by two processes.Methods The four products were detected for the antibody-dependent cell-mediated cytotoxicity(ADCC)by lactate dehydrogenase(LDH)method,measured for the complement dependent cytotoxicity(CDC)by CCK-8 assay,and detected for the affinity for binding with different T cell antigens(CD3,CD4,CD8)by double immunofluorescence staining method.Results Among the four products,only Thymoglobuline at the high concentration(1 mg/mL)had a weak ADCC effect on human peripheral blood mononuclear cell(PBMC).All products could induce the CDC effect in human PBMC in a dose-dependent manner,among which the effect of Thymoglobuline was higher than that of P-ATG or Grafalon preared by two processes,about 3 to 4 times,and the effect of Grafalon was comparable to that of P-ATG.In the proportion of four products binding with T cell surface antigens CD3 and CD4 was similar,However the proportion of Thymoglobuline and Grafalon binding with CD8 antigen was slightly lower than that of P-ATG.Conclusion The in vitro activity of the P-ATG prepared by Cohn ethanol fractionation and ammonium sulphate precipitation was in good agreement,and was not lower than that of imported products at the clinical dose.

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Surveillance of adverse events following immunization in Shaanxi Province,2010-2021

LV Yake

Chinese Journal of Biologicals.2024;37(6):696-702 . doi:10.13200/j.cnki.cjb.004241

Objective To analyze the surveillance quality and characteristics of adverse events following immunization(AEFIs)in Shaanxi Province,from 2010 to 2021.Methods The vaccine doses administered data and AEFI data reported from 2010 to 2021 in Shaanxi Province were collected through the National Immunization Program Information Management System and AEFI information management system,and descriptive epidemiological methods were used for analysis.Results A total of 24 057 AEFIs were reported from 2010 to 2021 in Shaanxi Province.The average annual reported incidence of AEFI in 2014-2021 was 18.998 per 100 000 doses,with 17.998,0.702,and 0.112 per 100 000 doses for common vaccine reactions,rare vaccine reactions,and serious rare vaccine reactions,respectively.The incidence of AEFI was high from May to August,with an average of 26.304 per 100 000 doses.There were 69.83% of the cases reported by the children younger than 1 year old.In Expanded Program on Immunization vaccines,the incidence of AEFI in DTaP(diphtheria,tetanus and acellular pertussis combined vaccine)was the highest(57.948 per 100 000 doses),and the incidence of rare vaccine reactions in MR,MMR and MM(measles and rubella vaccine,measles mumps and rubella vaccine,measles and mumps combined vaccine)was the highest(1.875 per 100 000 doses).In non-Expanded Program on Immunization vaccines,the incidence of AEFI in DTaP-Hib(diphtheria,tetanus and acellular pertussis,inactivated poliomyelitis and haemophilus influenzae type b conjugate combined vaccine)was the highest(37.073 per 100 000 doses),and the incidence of rare vaccine reactions in MPV-ACYW135(group A,C,Y and W123 meningococcal polysaccharide vaccine)was the highest(2.392 per 100 000 doses).In common vaccine reactions,the fever/local redness and swelling/local induration were the main clinical diagnosis(22.481 per 100 000 doses).In rare vaccine reactions,allergic rash was the main clinical diagnosis(0.473 per 100 000 doses).Conclusion The sensitivity of AEFI surveillance system in Shaanxi Province is increasing year by year,and the vaccines have good safety,with common vaccine reactions as the main ones and serious rare vaccine reactions as the rare ones.There were differences in AEFI surveillance levels in different prefecture-level cities,so it is still necessary to strengthen the training.

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