2020年12期目次

  • Immune protection of LTB-2xSTN12S fusion protein combined with cyclic guanosine monophosphate-adenosine monophosphate and double mutant heat-labile enterotoxin adjuvant in mice

    CHEN Jing;ZHANG Yan-bin;LIU Yu;WANG Xue-wei;ZHONG You-xiu;WANG Ping;LIU Mei-ying;National Vaccine & Serum Institute;

    Objective To express the fusion gene of heat-labile enterotoxin B subunit(LTB)and heat-stable enterotoxin mutant(STN12 S)of enterotoxigenic coli(ETEC)in prokaryotic system and preliminarily evaluate the protective effect and analyze the immune response of the protein combined with cyclic guanosine monophosphate-adenosine monophosphate(cGAMP)and double mutant heat-labile enterotoxin(dmLT)adjuvant in mice. Methods Expression vector pET28α-LTB-2 xSTN12 Swas constructed,transformed to E. coli BL21(DE3) and induced with IPTG. The expressed fusion protein was purified by nickel ion affinity chromatography. BALB/c mice were randomly divided into four groups. The mice in blank control group were untreated,while those in antigen group were immunized s. c. with purified LTB-2 xSTN12 Sprotein,and those in antigen + adjuvant group with the purified LTB-2 xSTN12 Sprotein in combination with dmLT and cGAMP,for 2 times at an interval of 2 weeks,and challenged i. n. with clinically isolated ETEC-HN strain(LT+/ST+)2 weeks after the last immunization. However,the mice in infection control group received no immunization before challenge. The mice in various groups were weighed 2 weeks after challenge,of which the venous blood and splenic lymphocytes were collected to analyze the humoral and cellular immune responses. Results Gene sequencing proved that recombinant plasmid p ET28-LTB-2 xSTN12 Swas constructed correctly. The purified recombinant LTB-2 xSTN12 Sprotein,with a relative molecular mass of about 20 000,reached a purity of 93% and showed specific reactions with polyclonal antibodies against cholera toxin(CT)and ST. The immune protection levels induced by purified LTB-2 xSTN12 Sprotein in combination with dmLT and cGAMP was higher than that in infection control group and that by purified LTB-2 xSTN12 Sprotein alone(each P < 0. 05),while the serum IgG antibody levels against LTB-2 xSTN12 Sas well as LTB and ST antigens were higher than those in other groups(each P < 0. 05). The IgG antibody levels against LTB-2 xSTN12 Sand LTB in culture supernatant of splenic lymphocytes were significantly higher(each P < 0. 01),while the number of cells secreting antigen-specific cytokines including IL-4,IL-6,IL-17 and IFNγ was larger(each P < 0. 05),than those in other groups. Conclusion LTB-2 xSTN12 Sfusion protein was successfully expressed by procaryotic expression system and purified,which showed good LTB and ST immunogenicity,provided protection against the challenge with ETEC(LT+/ST+)clinical isolate in combination with cGAMP and dmLT as adjuvant and induced significantly humoral and cellular immune responses.

    2020 12 v.33 [Abstract][OnlineView][Download 746K]

  • Analysis of consistence and components of various batches of bulks of freeze-dried rabies vaccine(Vero cells)for human use

    LI Chun-yan;ZHANG Rui;LIN Hong-juan;YANG Xue;MA Kun;LIU Yue;ZHENG Mei-hui;ZHU Bao-gang;XIE Hui;CHAI Bo-ya;Changchun Institute of Biological Products Co.,Ltd.;

    Objective To analyze the consistence and identify the components of various batches of bulks of human albumin-free freeze-dried rabies vaccine(Vero cells)for human use. Methods The contents of glycoprotein,total protein,residual DNA and endotoxin in four batches of bulks of human albumin-free freeze-dried rabies vaccine(Vero cells) for human use were determined. The purity of bulk was determined by SEC-HPLC. The component of peak 2 was alkylagted,enzymaticed,subjected to LTQ orbitrap velos mass spectrometry and analyzed by using Proteo Wizard software. Results The mean rabies virus glycoprotein content in the four batches of bulks was 31. 78%,with a relative deviation of 6. 44%,while the mean total protein content was 167. 04%,with a relative deviation of 2. 73%. However,all the residual DNA and endotoxin contents in the four batches were less than 200 pg/dose and less than 100 EU/mL respectively. The retention time of SEC-HPLC profile showed little change,on which the mean relative peak area of peak 1 was 93. 1%,with a relative deviation of 2. 5%,while that of peak 2 was 34. 0%,with a relative deviation of 6. 9%. The main components of peak 2 were lysate of rabies virus particles,bovine serum albumin,human serum albumin,as well as ubiquitin from Vero cells and poxvirus. Conclusion The bulks of human albumin-free freeze-dried rabies vaccine(Vero cells) for human use of various batches showed high consistence,and the components of peak 2 was successfully identified.

    2020 12 v.33 [Abstract][OnlineView][Download 244K]

  • Identification and analysis of nonstructural protein 1 gene of dengue virus type 2 isolated in Hunan Province,China in 2018

    FENG Shi-jun;LIU Yu;CHEN Jun;DUAN Su-qin;RAO Qing;CHEN Jun-ying;GUAN Jiao-qiong;Qiyang County People's Hospital Affiliated to Changsha Medical College;

    Objective To identify and analyze the nonstructural protein 1(NS1)gene of dengue virus type 2(DENV-2)isolated in Hunan Province,China in 2018. Methods RNA was extracted from sera of patients with dengue fever by kit,while the virus type was identified by RT-PCR,and the NS1 genes of 11 isolates were amplified and sequenced. The nucleotide and amino acid sequences of NS1 gene of the isolates and DENV-2 standard strain(KM204118)were analyzed by MEGA 7. 0 software,and the phylogenetic tree of NS1 gene was constructed by the maximum likelihood method(1000 boot strap replicate). Results The NS1 gene of 11 isolates were obtained. It was found that,except those of isolates HNN201804 and HNNS201810,the bases at site 585 of NS1 gene of the other isolates changed from T to C(nonsense mutation),and that at site 936 of isolated HNNS201806 from C to T(nonsense mutation). However,the other mutation sites of NS1 genes were identical in the 11 isolates,and there were 68 base mutations including 9 meaningful mutations.The 11 isolates were closely related to strains China-ZJ(2017-MH110594),China-GZ(2018-MK564485,2015-KX62-1245,2017-MK564483),Thailand(2017-LC410191),Singapore(2016-MF314189),and Philippines(2015-KU517-847). Conclusion All the 11 strains isolated in Hunan Province in 2018 were DENV-2,which might be imported from Zhejiang or Guangzhou,or from Thailand,Singapore,Philippines and other Southeast Asian countries.

    2020 12 v.33 [Abstract][OnlineView][Download 758K]

  • Inhibition and toxicity of a novel small peptide ωKWR to platelet aggregation

    KANG Zhi-ming;ZHAO Qi;ZHENG Jin-xiu;YANG Ting;YANG Li-jun;YANG Tao;Department of Biochemistry and Molecular Biology, Shanxi Medical University;

    Objective To investigate the inhibitory effect of a novel small peptide ωKWR on platelet aggregation and analyze its toxicity. Methods Blood was taken from ear marginal vein of rabbits,from which platelet-rich plasma(PRP)was isolated to explore the resistance of ωKWR on platelet aggregation in vitro. The antiplatelet aggregation of ωKWR in vivo was evaluated by pulmonary artery embolization experiment in mice. The effect of ωKWR on physiological hemostasis was evaluated via tail bleeding model test. The acute,teratogenic and mutagenic toxicities of ωKWR to mice as well as subacute toxicity to rats were analyzed by acute and subacute toxcity tests,mouse bone marrow micronucleus test and mouse sperm abnormality test. Results Platelet aggregation test in vitro showed that the maximum effective concentration of ωKWR was 40 μmol/L. Pulmonary embolism test confirmed good inhibitory effect of ωKWR on platelet aggregation in vivo. Tail bleeding time showed that ωKWR did not extend the physiological hemostasis time within the effective concentration range. Acute toxicity test showed no acute toxicity of ωKWR. Subacute toxicity test showed no significant effect of ωKWR on the organs and biochemical indicators of experimental animals. Mouse bone marrow micronucleus test and mouse sperm abnormality test showed no influence of ωKWR on reproductive system. Conclusion The novel antithrombotic peptide ωKWR may inhibit pathological platelet aggregation while has no effect on the physiological hemostasis. Toxicological experiments suggest high safety of ωKWR.

    2020 12 v.33 [Abstract][OnlineView][Download 2409K]

  • Secretory expression of hepatitis B virus core protein in Bacillus subtilis

    PING Fang;YANG Xun;JIN Jia-pei;CAI Xue-fei;Key Laboratory of Molecular Biology of Infectious Diseases of Ministry of Education,Chongqing Medical University;

    Objective To investigate the feasibility of secretory expression of hepatitis B virus core protein(HBc) in Bacillus subtilis. Methods HBc gene was amplified by PCR and inserted into the MCS region of pBE S-DNA shuttle plasmid. The constructed recombinant plasmid pBE S-DNA/HBc was linearized,ligated to the signal peptide(SP)DNA and transformed to competent E. coli Stellar cells. The positive colonies were collected,from which mixed plasmids were extracted and transformed to competent B. subtilis cells. The positive colonies for secretory expression of HBc protein were screened by ELISA,of which the one with the highest expression level was subjected to expansion culture. The culture supernatant was purified by affinity chromatography,and the expressed product was identified by SDS-PAGE and Western blot. Results Restriction analysis and sequencing proved that recombinant plasmid pBE S-DNA/HBc was constructed correctly. Of the 50 randomly selected recombinant B. subtilis strains,16 contained genes,including 4 ones in which HBc protein was expressed in culture supernatant. SDS-PAGE showed that recombinant HBc protein existed in monomer,dimer and polymer in supernatant. Western blot showed specific binding of recombinant HBc protein to HBc antibody.Conclusion Recombinant HBc protein with antigenic activity may be expressed in a secretory form in B. subtilis.

    2020 12 v.33 [Abstract][OnlineView][Download 1224K]

  • Stability of 146S antigen standard of inactivated foot-and-mouth disease virus particles

    XU Yuan;ZOU Xing-qi;WAN Jian-qing;WANG Zhao;LI Cui;HE Tian-ci;XIA Ying-ju;YANG Yan-li;SONG Yan-min;ZHANG Song-ping;ZHU Yuan-yuan;ZHAO Qi-zu;China Institute of Veterinary Drug Control;

    Objective To investigate the stability of 146 S antigen standard of inactivated foot-and-mouth disease virus(FMDV)particle. Methods Size-exclusion high-performance liquid chromatography(SE-HPLC)was used to determine the 146 S antigen content of the whole 146 S antigen standard of inactivated FMDV particle,monitor the content changes of the standard under various conditions for storage,including storage at-80 ℃ for 180 d,at 4 ℃ for 180 d,at 4 ℃ in oscillation for 0 ～ 7 d,at-80 ℃ for 0 ～ 150 d with 5 times of freezing-thawing with an interval of 30 d. The monitoring data were analyzed statistically by linear fitting method. Results The standard curve equation of the tracking 146 S antigen standard was acquired as y = 65 010 x-29 389,R2 = 0. 998 8. Analysis of the content changes of 146 S antigen standard of inactivated FMDV particle under various conditions for storage showed that the linear fitting equations after storage at-80 ℃ for 180 d,at 4 ℃ in oscillation for 0 ～ 7 d and at-80 ℃ for 150 d with 5 times of freezing-thawing were y =-0. 159 5 x + 63. 215,y =-0. 079 1 x + 63. 697 and y =-0. 635 7 x + 63. 881 respectively,all of which met the requirement of | β1| < t0. 95,( n-2)·s ( β1),so that the slopes were insignificant,indicating that the instability were not observed. However,the linear fitting equation of the standard after storage at 4 ℃ for 180 d was y =-2. 278 2 x +64. 801,of which the | β1| was more than t0. 95,(n-2)·s (β1),so that the slope was significant,indicating that the instability was observed. Conclusion The prepared 146 S antigen standard of inactivated FMDV particle met the needs for long-term storage and application after freezing-thawing at-80 ℃ and the needs for short-term refrigerated transport at 4 ℃. This study provides technical data for the development of 146 S antigen standard.

    2020 12 v.33 [Abstract][OnlineView][Download 399K]

  • Construction of lentivirus expression vector for UL27 and UL29 double genes of herpes simplex virus type 2 and its expression in HEK293 cells

    PAN Xiao-yu;LIN Jing-xian;HAN Jin-hui;ZHU Jun-fang;LUO Yan-na;Guangdong Food and Drug Vocational College;

    Objective To construct the lentivirus vector for coexpression of UL27 and UL29 genes of herpes simplex virus type 2(HSV-2) and determine its expression in HEK293 cells. Methods The optimal shRNA interference fragments to HSV-2 UL27 and UL29 genes were screened to construct a double gene co-expression lentivirus vector which was transfect to HEK293 cells. The untransfected HEK293 cells were served as blank group,while those transfected with lentivirus vector non-specific to any genes as negative control(NC). HSV-2 was inoculated 48 or 24 h after transfection,and the expression levels of target genes and proteins in cells of various groups were determined by RT-PCR and Western blot respectively. The survival rate of cells was determined by MTT assay. Results The inhibition rates of expressions of UL27 and UL29 mRNAs in test group were(78. 61 ± 2. 14)% and(84. 86 ± 1. 52)% respectively,while the relative expression levels of gB and ICP8 proteins decreased significantly(each P < 0. 05),and the survival rate of HEK293 cells increased significantly(each P < 0. 05). Conclusion The constructed recombinant lentivirus vector for coexpression of UL27 and UL29 double genes was successfully expressed in HEK293 cells,which laid an experimental foundation of further study on the gene therapy of HSV-2.

    2020 12 v.33 [Abstract][OnlineView][Download 884K]

  • Comparative study and analysis of advantages of multi-dose recombinant human interferon α2b injection in cartridges and freeze-dried preparation

    WANG Ying;LIU Yu-lin;ZHU Qiu-mei;SUN Rui-xin;LIU Ying;YU Lu;LI Ji-hong;FU Rui-li;ZHENG Quan-li;CHANG Jun-liang;LIU Jing-hui;Changchun Institute of Biological Products Co.,Ltd.;

    Objective To investigate the advantages of multi-dose recombinant human interferon(rhIFN)α2 b injection in cartridges by the comparison of dosage forms. Methods Three batches of multi-dose rhIFNα2 b injection in cartridges and three batches of commercial freeze-dried rhIFNα2 b for injection,either manufactured by Changchun Institute of Biological Products Co.,Ltd.,were stored at(25 ± 2) ℃,protected from light,from which samples were taken 0,1,2,3 and 6 months after storage,concentrated,pretreated by affinity chromatography,and then analyzed by SDS-PAGE,SECHPLC,mass spectrometry peptide mapping and mass spectrometry disulfide bond assay. The protein polymerization,degradation as well as kind and change trend of modification products of the two dosage forms were compared. The untreated samples were analyzed for biological activity to compared the change trends of effectiveness indexes of the two dosage forms. Results SDS-PAGE and SEC-HPLC showed visible protein dimers in both the dosage forms in the accelerating comparison research cycle. A variety of protein polymer was observed in freeze-dried preparation,while only a single kind of protein polymer in liquid preparation. The electrophoretic and chromatographic purities of freeze-dried preparation decreased more obviously than those of liquid preparation. Mass spectrometry showed that acetylated modified products,oxidized modified products and disulfide bond mismatch products were produced in both the dosage forms,of which the change trends were in agreement. However,the biological activities of the two dosage forms showed no significant decrease,while the stabilities were similar. Conclusion Less polymers were produced in the multi-dose rhIFNα2 b injection in cartridges,manufactured by Changchun Institute of Biological Products Co.,Ltd.,within 6 months after storage at(25 ± 2) ℃,while the protein purity decreased slowly,and the biological activity showed no decreasing trend,indicating the advantage of dosage form.

    2020 12 v.33 [Abstract][OnlineView][Download 1075K]

  • Preliminary screening of formula of human monoclonal antibody against tetanus toxin

    WANG Jian-feng;YE Xing;NAN Jian-jun;AN Chen;SONG Lan-lan;MAO Xiao-yan;Lanzhou Institute of Biological Products,Engineering Research Center of Macromolecules Biological Drugs of Gansu Province,Vaccinal Engineering Technology Research Center of Gansu Province;

    Objective To preliminarily screen the formula of human monoclonal antibody(McAb)against tetanus toxin.Methods Design of Experiment(DOE) was applied to 10 formula using PBS as the buffer system,polysorbate-80(Tween-80)as the surfactant,EDTA as the antioxidant at different pH values. Accelerated stability test was carried out at40 ℃(0,1,7 and 14 d). Optimal formula was preliminary determined by analyzing the changes of critical quality attributes of McAb drugs,including monomer purity by size-exclusive-high performance chromatography(SEC-HPLC)coupled with capillary electrophoresis-sodium dodecyl sulfate(CE-SDS),charge variation by capillary isoelectric focusing(cIEF)and antibody efficacy by animal experiment. Results High purities of McAbs of formula 1,2,9 and 10 were proved by SEC-HPLC,while those of formula 1,2 and 7 ～ 10 by CE-SDS. Analysis of charge variation by cIEF showed good protective effects of formula 2 and 9. However,animal experiment showed high antibody titers of formula 1,2 and7 ～ 10. Formula 2 and 9 were preliminarily determined as the optimal ones. Conclusion Two formula of McAbs against tetanus toxin were preliminarily screened,while Tween-80 concentration and pH value of solution might be the main influencing factors of antibody stability. This study provided a basis for further optimization of McAb drugs.

    2020 12 v.33 [Abstract][OnlineView][Download 668K]

  • Comparative study of HpaA-IgY and HP1188-IgY for prevention and treatment of Helicobacter pylori infection in mice

    HAN Fei;ZHANG Shuai-Shuai;Department of Medical Laboratory,Three Gorges Hospital of Chongqing University;

    Objective To compare the minimum concentrations of immunoglobulin yolk(IgY)against Helicobacter pylori adhesin A(HpaA)and H. pylori HP1188 in prevention and treatment of H. pylori infection in BALB/c mice and evaluate the curative effect. Methods Western blot was used to analyze the specificities of HpaA-IgY and HP1188-IgY antigens.H. pylori suspension at a concentration of 1 × 109 CFU/mL was prepared. BALB/c mice were randomly divided into various groups. The mice in positive control group were fed with 0. 5 mL H. pylori suspension,while those in negative control group with Brinell broth,those in prevention groups with 1,3 and 5 mg/mL HpaA-IgY,HP1188-IgY and PBS respectively,1 mL for each,followed by 0. 5 mL H. pylori suspension,and those in treatment groups with 0. 5 mL H. pylolri suspension followed by 1,3 and 5 mg/mL HpaA-IgY,HP1188-IgY and PBS respectively,1 mL for each. The gastric tissues of mice in various groups were subjected to H. pylori culture,rapid urease test(RUT) and histological examination to observe the H. pylori colonization and inflammatory reaction. Results Western blot showed specific binding of HpaA-IgY and HP1188-IgY to recombinant HpaA and HP1188 proteins respectively. Both the infection rates of mice in positive and negative control groups were 100%. The infection rate as well as scores for RUT,H. pylori colonization and inflammatory reaction in the mice prevented and treated with 5 mg/mL Hpa-IgY and 3 mg/mL HP1188-IgY decreased significantly as compared with those with PBS(each P < 0. 05),indicating satisfactory preventive and curative effects. Conclusion Both HpaA-IgY and HP1188-IgY showed preventive and curative effects on H. pylori infection in mice,while the later was superior to the former in dose selection.

    2020 12 v.33 [Abstract][OnlineView][Download 489K]

  • Development of national reference panel for detection kit for enterovirus 71 nucleic acids

    TIAN Ya-bin;ZHOU Hai-wei;LIU Dong-lai;SHEN Shu;XU Si-hong;ZHANG Chun-tao;Division Ⅱ of In Vitro Diagnostics for Infectious Diseases,National Institutes for Food and Drug Control;

    Objective To develop the national reference panel for detection kit for enterovirus 71(EV71)nucleic acids and apply to evaluate the kits of the same kind. Methods Various EV strains,including EV71 strains of different subtypes and Coxsackievirus strains of different types,were collected and cultured. After identification by sequencing,all the samples were tested and reconfirmed by commercial EV71 nucleic acid detection kits,then were filled and lyophilized to obtain the national reference panel. EV71 with genotype C4,the major circulating strain in China,was quantified by droplet digital PCR and used to evaluate the limit of detection of assays. The requirement for the national reference panel was developed by collaborative calibrations with the reagents from various manufacturers. Meanwhile,the stability of the national reference panel was evaluated. Results The national reference panel for EV71 nucleic acids consisted of six positive samples,eight negative samples and two samples for assessment of precision and the limit of detection respec-tively. The requirement for the national reference panel was as follows:the coincidence rate of positive samples was 6/6;the coincidence rate of positive samples was 8/8;the limit of detection was not more than 3. 6 × 103 copies/mL;all the10 repeat test results of the sample for assessment of precision were positive;the coefficient of variation(CV)of Ct value was less than 5. 0%(n = 10). The positive samples were stable after storage at room temperature for 1 d and after free-zing and thawing for 3 times. Conclusion A national reference panel was successfully developed for assessing the performance of detection kit for EV71 nucleic acids.

    2020 12 v.33 [Abstract][OnlineView][Download 406K]

  • Analysis and evaluation of 670 convalescent plasma samples from patients with COVID-19 infection

    WANG Yue;YU Jian-hong;YU Xue-hua;DUAN Kai;LIU Ying;HUANG Shi-he;GONG Qin;DENG Kun;HU Yong;XIE Yong;WU Xiao;LUO Yan;HAN Ren;ZHANG Lin-lin;ZHENG Xiao-bei;LI Pu;ZOU Xia;ZHANG Ya;HE Yan-lin;LI Ce-sheng;YANG Xiao-ming;Sinopharm Wuhan Plasma-derived Biotherapies Co.,Ltd;

    Objective To systematically analyze the 670 convalescent plasma(CP) samples from patients with coronavirus disease 2019(COVID-19). Methods The plasma samples were analyzed and evaluated for routine test items including hepatitis B virus surface antigen(HBsAg),hepatitis C virus(HCV)antibody,human immunodeficiency virus(HIV)-1/HIV-2 antibody,Treponema pallidum(TP)and alanine aminotransferase(ALT)as well as blood group,nucleic acid of severe acute respiratory syndrome coronavirus 2(SARS-CoV-2),IgG antibody,methylene blue residue and sterility. Results A total of 121 substandard plasma samples were detected from 670 convalescent plasma samples,of which substandard IgG antibody titer accounted for the highest proportion of 7. 91%. In the turn of proportions,the blood groups were A(32. 52%),B(29. 94%),O(28. 886%)and AB(8. 66%). All the test results of nucleic acids of SARSCoV-2 were negative. A total of 485 samples were from Wuhan,of which the highest proportion(21. 95%)were from the donors at ages of > 30 ～ 35 years,including 264 males and 221 females. Of the high titer plasma,those at titers of not less than 1 ∶ 640 accounted for the highest proportion(77. 43%). Most of the IgG titers in plasma of common patients were not less than 1 ∶ 640 > 10 ～ 20 d,while were less than 1 ∶ 160 3 ～ 10 d,after hospitalization. However,35 plasma samples were negative for IgG antibody(at titers of less than 1 ∶ 80),in 9 of which other pathogens were detected.Conclusion Unqualified IgG titer was the main reason for unqualified CP. The proportion of CP of group O was lower than that of the group in healthy population. The highest proportion of plasma donors in Wuhan was in the populations at ages of > 30 ～ 35 years,which was higher in males than in females. Satisfactory immune responses were induced in most of patients in convalescence period,which removed the virus in vivo effectively. High antibody titers were induced > 10 ～ 20 d after hospitalization,making the common cases were not easy to change into severe ones. It was speculated that patients negative for IgG antibody might be infected with other pathogens.

    2020 12 v.33 [Abstract][OnlineView][Download 598K]

  • Applicability of multiplex PCR for serogrouping of Neisseria meningitidis

    ZHANG Li-zhi;BAI Gui-jie;YANG Ying-ying;REN Ke-ming;LIU Fang-lei;LUO Shu-quan;SHEN Rong;First Department of Research,Lanzhou Institute of Biological Products Co.,Ltd.,Center for Gansu Provincial Vaccine Engineering Research;

    Objective To identify five serogroups of Neisseria meningitides(Nm),A,B,C,W135 and Y,by multiplex PCR,verify the applicability of the method for identification of serogroups of bacterial strains for vaccines at gene level and compare the result with those by classical laboratory bacterial culture and serum agglutination test. Methods The genomic DNAs of standard Nm strains of serogroups A,B,C,W135 and Y were extracted,based on which the target gene fragments were amplified by multiplex PCR according to the specific sequence of synthetic capsular polysaccharide genes of five serogroups. The serogroups of Nm strains were distinguished according to the size of base fragments of PCR products. The serogroups of seven Nm isolates were identified by PCR,and the results were compared with those by classical laboratory bacterial culture and serum agglutination test. Results Multiplex PCR was able to be used for the identification and serogrouping of Nm,which showed high specificity and reproducibility,with a minimum detection limit of 0. 1 ng of genomic DNA. All the test results of seven Nm isolates were consistent with those by classical laboratory bacterial culture and serum agglutination test. One of the seven strains was of serotype A,while one was of serotype B,two were of serotype C,one was of serotype W135,and two were of serotype Y. Conclusion The specificity and sensitivity of multiplex PCR for detection of Nm were higher than those of classical laboratory bacterial culture and serum agglutination test. The species-and serogroup-specific genes of Nm strains were detected at the same time,which improved the accuracy of serogrouping. In addition,the test results were objective,persistent and traceable,which was beneficial to the quality control of vaccine strains.

    2020 12 v.33 [Abstract][OnlineView][Download 1848K]

  • Development and validation of liquid chromatography tandem mass spectrometry for quantitative analysis of tracheal cytotoxin in combined vaccines based on DTacP

    WEI Chen;WU Yan;LONG Zhen;HUANG Yuan-li;WANG Li-chan;MA Xiao;National Institutes for Food and Drug Control,Key Laboratory of the Ministry of Health for Research on Quality and Standardization of Biotech Products;

    Objective To develop,validate and preliminarily apply a liquid chromatography tandem mass spectrometry(LC-MS/MS)method for quantitative analysis of tracheal cytotoxin(TCT)of B. pertussis in combined vaccine based on DTacP. Methods The condition for HPLC was as follows:BioC18 chromatographic column was adopted,serving 0. 1%(V/V)formic acid-aqueous solution as mobile phase A and 0. 1%(V/V)formic acid-acetonitrile solution as mobile phase B. The sample load was 5 μL,while the column temperature was 30 ℃,and the flow rate was 0. 3 mL/min.However,the condition for MS was as follows:positron mode with electrospray ion source was adopted. The voltage was3. 0 kV,while the temperatures of ion source and desolvent tube were 300 and 250 ℃,and the flow rates of atomizer and dry airflow were 3 and 10 L/min,respectively. The TCT was determined by multi reaction monitoring mode(MRM). The developed method was validated for linear range, sensitivity, precision and accuracy, and used for screening and quantitative analysis of TCT in 33 batches of final product,63 batches of bulk and 80 batches of purified protein of domestic and imported DTacP. Results The concentration of TCT standard at a range of 20. 66 ～ 661 ng/L showed good linear relationship to the peak area of quantitativeion channel 922. 3 > 719. 3,with a linear equation of Y = 3 555. 9 X-3 368 and a R2 value of 0. 996. The detection limit and quantitative limit of the developed method were 2. 58 and 10. 33 ng/L respectively. The RSDs of determination results of peak areas of TCT standard solutions at concentrations of 20. 65,82. 625 and 330. 5 ng/L in five tests were 2. 23%,4. 61% and 4. 40%,while the recovery rates of spike samples at concentrations of 66. 1 and 33. 05 ng/L were 104. 1% and 94. 4%,respectively. No TCT was detected in all the final products and bulks of DTacP,while only 65. 5 ng/L TCT was detected in the lysed bacterial concentrate during the purification of one batch of PRN protein from one manufacturer. However,no TCT was detected in the subsequent process of the protein. Conclusion The LC-MS/MS method for quantitative analysis of TCT in combined vaccine based on DTacP was successfully developed,which showed high sensitivity,accuracy,precision and linearity.

    2020 12 v.33 [Abstract][OnlineView][Download 376K]

  • Development and validation of real-time fluorescent quantitative PCR method for determination of residual host DNA in recombinant norovirus vaccine

    JIANG Ying;LEI Qing;ZHANG Qiao-ling;CHEN Ji-jun;YANG Jun-jie;The Fourth Department of Research,Lanzhou Institute of Biological Products;

    Objective To develop and validate a real-time fluorescent quantitative PCR(qPCR)method for determination of residual host DNA in recombinant norovirus vaccine expressed in Hansenula polymorpha. Methods Genomic DNA of H. polymorpha was extracted with magnetic beads and used as a DNA reference,and the standard curve for qPCR was established and validated for specificity,linearity,accuracy,precision and durability. Results Specify amplification curve of qPCR was obtained by using H. polymorpha DNA rather than the controls(water for injection,buffer as well as DNAs from culture supernatant of Vero cells and CHO cells). All the correlation coefficient(R2)in 5 validation tests for linearity were more than 0. 98. The recovery rates of spiked sample at various concentrations were 70% ～ 130% in accuracy validation test. The RSDs in validation tests for reproducibility and intermediate precision were less than 30%.However,in the durability test,the RSD of determination results of samples digested with Proteinase K at various temperatures was 20. 75%,while that of samples at various protein concentrations was 27. 11%,which met the requirements in Chinese Pharmacopoeia(Volume Ⅲ,2015 edition). Conclusion The specificity,linearity,precision,accuracy and durability of the developed could met the relevant requirements,indicating that the method may be used for the determination of residual DNA in norovirus VLP vaccine.

    2020 12 v.33 [Abstract][OnlineView][Download 355K]

  • Application of handheld Raman spectrometry in control tests on excipients for production of live attenuated Japanese encephalitis vaccine

    XIE Shan-shan;LI Xin;ZHU Xiao-yan;ZHANG Du-juan;ZHAO Qing;WANG Wei;Quality Control Department,Chengdu Institute of Biological Products Co.,Ltd.;

    Objective To analyze the feasibility of rapid identification of excipients for the production of live attenuated Japanese encephalitis vaccine(JEV)by Raman spectroscopy. Methods Raman spectrometer was used for scanning the excipients(sucrose,lactose,urea,potassium dihydrogen phosphate and disodium hydrogen phosphate dodecahydrate)for production of live attenuated JEV. According to the multiple acquisition of different parts of the excipients, the corresponding Raman spectrum method and the library were established. The excipients were used for verification test.The materials with similar chemical structure to that of excipients for production of live attenuated JEV,such as glucose and disodium hydrogen phosphate dehydrate,were selected for verification of specificity. Results No deviation was found in identification of the excipients for production of live attenuated JEV by using handheld Raman spectrometer. The spectrum of test sample was consistent with that in standard spectrum library,while the identity test result was consistent with that of chemical identification in Chinese Pharmacopoeia(Vol Ⅳ,2015 edition). However,when the materials with similar chemical structure to that of the excipients were tested,the system presented the message "Failed",indicating high accuracy,specificity and reliability of the method developed based on Raman spectrometer. Conclusion Raman spectroscopy meets the relevant requirements,which may be used for the rapid identification of excipients for production of live attenuated JEV.

    2020 12 v.33 [Abstract][OnlineView][Download 716K]

  • Comparative analysis of PCR and method in Chinese Pharmacopoeia for mycoplasma examination

    ZHANG Li-jun;YU Jia;WEI Qin-qin;SHI Yan;WEI Ran;LIU Xiao-fan;Quality Assurance Department,Lanzhou Institute of Biological Products Co.,Ltd.,Center for Gansu Provincial Vaccine Engineering Research;

    Objective To investigate the feasibility of PCR technique used for mycoplasma examination in the vaccine production process. Methods GP03 and MGSO were selected as primers,of which the sequences were compared with the 16 s r RNA sequences of M. pneumonia,M. orale and M. hyorhinis specified in Chinese Phamacopoeia(Volume Ⅲ,2015 edition) for mycloplasma examination. The 16 s r RNA sequences of three mycoplasma positive strains were amplified by PCR using the two primers to determine the primer suitability. Concentration gradient test and amplification test of dif-ferent passages of mycoplasma were carried out using the mycoplasma positive strains as templates to confirm the detection limit and precision of PCR method respectively. Twenty cell cultures in the production process of oral live attenuated ro-tavirus vaccine were detected by PCR method,liquid culture method and solid culture method respectively,and the results were compared. Results GP03 and MGSO were suitable for mycoplasma examination. The detection result by PCR method was consistent with those by liquid and solid culture methods. However,PCR method was rapid and easy to handle. Conclusion PCR may be used as a method for mycoplasma examination in vaccine production.

    2020 12 v.33 [Abstract][OnlineView][Download 1022K]

  • Evaluation of effectiveness of purification process of human albumin by capillary electrophoresis

    LV Xing-kai;WANG Yin;REN Ke-yun;HU Shang-jie;HE Kun;CHEN Ai-fang;ZHANG Fan;Wuhan Optics Valley Humanwell Biological Medicine Co.LTD;

    Objective To evaluate the effectiveness of purification process of human albumin by capillary electrophoresis. Methods Human plasma was purified by Blue Bestarose FF affinity chromatography,EzFast Phenyl FF hydrophobic chromatography,EzFast CM FF cation exchange chromatography and EzFast DEAE FF anion exchange chromatography. The purities of commercial human albumin as well as the purified products in various steps were determined by capillary electrophoresis,based on which the recoveries were calculated. Results The commercial human albumin consisted for dimer,monomer and polymer,of which the purity was 96. 6%,and the time of appearance of peak was 19. 68 min. The times of appearance of peaks of affinity,hydrophobic,cation exchange and anion exchange chromatography were 19. 667,19. 867,19. 867 and 20. 075 min respectively,which were basically consistent with those of commercial human albumin. However,the purities of purified products in the four steps were 90. 0%,93. 4%,99. 9%and 99. 9%,while recoveries were 49. 59%,35. 49%,24. 12% and 17. 56%,respectively. Conclusion The purification process of human albumin was effective,while the obtained albumin showed high purity but low recovery.

    2020 12 v.33 [Abstract][OnlineView][Download 362K]

  • Progress in research on polyrimidine tract binding protein 1gene and relevant diseases

    CHEN Yun-cheng;ZENG Qiong-yao;ZHANG Wen-jing;College of Medical Science,Kunming University of Science and Technology;

    Polyrimidine tract binding protein 1(PTBP1) is an alternative splicing protein which can produce diverse mRNA through selectively splicing pre-mRNA to mediate transcriptional regulation of multi-genes,thus plays an important role in the survival,proliferation and metastasis of tumor cells. Both the expression level and biological function of PTBP1 are different in various types of malignant tumors,of which one of the mechanisms may lie in the different binding interaction proteins. In addition,PTBP1 is related to neurodegenerative diseases and hypertension,and is also involved in immune system and cell senescence. This paper reviews the structure and function of PTBP1 as well as its relationship to the diseases.

    2020 12 v.33 [Abstract][OnlineView][Download 197K]

  • Role of stem cells in depression therapy

    JIANG Ru-ru;LI Xin;HA Xiao-qin;Clinical Medical College,Gansu University of Chinese Medicine;

    Depression is a common mental disorder worldwide. Psychological treatments and antidepressant medication are the usual therapies for depression. However, since most of the patients are resistant to treatments at the late stage of therapy,it is urgent to develop a novel method for depression therapy. In recent years,with the wide application of stem cells in establishment of disease model and cell therapy,the technology of mimicking human neural system in vitro by neural stem cells(NSCs) and induced pluripotent stem cells(i PSCs) is more and more mature,which allows a better understanding of depression in pathogenesis,pathophysiology as well as the mechanism of antidepressant medicines. This paper reviews the pathogenic mechanism of depression as well as the strategies for depression modelling and cell therapy by NSCs and i PSCs,so as to provide a new understanding of antidepressant treatment.

    2020 12 v.33 [Abstract][OnlineView][Download 190K]

  • Progress in research on insect cell-baculovirus expression system

    ZHANG Yi-chi;LI Yuan-yuan;Department 7 of Research,National Vaccine & Serum Institute;

    Baculovirus is an arthropod-specific large circular double-stranded DNA virus,of which the genes are in close array. It plays an important role in pest control because of its high specificity. In 1983,the first recombinant baculovirus vector was constructed,and interferon β protein was successfully expressed. Afterwards,baculovirus expression vector(BEV)system is continuously optimized in the production of recombinant virus as well as expression and improvement of stability,yield and post-translational modification of recombinant protein. Insect cell-baculovirus expression system has become one of the four routine expression systems. This article mainly introduces the novel methods and key elements for optimization of BEV system.

    2020 12 v.33 [Abstract][OnlineView][Download 256K]

  • Progress in research of biological drugs and advanced therapies for rheumatoid arthritis

    ZHAO Jing;PAN Zhu-ping;Center for Drug Evaluation,National Medical Products Administration;

    Rheumatoid arthritis(RA)is a serious chronic systemic autoimmune inflammatory disease with high disability rate,which will bring huge economic burden to patients and their families. With the expansion of clinical application,the side effects of the current drugs,such as infection and cancer incidence,are gradually exposed. In addition,some patients have no response or no continuous response to the drugs. Therefore,the development of drugs against RA has been one of the obstacles in research of drugs against autoimmune-related diseases. This paper mainly reviews the action mechanism and research progresses of biological drugs targeting cytokines and immune cells and innovative therapies so as to provide ideas for the treatment of RA and drug development in the future.

    2020 12 v.33 [Abstract][OnlineView][Download 223K]


@2012 Editorial Department of "Chinese Journal of Biologicals"