2020年08期目次

  • Thermal stability of imported rotavirus vaccine RotaTeq

    DU Jia-liang;LIU Yan;JIAO Yang;YU Qing-chuan;LIU Yue-yue;ZHAO Rong-rong;GAO Jia-mei;FAN Xing-liang;GUO Tai;National Institutes for Food and Drug Control;

    Objective To analyze the thermal stability of an imported rotavirus vaccine RotaTeq(RV5). Methods The virus titers of RV5 vaccine stored at 22 ～ 25 ℃ and 37 ℃ for various time durations were determined by real-time PCR,based on which the titers of vaccine strains of five types(G1,G2,G3,G4 and P1) and the total titer were calculated using the titer of reference vaccine as standard. The VP7 sequence of RV5 vaccine strain of type G3 was amplified and compared with that of prototypic strain r G3 in GenBank. Results The decrease rates of titers of vaccine strains of types G1,G2,G3,G4 and P1 and the total titer were 3. 51%,2. 26%,3. 19%,5. 22%,3. 18% and 3. 61% per day respectively at 25 ℃,which showed no significant difference between various types(P > 0. 05). However,at 37 ℃,the decrease rate of vaccine strain of type G3(31. 20% per hour) was significantly higher than those of other types(G1:0. 75%;G2:0. 31%;G4:1. 08%;P1:2. 07%)(P < 0. 05). The half life of titer of vaccine strain of type G3 at 37 ℃was only 0. 07 d,while that of its prototypic strain rG3 was 2. 10 d. The RV5 vaccine strain of type G3 was different from its prototypic strain in scattered amino acids(F32 L,T69 A,L83 F,I118 T,D151 G and P275 L)as well as nucleotides in5′ untransplated region(UTR)(T25 C) and 3′ UTR(G1041 A,A1045 G,G1046 A,G1047 A,C1048 T and A1053 T).Conclusion The RV5 vaccine strains of various types showed high thermal stability at 25 ℃. However,at 37 ℃,the low thermal stability of vaccine strain of type G3 showed significant impact on the thermal stability of RV5 vaccine by a possible mechanism of nucleotide mutation in VP7 UTR.

    2020 08 v.33 [Abstract][OnlineView][Download 681K]

  • Stability of types Ⅰ and Ⅲ oral live attenuated poliomyelitis vaccine(human diploid cells)in actual storage and transportation environment

    MA Ru-fei;FU Yu-ting;LI Guo-liang;SHI Hong-yuan;CHEN Shi-yi;ZHAO Yu-ping;LI Ying;WANG Jing-dong;CHEN Qiu-yu;SHI Xiao-jun;LIU Xi-ping;YANG Xiao-lei;YANG Jing-si;Institute of Medical Biology,Chinese Academy of Medical Sciences & Peking Union Medical College;

    Objective To investigate the stability of typesⅠand Ⅲ oral live attenuated poliomyelitis vaccine(bOPV)in liquid and sugar pill forms in storage and transportation and at different temperatures. Methods Three batches of liquid b OPV were tested for stability in transportation and after repeated freezing and thawing for 5 times according to the requirements for land transportation during which the course of opening the door for unloading and warehousing was simulated for 10 times as well as the requirements for air transportation. Meanwhile,three batches of bOPV sugar pills were tested for stability in transportation according to the requirements for land transportation during which the course of opening the door for unloading a nd warehousing was simulated for 10 times then stored at-20 ℃,from which samples were taken for determination of virus titer 6,12 and 18 months,and for overall control tests 24 month after storage. Six batches of liquid bOPV and six batches of bOPV sugar pills were stored at-20 ℃ for 36 months,at 2 ～ 8 ℃ for 12 months,at 25 ℃ for 4 weeks and at 37 ℃ for 8 d separately,from which samples were taken at various time points and determined for virus titer. The titers of bivalent virus and each virus type were determined by culture infective does 50%(CCID50) method to evaluate the stability of vaccine potency according to the internal requirements for preparation and control of vaccine by the manufacturers. Results All the quality indexes of liquid bOPV/bOPV sugar pills after transportation at 2 ～ 8 ℃ and of liquid bOPV after repeated freezing and thawing for 5 times and storage at-20 ℃ for 24 months met the requirements for quality control. However,the virus titer of vaccine after storage at-20 ℃ for 36 months,at 2 ～ 8 ℃ for 9 months,at 25 ℃ for one week and at 37 ℃ for 2 d met the internal requirements for preparation and control of vaccine by the manufacturers. Conclusion The bOPV showed good stability at normal conditions for storage and transportation,while repeated freezing and thawing showed limited impact on the stability of liquid bOPV. However,the temperature and time for storage showed effect at different degrees on the stability of bOPV. In actual storage and transportation process,long-term exposure to temperature deviation should be avoided.

    2020 08 v.33 [Abstract][OnlineView][Download 1538K]

  • Effect of moisture diffusion block on appearance collapse of freeze-dried vaccine in vials

    WANG Wei;YAN Jun-xi;GOU Peng;TAN Shen-wei;TIAN Li;GU Yong-jun;ZHANG Cai-li;SONG Xiang-rong;National Key Laboratory of Biotherapy,Sichuan University;

    Objective To investigate the effect of moisture diffusion block on appearance collapse of freeze-dried vaccine in vials. Methods Live attenuated Japanese encephalitis(JE)and groups A and C meningococcal polysaccharide vaccine at various filling quantities(0. 4,0. 6,0. 8,1. 0 and 1. 2 mg/vial)and frozen by rapid and slow freeze crystallization modes were lyophilized by LSC lyophilizer and observed for appearance collapse. JE vaccine was filled into vials,0. 6 m L for each,and lyophilized by LYO-20(CIP and SIP) lyophilizer,determined for residual moisture by pressure rise test and observed for the appearance collapse when the incubation times for primary drying were 13,14 and 15 h. Results Appearance collapses were observed in the both the vaccine lyophilized by rapid freeze crystallization at filling quantities of more than 0. 6 mL,of which the heights increased with the increasing filling quantity,while the heights of appearance without collapse were in agreement. Appearance collapse was also observed in groups A and C meningococcal polysaccharide vaccine lyophilized by slow freeze crystallization at larger filling quantities. However,appearance collapses were observed in JE vaccine at various filling quantities,of which the height increased with the increasing filling quantity.A little residual moisture was observed in JE vaccine after drying for 13,14 and 15 h,while no appearance collapse was observed after the vaccine was heated for desorption drying. Conclusion Moisture diffusion block is one of the factors causing appearance collapse of vaccine filled in vials.

    2020 08 v.33 [Abstract][OnlineView][Download 143K]

  • Genetic characteristics and differential analysis of Coxsackievirus A group 10 isolated from patients with hand,foot and mouth disease and herpes angina

    RAO Qing;ZHAO Yue-li;LI Ren-qiu;JIANG Hong-chao;ZHANG Zhen;SUN Qiang-ming;Children's Hospital Affiliated to Kunming Medical University;

    Objective To compare the genomic differences and genetic characteristics of Coxsackievirus group 10(CA10)isolated from the stool samples of patients with hand,foot and mouth disease and herpes angina and investigate the differences in clinical symptoms caused by gene mutation. Methods The stool samples of patients with HFMD and HA patients,which were positive for CA10 as proved by real-time quantitative PCR(RT-qPCR),were collected from Kunming Children′ s Hospital,then cultured in sensitive human rhabdomyoma(RD) cells,from which CA10 was isolated.Specific primers were designed,with which the whole gene sequence of CA10 was amplified. The nucleotide and amino acid sequences of CA10 gene isolated from the patients with HFMD and HA were compared by using BioEdit software. The CA10 sequence was downloaded from NCBI gene library and served as the reference sequence,and phylogenetic analysis was carried out to discuss the evolution and source of CA10 epidemic strain isolated in this study. Results The homology of nucleotides in VP1 regions of CA10 isolated from the patients with HFMD and HA was 99. 1%,while that of amino acids was 99. 8%,with two non-synonymous mutation sites. However,the homology of nucleotides of whole gene sequence was 98. 9%,while that of amino acids in coding region was 99. 5%,with 12 non-synonymous mutation sites. Phylogenetic analysis showed that both CA10 isolates were located in G gene lineage and were most closely related to KF999765(Beijing) and JX473455(Shenzhen) strains. Conclusion CA10 virus isolates from the patients with HFMD and HA showed high similarity at nucleotide and amino acid levels,which belonged to the same gene lineage. However,the amino acid variation caused by gene mutation may affect the virulence of virus and lead to the difference in patients′ symptoms.

    2020 08 v.33 [Abstract][OnlineView][Download 3275K]

  • Correlation of KRAS and NRAS gene mutations to MSI in colorectal cancer

    LIANG Jing;LI Ling-min;BAI Wen-qi;XI Yan-feng;BU Peng;GAO Ning;Department of Pathology,School of Basic Medical Sciences,Shanxi Medical University;

    Objective To investigate the correlation of KRAS and NRAS gene mutations to microsatellite instability(MSI) by determination of the mutations and MSI in colorectal cancer(CRC). Methods The expressions of four mismatch repair(MMR) proteins in 210 cases of CRC were determined by immunohistochemistry(IHC),while the distribution of MSI by fluorescence multiplex PCR-capillary electrophoresis,and the mutations of KRAS and NRAS genes in patients with CRC by amplification refractory mutation system(ARMS)-fluorescent PCR. Results The total deletion rate of MMR protein expression was 9. 5%(20/210),while the expression deletion rates of MLH1,PMS2,MSH2 and MSH6 were 6. 7%(14/210),6. 2%(13/210),1%(2/210) and 1. 9%(4/210) respectively. The incidences of high-frequency microsatellite instability(MSI-H)and low-frequency microsatellite instability(MSI-L)in 210 cases of CRC were 10. 5%(22/210) and 0. 5%(1/210) respectively. The consistency of IHC and fluorescence multiplex PCRcapillary electrophoresis in test for MSI was 96. 2%. The mutation rates of KRAS and NRAS genes in CRC were 46. 7%and 2. 9% respectively. MSI was associated with age,tumor location and clinical stage(P < 0. 05). KRAS gene mutation was associated with gender,clinical stage and distant metastasis(P < 0. 05). There was no apparent association between NRAS gene mutation and clinicopathological features(P > 0. 05). The mutation rate of KRAS gene in MSS CRC was higher than that in MSI CRC(P < 0. 05). Univariate survival analysis showed that KRAS gene mutation,clinical stage and distant metastasis were the poor prognostic factors of CRC(P < 0. 05). However,multivariate COX regression analysis showed that distant metastasis and KRAS gene mutation were independent prognostic risk factors for CRC.Conclusion MSI is negatively correlated with KRAS gene mutation in CRC. KRAS gene mutation is an independent prognostic risk factor for CRC;IHC and fluorescence multiplex PCR-capillary electrophoresis showed a high consistency in test for MSI.

    2020 08 v.33 [Abstract][OnlineView][Download 516K]

  • Prokaryotic expression and immunogenicity of HEV ORF2 truncated fusion protein

    ZHOU Yong-fei;QIAO Hong-lei;ZHAO Dan-ying;TANG Jian-guang;CHANG Dong-ying;HAN Shun-zi;CHANG Jun-liang;ZHANG Jian;LIU Yu-lin;CAO Yu-feng;Changchun Institute of Biological Products;

    Objective To express the truncated fusion protein HEV3-179-Fe of open reading frame 2(ORF2)of hepatitis E virus type 3(HEV3)in prokaryotic cells and determine its immunogenicity. Methods HEV3-179 and Fe gene fragments were amplified separately then linked and amplified by overlap PCR,and inserted into vector pET-28 a. The constructed recombinant plasmid pET-28 a-HEV179-Fe was transformed to competent E. coli BL21(DE3),and the positive colonies were screened and induced with IPTG. The expressed fusion protein HEV3-179-Fe was analyzed by SDS-PAGE,purified by Ni-Agarose affinity chromatography,added with ammonium sulphate for reconstruction of virus-like particles(VLPs),and observed by electron microscopy. The HEV3-179-Fe protein was adsorbed to aluminium hydroxide adjuvant and immunized to mice,100 μL for each,at weeks 0,2 and 4 respectively. The serum samples of mice were collected at week5 after the first immunization and determined for titer by ELISA to evoluate the immunognicity of truncated HEV ORF2 fusion protein. Results Colony PCR and sequencing confirmed that recombinant plasmid pET-28 a-HEV3-179-Fe was constructed correctly. The expressed recombinant protein,with a relative molecular mass of about 40 000,mainly existed in a form of inclusion body and contained more than 35% of total somatic protein,which reached a purity of about 95% after purification and showed specific reaction with mouse polyclonal antibody against HEV3. VLPs at diameters of about 20 nm were observed under electron microscope after reconstruction with ammonium sulphate. The antibody titer in sera of mice immunized reached more than 1 ∶ 4 800 000. Conclusion The HEV3-179-Fe were expressed in prokaryotic cells,which showed good immunogenicity. It laid a foundation of development of recombinant VLP vaccine based on HEV ORF2-179 protein.

    2020 08 v.33 [Abstract][OnlineView][Download 886K]

  • Prokaryotic expression and sequence analysis of LGALS1 gene in pigs

    CUI Yi-long;YANG Da-han;SHI Yun;FAN Pei-chao;XUE Jiang-dong;MA De-hui;College of Animal Science and Technology,Inner Mongolia University for Nationalities;

    Objective To express LGALS1 in prokaryotic cells and analyze its sequence. Methods The total RNA of porcine alveolar macrophages was extracted,with which the cDNA sequence of LGALS1 was amplified by RT-PCR and inserted into vector pMD18-T. The constructed recombinant plasmid pMD18-T-LGALS1 was sequenced,and the homology of target gene sequence was analyzed by bioanalytical software,based on which a phylogenetic tree was plotted. The correctly constructed plasmid pMD18-T-LGALS1 was subcloned into prokaryotic expression vector pET-30 a(+),and the obtained recombinant plasmid pET-30 a-LGALS1 was transformed to E. coli BL21(DE3) and induced with IPTG. The expressed product was purified by nickel ion affinity chromatography and Western blot. Results A 408 bp cDNA sequence was obtained by PCR amplification,of which the similarity was 99% to the LGALS1 coding region sequence(NM_001001867. 1) registered in GenBank. Nucleotide sequence analysis showed the highest homology of the cloned gene sequence to porcine LGALS1 gene. Plasmid pET-30 a-LGALS1 was confirmed to be correctly constructed by PCR and restriction analysis. The expressed LGALS1,with a relative molecular mass of about 21 000,showed reaction with LGALS1 antibody,indicating a reactogenicity. Conclusion Porcine LGALS1 was successfully expressed.

    2020 08 v.33 [Abstract][OnlineView][Download 1013K]

  • Prokaryotic expression and purification of recombinant C-terminal protein of human anti-Müllerian hormone

    LIN Li-shu;LONG Yun-hong;XU Li-hui;FENG Zheng-tong;XIE Chen-ying;CHEN Rui;WANG Ge-fei;Department of Clinical Laboratory,Second People′s Hospital of Futian District,Shenzhen City;

    Objective To express the C-terminal protein of human anti-Müllerian hormone(hAMH)in prokaryotic cells and purify the expressed product. Methods The gene encoding C-terminal protein of hAMH was amplified by PCR and inserted into vector pET-28 a. The constructed recombinant plasmid p ET-28 a-hAMH C was transformed to E. coli BL21(DE3)and induced with IPTG at a final concentration of 1. 0 mmol/L. The expressed protein was subjected to expanded culture,then purified by Ni affinity chromatography and analyzed by 12% SDS-PAGE. Results Colony PCR and sequencing proved that recombinant plasmid pET-28 a-hAMH C was constructed correctly. Target protein band with a relative molecular mass of about 12 500 was observed in both expressed and purified proteins,of which the purity reached more than 90%. Conclusion Recombinant hAMH C-terminal protein was expressed in prokaryotic cells,which reached a high purity after purification.

    2020 08 v.33 [Abstract][OnlineView][Download 361K]

  • Construction of eukaryotic expression vector for bovine infectious rhinotracheitis virus gD gene and its expression in MDBK cells

    JIA Xiao-xue;LIU Hui-zhen;ZHANG Fan;WU Wen-bo;MA Jun-yan;NI Hong-bo;College of Animal Science and Veterinary Medicine,Heilongjiang Bayi Agricultural University;

    Objective To construct a eukaryotic expression vector for bovine infectious rhinotracheitis virus(IBRV) gD gene express in MDBK cells. Methods According to the sequence of BHV-1-gD gene in GenBank,the target gene was synthesized with the primers to which Kpn Ⅰ/BSTB Ⅰ restriction sites were introduced upstream and downstream,and inserted into eukaryotic expression vector pcDNA4-myc-His. The constructed recombinant plasmid pcDNA4-gD-His was identified by restriction analysis and sequencing and transfected to MDBK cells. The culture supernatant was collected,from which the recombinant gD protein with His tag was purified by nickel ion column chromatography. The expressed and purified proteins were identified by indirect IFA and Dot blot. Results Restriction analysis and sequencing proved that recombinant plasmid pcDNA4-gD-His was constructed correctly. The expressed recombinant protein,with a relative molecular mass of about 61 000,reached a protein concentration of 0. 85 mg/m L after purification. Both the expressed and purified recombinant g D protein showed reactions with monoclonal antibody against IBRV-gD,indicating a good reactogenicity. Conclusion Eukaryotic expression plasmid pcDNA4-gD-His was successfully constructed and expressed in MDBK cell line,and the recombinant protein showed good reactogenicity.

    2020 08 v.33 [Abstract][OnlineView][Download 474K]

  • Effect of up-regulation of expression of thioredoxin interacting protein on senescence of islet β cells and relevant mechanism

    HUO Hai-yan;WANG Jin;ZHANG Xu-mei;ZHANG Wen-ting;YUE Ji-ping;JIAO Xiang-ying;Department of Physiology,Shanxi Medical University;

    Objective To investigate the effect of up-regulation of the expression of thioredoxin interacting protein(TXNIP)on senescence of islet β cells as well as the possible mechanism. Methods Two stably transfected INS-1 isletβ cells,Ad-TXNIP-GFP for overexpressing TXNIP and TXNIP-ShRNA for silencing TXNIP,were constructed,in which the expression levels of p16,p21,Rb and p53 proteins and the phosphorylation of p38 MAPK were determined by Western blot. The cells were treated with p38 MAPK inhibitor SB203580 and p35 inhibitor Pifithrin-β,and determined for the expression levels of above-mentioned proteins by Western blot. Results The over-expression of TXNIP promoted the expressions of p21,p16,Rb and p53 proteins,the phosphorylation of p38 MAPK(P < 0. 01)and the senescence of INS-1 cells. However,TXNIP silencing inhibited the expressions of p21,p16,Rb and p53 proteins,the phosphorylation of p38 MAPK(P < 0. 05) and the senescence of INS-1 cells. Both the inhibitors of p38 MAPK and p53 inhibited the expressions of p21 and Rb proteins(P < 0. 05)and relieved the cell senescence,while the p38 MAPK inhibitor inhibited the expression of p53 protein(P < 0. 05). However,the p53 inhibitor showed no influence on the phosphorylation of p38 MAPK and the expression of p16(P > 0. 05). Conclusion TXNIP promoted the phosphorylation of p38 MAPK and upregulated the expressions of p16 and p53,and then increased the expressions of p21 and Rb protein so as to induce the senescence of INS-1 cells.

    2020 08 v.33 [Abstract][OnlineView][Download 841K]

  • Effect of naringin on Caspase-3 activity and IRE1α expression in myocardial cells injured by hypoxia/reoxygenation

    LIU Dan;XIONG Shu;MA Ling;MA Li-hua;ZHANG Yong-hui;Chongqing Three Gorges Medical College;

    Objective To investigate the effect of naringin(Nar) on Caspase-3 activity and IRE1α expression level in myocardial cells injured by hypoxia/reoxygenation(H/R). Methods H9 c2 cell model of H/R injury was established.The cells were randomly divided into five groups:normal control(C),hypoxia/reoxygenation(H/R)as well as H/R +low,moderate and high dose Nar groups. The cells in C group were normally cultured until the end of the experiment,while those in H/R group were treated by hypoxia for 4 h followed by reoxygenation for 24 h. However,the cells in H/R + low,moderate and high dose Nar groups were cultured with 10,20 and 40 μg/mL Nar 6 h before hypoxia respectively,followed by reoxygenation for 24 h. The activities of Caspase-3 in various groups were determined by colorimetric method,while the expression levels of IRE1α by Western blot. Results Compared with those in C group,both the activity of Caspase-3 and the expression level of IRE1α in H/R group increased significantly(each P < 0. 05).However,compared with those in H/R and H/R + low dose Nar group,the Caspase-3 activities and IRE1α expression levels in H/R + moderate dose Nar and H/R + high dose Nar groups decreased significantly(each P < 0. 05).Conclusion The pretreatment with Nar may decrease the activity of Caspase-3 and down-regulate the expression level of IRE1α.

    2020 08 v.33 [Abstract][OnlineView][Download 264K]

  • Preparation and application of human papillomavirus type-16 L1 protein antibody

    LIU Chuan-zhi;BIAN Wen-zhi;LIANG Yu-ping;College of Life Science and Technology,Changchun University of Science and Technology;

    Objective To prepare the antibody against human papillomavirus type 16(HPV-16)L1 protein and verify the characteristics of monoclonal antibody(McAb) from mouse origin. Methods BALB/c mice and New Zealand white rabbits were immunized with HPV-16 capsid protein L1 to prepare the mouse McAb and rabbit serum as polyclonal antibody(PcAb)respectively,based on which a double antibody sandwich ELISA method was developed. The specificity and thermal stability of mouse McAb of the developed method were identified,while the accuracy was verified. Results The titer of prepared mouse McAb against HPV-16 was 5. 5 × 106 at most,while that of rabbit PcAb was 2. 25 × 107. The mouse McAb showed positive reaction with only inactivated HPV-16,while no cross reactions with inactivated HPV-18,HPV-52 or HPV-58. However,the antibody showed high stability after storage at 37 ℃ for 6 d. The deviation rate of detection result of inactivated HPV-16 at a concentration of 1 ～ 50 μg/mL with mouse McAb by ELISA was less than5%. Conclusion The mouse McAb detection of HPV-16 were successfully prepared,which showed high specificity and thermal stability. The developed double antibody sandwich ELISA method was accurate,which provided a possibility for early clinical screening of HPV virus and evaluation of curative effect.

    2020 08 v.33 [Abstract][OnlineView][Download 171K]

  • Mumps antibody level in healthy population at ages of 0～19 years in Tongzhou District,Beijing

    ZHAO Chun-yan;ZHANG Guo-feng;SHI Jing;ZHANG Jian-ming;SUN Yuan-jie;ZOU Lin;DENG Yan-chun;GAO Jie;WANG Feng-jun;Center for Disease Prevention and Control of Tongzhou District;

    Objective To determine the antibody level against mumps virus(MuV) in healthy population in Tongzhou District,Beijing and provide a scientific basis for prevention and control of mumps. Methods Healthy people at ages of0 ～ 19 years in 10 communities,who lived there for 6 months or more,were selected by randomly sampling and investigated by questionnaire,of whom the vaccination certificates of the peoples were checked up,the basic data and vaccination history were collected from Beijing Immunization Program Information Management System,and blood samples were collected and determined for IgG against MuV by ELISA. Results A total of 481 people were investigated,of whom the total positive rate of MuV IgG was 65. 70%(316/481),the median concentration of MuV IgG was 50. 02 IU/L.The positive rates of MuV-IgG in various age groups ranged from 4. 95% to 91. 2%,while the median concentrations from3. 51 to 109. 47 IU/L. The antibody level was the lowest in the age group of 0 ～ 8 months,while was the highest in the age group of 5 ～ 9 years,which showed increasing tendency at first then decreased. The antibody levels in various age groups showed significant difference(P < 0. 05),which was higher in the people with vaccination history than in that without vaccination history(P < 0. 05). Conclusion The mumps antibody level in the population at ages of 18 months ～15 years in Tongzhou District was high,so the people at ages of more than 15 years need to be paid more attention. A booster dose of mumps vaccine is recommended to the people at high school age.

    2020 08 v.33 [Abstract][OnlineView][Download 146K]

  • Rapid preparation and application of high titer neutralizing antiserum against SARS-Co V-2

    XU Kang-wei;WANG Jian-feng;QUAN Ya-ru;SHAO Ming;ZHAO Hui;LI Chang-gui;WANG Jun-zhi;Division of Respiratory Virus Vaccines,National Institutes for Food and Drug Control;

    Objective To prepare high titer neutralizing antiserum against SARS-CoV-2. Methods SPF rabbits were immunized with RBD protein of SARS-CoV-2,expressed in 293 cells,as an immunogen. The prepared antiserum was determined for titer by ELISA and microneutralization test,and distributed to several manufacturers for development of inactivated SARS-CoV-2 vaccine for determination of titer. Results After 3 times of immunization,the antiserum titer reached more than 1 600 000,while the neutralizing titer was 8 192. The geometric mean titer(GMT) determined by microneutralization test in five manufacturers was 6. 950. Conclusion High titer neutralizing antiserum against SARSCoV-2 was successfully prepared and distributed to several manufacturers of inactivated SARS-CoV-2 vaccine candidate for virus identification and adventitious virus agent test,which broke through the technical bottleneck restricting the rapid development of SARS-CoV-2 vaccine.

    2020 08 v.33 [Abstract][OnlineView][Download 185K]

  • Development of a microbiological method for determination of residual tetracycline content in Lispro Insulin Crystals

    MA Bu-fang;YANG Long-hua;YAO Shang-chen;CHANG Yan;National Institutes for Food and Drug Control;

    Objective To develop a microbiological method for quantitative determination of residual tetracycline content in Lispro Insulin Crystals. Methods The content of tetracycline in Lispro Insulin Crystals was determined by tube-disc method,based on which a standard curve was plotted with the logarithm of tetracycline concentration against the square of inhibition zone radius. The method was verified for homogeneity,minimum detection limit and accuracy,by which the residual tetracycline contents in three batches of materials of Lispro Insulin Crystals were determined. Results The accuracy of the method for quantitative determination of tetracycline residues was not influenced when the concentration of Lispro Insulin Crystals was 4 mg/mL,while was influenced by the matrix interference when the concentration was 10 mg/m L.The minimum limit of detection of the developed method was 0. 12 μg/mL,while the minimum limit of quantification was 0. 24 μg/mL. The recovery rates of standard tetracycline solution at concentrations of 0. 60,0. 84 and 0. 96 μg/mL were 95. 59%,98. 78% and 96. 17% when the matrix concentration was 4 mg/m L,while were 82. 55%,89. 38% and82. 30% when the matrix concentration was 10 mg/mL,respectively. No residual tetracycline was detected in three batches of materials of Lispro Insulin Crystals. Conclusion A microbiological method suitable for quantitative determination of residual tetracycline content in Lispro Insulin Crystals was developed,which showed high accuracy.

    2020 08 v.33 [Abstract][OnlineView][Download 191K]

  • Development and validation of a sandwich ELISA for residual host cell protein of CHO cells in recombinant human anti-TNFα monoclonal antibody

    DENG Chun-ping;MEI Xiong;CHEN Hang;HE Shui-hua;LIU Cui-hua;Bio-Thera Solutions,Ltd;

    Objective To develop and validate a sandwich ELISA for residual host cell proteins(HCP)of CHO cells in recombinant human anti-TNFα monoclonal antibody. Methods The coverage of product-specific CHO HCP population of recombinant human anti-TNFα monoclonal antibody recognized by rabbit(Cat. NO AB000103-A and AB000103-C) and goat(Cat. NO 3 G-0016-PA)anti-CHO HCPs polyclonal antibodies was determined by two-dimensional electrophoresis with Western blot(2 D-Western blot). The polyclonal antibody with the highest coverage was selected as the detection antibody,based on which a sandwich ELISA method for detection of residual HCP in product was developed and validated for linearity,matrix interference,dilution linearity,sensitivity,precision and accuracy. Results Rabbit anti-CHO HCP polyclonal antibody with a coverage of 57% was screened as the detection antibody. Since the matrix of samples interferred in the detemination of HCP,the samples needed to be diluted by 6 folds. The linear range of the developed sandwich ELISA was 3. 33 ～ 810 ng/mL(R2 = 1). Both the CVs in intra-and inter-assays were less than 12%,while the detection limit and quantitative limit in validation for sensitivity were 2. 4 and 20 ng/mL respectively. The recovery rates of spike samples at concentrations of 20,150 and 300 ng/mL were 99. 5%,97. 1% and 100. 3% respectively.Conclusion A sandwich ELISA method was successfully developed,which showed high sensitivity,accuracy,precision and good linearity,and was suitable for detection of residual HCP in recombinant human anti-TNFα monoclonal antibody.

    2020 08 v.33 [Abstract][OnlineView][Download 230K]

  • Characteristics of virus-like particles of human papilloma virus 18

    CAO Huan-huan;Xiamen Innovax Biotech Company,Ltd;

    Objective To analyze the characteristics of virus-like particles(VLPs)of human papilloma virus(HPV)18.Methods HPV18 VLPs were obtained by self-assembly of L1 protein expressed by recombinant E. coli expression system and analyzed for primary structure by mass spectrometry,for secondary structure by circular dichroism technique,and for tertiary and quaternary structures by high performance liquid chromatography,transmission electronic microscopy and dynamic light scattering technique. Meanwhile,the immunogenicity of HPV18 VLPs in animals was analyzed by pseudovirusbased neutralization assay,while the purity by SDS-PAGE. Results The post-translational modification of HPV18 VLPs mainly included oxidation and deamidation,while the relative molecular mass of intact L1 monomer was about 56 000.The proportions of helix,fold,angle and irregular coil in secondary structure were 8. 4%,46. 1%,18. 5% and 30. 2%respectively. The VLPs,with a purity of more than 99%,were full and intact in shape,even in size and at a diameter of about 60 nm,by which the GMT of neutralizing antibody induced in mice reached more than 3 000. However,the content of intact L1 monomer in HPV18 VLPs was more than 95%. Conclusion HPV-18 VLPs were intact in structure and even in size,which reached a high purity and showed good immunogenicity. It laid a foundation of evaluation on safety and effectiveness of cervical cancer vaccine.

    2020 08 v.33 [Abstract][OnlineView][Download 543K]

  • Liquid chromatography-mass spectrometry-based analysis of change of serum metabolite during development of chick embryo

    PENG Meng-ling;HU Wen-ye;WANG Ju-hua;ZHANG Su-zi;ZHOU Jie;College of Animal Science and Technology,Anhui Agricultural University;

    Objective To analyze the changes of serum metabolites during development of chick embryo by liquid chromatography-mass spectrometry(LC-MS). Methods A total of 120 fertilized eggs from Ross 308 hens were randomly divided into two groups,20 for each in triplicates. Serum samples were collected on day 14 after incubation(E14) and day 1 after being hatched(H1). LC-MS quadrapole time of flight system(LC/MS-QTOF) combined with principal component analysis(PCA),partial least squares-discriminate analysis(PLS-DA)and variable important for the projection(VIP)were applied to determine the differentially changed metabolites,and metabolite set enrichment analysis(MSEA)to analyze the pathways involved in. Results Thirty-eight serum metabolites showed significant changes on H1 as compared with those on E14,of which one increased significantly and 37 decreased significantly. The differential metabolites were mainly enriched in 22 metabolism pathways,of which L-threonine,L-leucine,L-isoleucine and Lmethionine were mainly enriched in protein synthesis pathway,while L-lactic acid,α-ketoglutarate and β-D-glucose in gluconeogenesis pathway,and L-isoleucine,L-leucine and lipoamide in degradation pathway of valine,leucine and isoleucine. However,all the contents of the differential metabolites enriched in various metabolic pathways on H1 decreased significantly as compared with those on E14. Conclusion The metabolites in sera on E14 and H1 showed significant difference,which showed a certain effect on the development of chick embryo. The metabolites may be used as the additives of daily diet of chicks starting from H1 so as to meet the requirement for growth of broilers,improve the production performance of broilers,save the protein forage and decrease the cost.

    2020 08 v.33 [Abstract][OnlineView][Download 532K]

  • Progress in research on action mechanism of LncRNA in Alzheimer disease

    LIU Hui;ZHANG Ya-lan;ZHANG Wei;WANG Man-xia;Department of Neurology,The Second Hospital of Lanzhou University;

    Alzheimer disease(AD)is a typical neurodegenerative disease,of which the course is progressive,and the main clinical manifestations are progressive memory impairment,cognitive impairment,personality change and language impairment. Pathological features of AD include extracellularβ-amyloid deposition, neurofibrillary tangles(NFTs),neuronal loss,synaptic and vegetative neuritis. However,the pathogenesis and molecular spectrum of the above pathological changes are still unclear,which brings great challenges to the early diagnosis and treatment of AD. Long noncoding RNA(LncRNA) is a type of RNA with a length of more than 200 nucleotide units,which is involved in the occurrence and development of many neurodegenerative diseases,such as Parkinson disease(PD) and AD. More and more studies have found a close relationship between LncRNAs and AD. Therefore,in order to further understand the role and mechanism of LncRNA in AD,this paper reviews the role of LncRNAs such as BACEL-AS,NEAT1,51 A,BC200 and EBF3-AS in AD,so as to provide a basis for early diagnosis and clinical treatment of AD.

    2020 08 v.33 [Abstract][OnlineView][Download 184K]

  • Progress in research on production procedure of virus-based biologicals

    YUAN Su-ying;TANG Rong-hong;YU Hong-wei;Tianjin Ringpu Biotechnology Co.,Ltd.;

    Viruses consist of protein envelope and internal genetic material(DNA or RNA),which are replicated themselves by using host cell system,without the capacity of growing and replicating independently. Viruses may infect almost all the organisms with cell structure,resulting in various diseases. At present,there are no specially effective drugs for most of diseases caused by virus infection,so vaccination is an effective measure to prevent the diseases. Isolation and purification of viral protein are the key technological steps of production of virus-based biologicals. This paper reviews the progress in research on virus-based biologicals and the production procedure of the biologicals applied successfully in the aspects of expression system,procedure development and test technique.

    2020 08 v.33 [Abstract][OnlineView][Download 201K]

  • Advances in research of γδT cells in therapy of cancer

    QIN Jie-chen;LIU Xin-yang;ZOU Cheng;School of Pharmaceutical Science & Yunnan Key Laboratory of Pharmacology for Natural Products,Kunming Medical University;

    The γδT cells are a subset of T cells and an innate lymphocyte that is not restricted by MHC,which secret a large quantity of cytokines and are involved in a variety of physiological and pathological processes in vivo,and play an important role in immune surveillance and immune defense,producing an effective cytotoxic response to cancer cells.The current clinical trials show good application prospect of γδT cells in anti-tumor therapy. This paper reviews the classification and structural characteristics,action mechanism,isolation and activation methods as well as clinical application of γδT cells.

    2020 08 v.33 [Abstract][OnlineView][Download 225K]

  • Progress in research on correlation of estrogen receptor signaling pathway with osteoporosis

    SHAO Jia-le;ZHOU Jian;LI Zhi-zhong;CHEN Ke-ming;Lanzhou University of Technology;

    Osteoporosis is a systemic disease due to the increased bone fragility caused by osteopenia and loose bone tissue structure,leaving the bones vulnerable to fracture. The disease is mainly correlated with amenorrhoea,life style,nutrition,abnormal hormone metabolism and inheritance. Estrogen receptor(ER)consisting of ERα and ERβ is contained in several parts of female body,which may bind to estrogen and form dimer complex to activate various downstream signal cascade reaction,thus plays an important regulatory role in bone formation and bone resorption of osteoclasts and influences the osteogenic differentiation ability. This paper reviews the correlation of ER signaling pathway with osteoporosis.

    2020 08 v.33 [Abstract][OnlineView][Download 176K]

  • Analysis on design and application of monocyte pyrogen assay

    HE Qing;GAO Hua;National Institutes for Food and Drug Control;

    Pyrogen detection is a key technology to ensure drug safety. With the development of innovative biotechnological drugs,rabbit pyrogen test and bacterial endotoxin test as traditional pyrogen tests to control the safety of those products are facing severe challenges. It is a trend to develop monocyte pyrogen assay instead of the above-mentioned tests. The monocyte pyrogen assay will be included into the Chinese Pharmacopoeia(Vol Ⅳ,2020 edition). This paper mainly introduces the design and application of monocyte pyrogen assay from the aspects of selection,design,materials,steps,test effectiveness and method variation,so as to provide a reference for the quality of relevant products by the method.

    2020 08 v.33 [Abstract][OnlineView][Download 170K]


@2012 Editorial Department of "Chinese Journal of Biologicals"