2020年09期目次

  • Immunogenicity of recombinant hepatitis B vaccine for nasal spraying in mice

    SHAN Pu;DENG Xia;WANG Zhi-biao;ZHANG Yu;ZHAO Jing;WU Jian-hua;XU Jing;National Vaccine and Serum Institute;

    Objective To evaluate the immunogenicity of recombinant hepatitis B(HB) vaccine for nasal spraying in mice. Methods Mice were immunized with aluminum adjuvant-containing HB vaccine by intramuscular injection and with recombinant HB vaccine for nasal spraying,at various dosages,respectively,and determined for anti-HBs level in sera by ELISA and for IL-2 and IFNγ levels in splenocytes by ELISPOT. Mice were immunized with recombinant HB vaccine for nasal spraying at various dosages,and determined for sIgA titer in lungs and genital tract washes by indirect ELISA. Mice were immunized with aluminum adjuvant-containing HB vaccine by intramuscular injection in combination with recombinant HB vaccine for nasal spraying or with the vaccine for intramuscular injection alone,and the effects of various immunization schedules(intramuscular injection followed by nasal spraying and nasal spraying followed by intramuscular injection)on the anti-HBs level in sera and IL-2 and IFNγ levels in splenocytes,as well as the persistence of anti-HBs level induced by intramuscular injection followed by nasal spraying,were investigated. Results The anti-HBs level induced by recombinant HB vaccine for nasal spraying was significantly lower than that by the vaccine for in-tramuscular injection at the same dosage(P < 0. 001). However,the recombinant HB vaccine for nasal spraying at a 4-fold increased dosage induced the anti-HBs level equivalent to that by intramuscular injection. The vaccine for nasal spraying also induced a certain sIgA level in genital tract mucosa. Compared with that induced with aluminum adjuvant-containing HB vaccine by intramuscular injection alone,the anti-HBs level induced with the vaccine by intramuscular injection followed by nasal spraying showed no significant difference(P > 0. 05),while the IL-2 and IFNγ levels in splenocytes increased significantly(P < 0. 001),and the high anti-HBs level was maintained for at least 12 weeks. Conclusion The recombinant HB vaccine for nasal spraying showed good immunogenicity, which induced high anti-HBs level in combination with aluminum adjuvant-HB vaccine,enhanced the cellular immunity and showed immune persistence.

    2020 09 v.33 [Abstract][OnlineView][Download 518K]

  • Construction and immunogenicity of recombinant Mycobacterium smegmatis expressing neutralizing epitope fusion gene of enterovirus 71

    LIANG Si-jia;WU Yue;SHI Chun-wei;LIU Hong-bo;Department of Clinical Laboratory,Second Affiliated Hospital,Guilin Medical College;

    Objective To construct a recombinant Mycobacterium smegmatis(MS)for expression of a fusion gene HBcE1/2 consisting of chimeric VP1-SP70(E1)and VP2-141(E2)neutralizing epitopes of enterovirus(EV)71 and evaluate its immunogenicity in mice. Methods HBc-E1/2 gene was amplified by PCR and cloned into E. coli-Mycobacterium shuttle plasmid pMV261. The constructed recombinant plasmid pMV261-HBc-E1/2 was transformed to mc2155,a mutant of wild MS ATCC607 strain,by electroporation,and the obtained recombinant bacterial strain pMV261-HBc-E1/2/mc2155 was inoculated i.p. into BALB/c mice. The serum antibody titer of mice was determined by indirect ELISA. Results Restriction analysis and sequencing proved that recombinant plasmid pMV261-HBc-E1/2 was constructed correctly. The expressed product showed specific binding to the sera of mice immunized with E1 and E2 polypeptides and inactivated EV71 vaccine,and showed specific binding bands with relative molecular masses of about 27 500 and 55 000. Both the titers of anti-E1 and E2 epitope antibodies in the sera of immunized mice were 1 ∶ 1 600. Conclusion A recombinant MS vaccine presenting HBc-E1/2 was successfully constructed,which induced specific antibody against EV71 in mice. It laid a foundation of the development of a novel vaccine against hand,foot and mouth disease(HFMD).

    2020 09 v.33 [Abstract][OnlineView][Download 710K]

  • Compatibility of inner packaging materials and 23-valent pneumococcal polysaccharide vaccine

    TIAN Ji-zhao;YANG Wen-jie;ZHU Jian-guo;GUO Ying;ZHUO Jin-rong;Chengdu Institute of Biological Products;

    Objective To investigate the compatibility of inner packaging materials(glass container and rubber stopper)and 23-valent pneumococcal polysaccharide vaccine. Methods Under the mandatory experimental condition,the extracts of vials made of borosilicate glass and stoppers made of halogenated butyl rubber were prepared with three batches of 23-valent pneumococcal polysaccharide vaccine. The contents of harmful metal ions(arsenic,antimony,lead and cadmium)in extract of vials were determined by inductively coupled plasma optical emission spectrometer(ICP-OES),while the 2,6-tert-butyl-4-methylphenol(BHT)content in extract of stopper by HPLC. The change of inner surface of flaking of vials was observed by scanning electron microscopy. Results The contents of arsenic,antimony,lead and cadmium ions in three batches of extracts of vials were lower than the corresponding limits of detection,while were far lower than the permitted daily exposure(PDE)values. No BHT was detected in the three batches of extracts of stoppers,indicating that the BHT content was far lower than the PDE. No pit or erosion was observed on the inner surface of flaking of vials.Conclusion The vials and stoppers showed high safety and good compatibility with 23-valent pneumococcal polysaccharide vaccine.

    2020 09 v.33 [Abstract][OnlineView][Download 740K]

  • Isolation,identification and G gene sequence analysis of rabies from the first suspected patient in Fujian Province,China in 2018

    MENG Sheng-li;LI Qian;YAN Chang-hu;ZHUANG Hong-zhi;MAI Jian-yi;WU Jie;WANG Wen-hui;WANG Ze-jun;National Engineering Technology Research Center for Combined Vaccines, Wuhan Institute of Biological Products Co.Ltd;

    Objective To isolate and identify rabies virus from the first suspected patient in Jinjiang Ciy,Fujian Province,China in 2018 and analyze the sequence of glycoprotein(G)gene. Methods The blood,saliva and cerebrospinal fluid samples of suspected patients with rabies submitted from Fujian Province were subjected to laboratory test. Kunming suckling mice were inoculated i. c. with the saliva and cerebrospinal fluid samples,and determined for rabies virus(RABV) antigen in brain tissue by ELISA,and for neutralizing antibody against RABV in blood by rapid fluorescent focus inhibition test(RFFIT). RNAs were extracted from the brain suspension of suckling mice inoculated with saliva,with which the G gene sequence was amplified by RT-PCR and compared with those of vaccine strains at home and abroad as well as street virus strains isolated from Fujian and neighboring provinces to construct a phylogenetic tree by Neighbor-Joining(NJ)method and analyze the homology. Results No neutralizing antibody against RABV was detected in blood samples. RABV was only detected in saliva samples by intracerebral inoculation test in suckling mice and RT-PCR,confirming the patient as that with rabies. The newly isolated street strain was named as 18 FJ01,of which the open reading frame of G gene,at a full-length of 1 575 bp,encoded 524 amino acids. The strain belonged to rabies genotypeⅠ,which showed the closest relationship to vaccine strain CTN-33 isolated in Shandong Province and distant relationship to domestic vaccine strains PV,aG,HEP-Flury and ERA. The homologies of nucleotide and amino acid sequences of the isolated strain were 84. 3% ～ 97. 8% and 9 2. 9% ～ 98. 8% to those of street strains isolated in the neighboring provinces,while were 80. 1% ～ 93. 2% and 88. 9% ～ 96. 9% to the vaccine strains at home and abroad,respectively.Conclusion Virus strain was successfully isolated from the patient and identified,which showed high homology to vaccine strain CTN-33.

    2020 09 v.33 [Abstract][OnlineView][Download 804K]

  • Effect of osteogenic growth peptide on proliferation and osteogenic differentiation of mouse bone marrow mesenchymal stem cells

    SUN Shan-shan;LIN Jing-guang;The Second Affiliated Hospital of Jinzhou Medical University;

    Objective To investigate the effect of osteogenic growth peptide(OGP) on proliferation and osteogenic differentiation of mouse bone marrow mesenchymal stem cells(mBMSCs) as well as the relevant molecular mechanism.Methods The mBMSCs were isolated by mouse bone slice method,of which the characteristic molecules on surface were determined by flow cytometry. The mBMSCs were treated with 10-5 mol/L OGP10-14,using OGP10-14-free osteogenic induction medium as control. The effect of OGP10-14 on proliferation of mBMSCs was evaluated by MTS assay,while that on osteogenic differentiation of mBMSCs by Alizarin red staining. The expression levels of mRNA and protein of osteogenic differentiation related factors β-catenin,RUNX2,BSP and cell cycle related factors cyclin B1,CDK2 and c-myc in mBMSCs were determined by qPCR and Western blot respectively. Results CD29 and CD90 were highly expressed,while CD45 and CD11 b/c were lowly expressed,in isolated primary mBMSCs,which were consistent with the phenotypic characteristics of BMSCs. Compared with that in control group,the treatment with OGP10-14 for 24,48 and 72 h promoted the proliferation of mBMSCs(P < 0. 05). Alizarin red staining showed colony-like aggregative growth of partial cells in control group,while red layered mineralized nodules appeared with the increasing time for induction. However,the mineralized nodules of mBMSCs in OGP10-14 group showed an increasing trend compared with that in control group.Compared with those in control group,the mRNA and protein levels of β-catenin,RUNX2,and BSP increased in OGP10-14 group increased(P < 0. 05),while those of cyclin B1,CDK2,and c-myc increased significantly(P < 0. 01). Conclusion OGP10-14 promotes the proliferation of mBMSCs by promoting osteogenic differentiation and triggering the cell cycle pathway of cyclin B1/CDK2,thereby increases the bone formation.

    2020 09 v.33 [Abstract][OnlineView][Download 1721K]

  • Immunogenicity and immune protection of recombinant Staphylococcus aureus α-toxin

    YUAN Yu-lan;GUO Jing-rong;ZHOU Ji-wei;LIN Hai-tao;ZHANG Yun-tao;National Vaccine and Serum Institute;

    Objective To evaluate the immunogenicity of nontoxic alpha-toxin(AT) of Staphylococcus aureus and its immune protection against challenge with S. aureus strains 49521(type CP5) and 49525(type CP8). Methods Based on NCBI reference sequence NC_007795. 1,a nontoxic mutant mhla(hla H35 L) gene sequence was synthesized,and cloned into expression vector pET-28 a(+). The constructed recombinant plasmid pET-28 a-mhla was transformed to E. coli BL21(DE3)and induced with IPTG. The expressed mAT protein was purified by nickel ion affinity chromatography and analyzed for solubility. BALB/c mice were immunized s. c. with mAT protein + aluminum hydroxide adjuvant(test group)and aluminum hydroxide adjuvant(control group)respectively on days 0,14 and 28 d,of which the serum samples were collected 1 d before challenge and determined for the levels of IgG antibody and its subtypes IgG1,IgG2 a and IgG2 b.The mice were challenged i. p. with strains 49521 and 49525 at lethal dosages of 6. 0 × 108 and 3. 0 × 109 CFU respectively 10 d after the last immunization,of which the survival rate was calculated. The challenge test was repeated for 3 times. Results Restriction analysis and sequencing proved that recombinant plasmid pET-28 a-mhla was constructed correctly. Recombinant mAT,with a relative molecular mass of about 34 000,was expressed in a soluble form. The purified mAT induced IgG at a titer of 1 ∶ 580 000(mainly IgG1 at a titer of 1 ∶ 440 000)in sera of mice. The protection rates against challenge with strain 49521(type CP5)in three tests were 75. 0%,70. 0% and 83. 3%,while those against strain 49525(type CP8) were 80. 0%,60. 0%and 80. 0%,respectively,which were significantly higher than those in control group(P < 0. 05). Conclusion Recombinant nontoxic mAT of S. aureus shows good immunogenicity and immune protection,which may be used as a candidate antigen for multi-antigen component vaccine.

    2020 09 v.33 [Abstract][OnlineView][Download 718K]

  • Effect of extract FP103 from yeast on growth of CHO cells

    LI Qiu-xia;YAN Wei-hong;HE Hai-bo;ZOU Kun;FENG Min-lu;XU Hai-yan;CHENG Fan;LI Xiao;College of Biological and Pharmaceutical Sciences & Hubei Key Laboratory of Natural Products Research and Development,China Three Gorges University;

    Objective To investigate the effect of extract FP103 from yeast on growth of CHO cells. Methods CHO cells were cultured in DMEM/F12 medium(containing 1% FBS)added with 3,1 and 0. 125 g/L yeast extract FP103 respectively,and observed for growth by CCK-8 method. Meanwhile,extract FP103 and epidermal growth factor(EGF)were added into DMEM/F12 medium containing 0. 5% FBS,with which CHO cells were cultured and observed for growth by CCK-8 method,and for morphology by microscopy. Results The yeast extract FP103 at a concentration of 1 g/L significantly promoted the growth of CHO cells(P < 0. 05). However,FP103 in combination with EGF effectively promoted the normal growth of CHO cells at low serum concentration,while the cells were in a normal morphology without suspension. Conclusion Yeast extract FP103 can provide necessary nutrition for the growth of CHO cells instead of partial sera.

    2020 09 v.33 [Abstract][OnlineView][Download 645K]

  • Bioinformatics and prokaryotic expression of cutinase MGG＿09100 of Magnaporthe oryzae

    WANG Shuang;LI Meng-jian;HUANG Feng-lan;CHEN Yong-sheng;LI Guo-rui;College of Life Sciences and Food,Inner Mongolia University for Nationalities;

    Objective To analyze the bioinformatics of cutinase MGG_09100 of Magnaporthe oryzae and express in prokaryotic cells. Methods The conserved domain,signal peptide shear site and transmembrane domain of cutinase MGG_09100 were analyzed by online program,while the MGG_09100 gene was amplified by PCR and cloned into prokaryotic expression vector p ETM-10. The constructed recombinant plasmid pETM10-MGG_09100 was transformed to E. coli BL21(DE3)and induced with IPTG. The expressed recombinant protein was denaturalized and re-naturalized,then purified by Ni-NTA and molecular sieve chromatography. Results The cutinase belonged to the hydrolytic enzyme superfamily of abhydrolase,with signal peptide shear sites between the 18 th and 19 th amino acids and a possible transmembrane structure between the 1 st and the 20 th amino acids. Colony PCR and sequencing proved that recombinant plasmid p ETM10-MGG_09100 was constructed correctly. The expressed recombinant protein mainly existed in a form of inclusion body. After denaturalization,renaturation and purification,the relative molecular mass of soluble recombinant protein was about 24 000,while the concentration was 6 mg/mL. Conclusion Recombinant plasmid pETM10-MGG_09100 was successfully constructed,and the soluble cutinase MGG_09100 protein was finally obtained,which laid a foundation of further study on the function of the protein and the pathogenic mechanism of rice blast disease.

    2020 09 v.33 [Abstract][OnlineView][Download 1004K]

  • Screening of plasma for preparation of human intravenous immunoglobulin against cytomegalovirus

    GUO Bing-feng;ZHANG Jian-tao;LI Guan-jun;ZHANG Jing-jing;LI Guang-fei;ZHANG Xue-cheng;MA Xiao-wei;PAN Ruo-wen;Hualan Biological Engineering Inc.;

    Objective To screen high titer plasma and prepare human intravenous immunoglobulin against cytomegalovirus(CMV-IVIG). Methods The binding and neutralizing titers of 113 CMV IgG samples were determined by ELISA and microneutralization test respectively. CMV-IVIG was prepared with the high titer plasma screened by the two methods respectively,of which the neutralizing titer against CMV was determined by routine microneutralization test and compared with that of IVIG to prepare the final product of IVIG meeting the relevant requirements and determine the method for screening plasma. Results The 113 plasma samples were determined for titer,from which those with binding titer of not less than 20 IU/mL and neutralizing titer of not less than 1 ∶ 32 were collected separately and prepared into 5% CMVIVIG with lot numbers of 20190401 and 20190402 respectively. The neutralization titers of final product of CMV-IVIG with lot number of 20190401 was 498. 28 IU/mL,while that with lot number of 20190402 was 2 357. 56 IU/mL.However,the neutralizing titer of 5% IVIG was 259. 53 IU/mL. Conclusion The neutralizing titer of CMV-IVIG prepared with plasma screened by ELISA was 1. 92 times of that of common IVIG,which did not meet the requirement that the titer of specific immunoglobulin is not less than 4 times of that of common immunoglobulin. However,the neutralizing titer of CMV-IVIG prepared with plasma screened by microneutralization test was 9. 08 times of that of common IVIG,which met the requirement for titer,indicating that microneutralization test might be used for screening of plasma with high neutralizing titer against CMV. It provided qualified source plasma for preparation of CMV-IVIG.

    2020 09 v.33 [Abstract][OnlineView][Download 308K]

  • Preparation and identification of polyclonal antibody against major outer membrane protein of Chlamydia trachomatis serovar E

    LI Ming-yang;WEI He-ting;CHEN Tian-feng;HU Yu;SHEN Yu-ting;DONG Hai-yan;ZHU Shan-li;ZHANG Li-fang;Institute of Molecular Viruses and Immunology,Department of Microbiology and Immunology,Wenzhou Medical University;

    Objective To prepare the polyclonal antibody against major outer membrane protein(MOMP) of Chlamydia trachomatis(Ct)and determine its biological characteristics in vitro. Methods Recombinant plasmid pET21 a(+)/MOMP was constructed by molecular cloning method and transformed to E. coli BL21(DE3) for expression under induction of IPTG. The expressed recombinant MOMP was purified by nickel ion affinity chromatography and injected s. c. into Japanese rabbits in several sites to prepare polyclonal antibody. The prepared antibody was determined for titer and specificity by ELISA and Western blot respectively. Ct serovar E was propagated in HeLa cells and observed for formation of inclusion body by Lugo and Giemsa staining. The binding specificity of prepared polyclonal antibody against MOMP to natural MOMP of Ct was tested by indirect immunofluorescence assay(IFA). Results Ct MOMP was highly expressed in E. coli BL21(DE3). The prepared polyclonal antibody against MOMP reached an ELISA titer of 1 ∶ 256 000,which bound and recognized fusion and natural Ct MOMP specifically. Conclusion The prepared rabbit polyclonal antibody against Ct MOMP showed high titer and specificity,which laid a foundation of further study on the pathogenic mechanism of Ct and development of novel immunodiagnostic kit.

    2020 09 v.33 [Abstract][OnlineView][Download 2676K]

  • Preparation of national references for serotypes 1,4,5,7F and 23F pneumococcal polysaccharides

    CHEN Qiong;ZHANG Rui;LIU Ju;HAN Fei;YIN Shan-shan;SHI Ji-chun;YE Qiang;National Institutes for Food and Drug Control,Key Laboratory of Method and Standardization for Quality Control of Biotechnical Products;

    Objective To prepare the national references for serotypes 1,4,5,7 F and 23 F pneumococcal polysaccharide.Methods According to the requirements in European Pharmacopoeia(EP) 9. 0,serotypes 1,4,5,7 F and 23 F pneumococcal polysaccharide candidate reference was determined for the contents of nucleic acid,protein,total nitrogen,phosphorus,uronic acid,hexosamine,methyl pentose,O-acetyl group and bacterial endotoxin,measured for molecular size,and identified by 1 H nuclear magnetic resonance. Pneumococcal polysaccharide candidate references was coo-peratively calibrated by three laboratories by rate nephelometry using the reference provided by the manufacturer as a standard. Results The quality analysis results of candidate reference met the requirements in EP 9. 0. The1 H NMR result defined the candidate reference as serotypes 1,4,5,7 F and 23 F pneumococcal polysaccharide. All the RSDs of determination results of polysaccharide contents in five candidate references by three laboratories were less than 10%,while means were 370,320,330,432 and 422 g/mL respectively. Conclusion The candidate reference materials meet the requirements for the national reference materials,and may be used for determination of polysaccharide content of 23-valent pneumococcal polysaccharide vaccine.

    2020 09 v.33 [Abstract][OnlineView][Download 162K]

  • Epidemiological characteristics of hepatitis A in Nanchong City,Sichuan Province,China from 2005 to 2019

    ZHAO Lin;GAN Lin;Centre for Disease Prevention and Control of Nanchong City;

    Objective To understand the epidemiologic characteristics of hepatitis A(HA) in Nanchong City,Sichuan Province,China from 2005 to 2019 so as to compare the characteristics before and after inclusion of HA vaccine into the Expanded Program Immunization(EPI)and provide a scientific basis for developing the strategies and measures to prevent and control HA. Methods Descriptively epidemiological method was used to analyze the epidemiologic characteristics of HA in Nanchong from 2005 to 2019. Results From 2005 to 2019,a total of 3 407 cases of HA were reported in Nanchong City. The annual average incidence rate was 3. 52/100 000,which showed a decreasing tendency in general.However,the annual average incidence after inclusion of HA vaccine A into the EPI(2009 ～ 2019)was 2. 30/100 000,which was significantly lower than that before inclusion(2005 ～ 2008,6. 75/100 000,P < 0. 001),with a decreasing rate of 87. 24%. The annual average incidence rates in Langzhong City(6. 13/100 000) and Xichong County(5. 09/100 000) were relatively high. The peak of incidence before inclusion of HA vaccine into the EPI appeared in March to May,while the cases showed no obviously seasonal distribution after inclusion. There were no difference in the sex ratio of cases before and after inclusion of HA vaccine into the EPI(both 2. 06 ∶ 1,P > 0. 05). The peak of incidence appeared in the populations at ages of 31 ～ 58 years,while the incidence showed an increasing tendency with the increasing age and a prominent aggregation. The median age of incidence increased from 39 years before inclusion of HA vaccine into the EPI to the 50 years after inclusion,while the proportion of patients at ages of less than 15 years decreased significantly from 13. 46% to 2. 35%. However,the peak age moved from youth towards agedindistinctly in Shunqing District and Peng'an County,which increased to 46. 5 ～ 53 years in the other seven areas. Most of the cases appeared in farmers(2 253 cases,66. 13%) and students(384 cases,11. 27%). However,the proportion of farmers increased,while that of students decreased,year by year. Conclusion At present,the incidence of HA is low in Nanchong,while the free vaccination for children at school age after 2008 effectively controlled the incidence in the population at ages of less than 15 years,indicating significant effect of vaccination in prevention and control of HA. The people born around the 1970 s should be paid more attention,in whom a measure which encourages the combination of voluntary vaccination and health education should to taken to improve the vaccination coverage and the disease prevention awareness.

    2020 09 v.33 [Abstract][OnlineView][Download 265K]

  • Investigation on the first case of group B cerebrospinal meningitis in neonates in Nanchang City,Jiangxi Province,China in 2019

    ZHANG Yan-xia;XU Bo;WEN Hai-rong;PENG Shi-hui;The Collaboration Unit for Field Epidemiology of State Key Laboratory for Infectious Disease Prevention and Control,Nanchang Center for Disease Control and Prevention;

    Objective To investigate the pathogenic and epidemiological characteristics of the first case of group B cerebrospinal meningitis in neonates in Nanchang City,Jiangxi Province,China in 2019. Methods The medical record of the case was consulted,while an epidemiological investigation was carried out,and the guardians and family members were visited. Blood samples and cerebrospinal fluid samples of patient as well as the pharyngeal swabs of close contacts were collected for pathogen culture. Results Fever,slightly frothing with poor reaction,gazing with both eyes and cyanosis of mouth and lips were observed in a female neonate at age of 24 days. The blood sample of the neonate was positive for group B Neisseria meningitii(Nm). No Nm was detected in the cerebrospinal fluid sample of patient or in the swab samples of close contacts. Conclusion The prevention and control of encephalitis in Nanchang should be strengthened in order to grasp the changes of epidemic bacteria of encephalitis in the city.

    2020 09 v.33 [Abstract][OnlineView][Download 120K]

  • Preparation of the first batch of Vero cell protein standard

    LI Jia;SHI Lei-tai;WANG Ling;LIU Jing-jing;ZHANG Yue-lan;WANG Hui;LEI Ji-jun;GUO Zhong-ping;LI Yu-hua;National Institutes for Food and Drug Control;

    Objective To prepare the first batch of Vero cell protein standard for detection of residual Vero cell host pro-tein in rabies vaccine for human use. Methods Vero cell protein standard was prepared by using Vero cell secretory protein as the raw material and sucrose as the protective agent for lyophilization,of which the assignment was completed by two steps. The first step was the determination of protein content in bulk of Vero cell protein standard by Lowry method,while the second step was the assignment of protein content of Vero cell protein standard by ELISA method using the standard curve plotted with the bulk of the standard. Sixteen containers of Vero cell protein standard were randomly selected and determined for pH value to test the uniformity by analysis of variance. The standard was randomly selected and stored at various temperatures(4,25 and 37 ℃)for various times durations(1,2,3 and 4 weeks),from which three containers were taken at each time point and determined for antigen content by ELISA to test the stability by analysis of variance. Results Collaborative calibration showed that the stated amount of Vero cell protein standard was 2. 6 μg/mL,with a standard deviation of 0. 4 μg/mL and a 95% confidence interval of 2. 51 ～ 2. 78 μg/mL. This batch of standard showed good uniformity and stability. Conclusion At present,Vero cell protein standard has been approved for use,of which batch number is 250022-201901. The establishment of this standard is of great significance in improving the quality control of rabies vaccine for human use.

    2020 09 v.33 [Abstract][OnlineView][Download 217K]

  • Optimization of preparation process for spray-dried recombinant Lactobacillus casei

    ZHANG Lian-mei;CHEN Xiao-yan;BAI Yong-fei;LI Ying;CAI Chang-fu;LI Shu-dong;LIU Huan-huan;LI Yan;SUN Xiao-ying;YANG Yu-qing;HOU Xi-lin;YU Li-yun;College of Life Science and Technology,Heilongjiang Bayi Agricultural University;

    Objective To optimize the preparation process for spray-dried recombinant Lactobacillus casei. Methods Recombinant L. casei was mixed with whey protein as a suspension matrix for spray drying,while the addition amount of whey protein(10%,20%,30%,40%,50%,60%,70%,80% and 90%),inlet air temperature(60,80,100,120,140,160 and 180 ℃),air input(65%,70%,75%,80%,85%,90%,95% and 100%)and charging rate(2. 5,5. 0,7. 5,10. 0,12. 5 and 15. 0 mL/min)were optimized by single factor test. Orthogonal experiments were used to further optimize the process parameters using viable count and survival rate as indicators. The quality and storage stability of spray-dried recombinant L. casei prepared under the optimal condition were tested. Results The optimal addition amount of whey protein,inlet air temperature,air input and the charging rate were 20%,120 ℃,75% and 10 mL/min respectively. The relevant quality indicators of the spray-dried preparation met the relevant requirements,while the stability was satisfactory after storage at 4 ℃ in vacuum. Conclusion The preparation process for spray-dried recombinant L. casei was optimized,which laid a foundation of industrial production of the spray-dried preparation.

    2020 09 v.33 [Abstract][OnlineView][Download 477K]

  • Development and verification of a method for multiplex detection of specific human serum IgG antibodies against recombinant Staphylococcus aureus vaccine based on Luminex system

    CHEN Zhi-bin;WANG Bin;GU Jiang;LIANG Hao-yu;LU Xu;ZENG Ming;National Institutes for Food and Drug Control;

    Objective To develop and verify a method for multiplex detection of specific human serum IgG antibodies against recombinant Staphylococcus aureus(SA) vaccine based on Luminex system. Methods Five antigens in recombinant SA vaccine,i.e. mHla,IsdB-N2,SpA5,mSEB and MntC,were coupled to magnetic beads respectively. Eight serum samples containing high titer antibodies against the five antigens were collected from the volunteers 21 d after the3 rd immunization with recombinant SA vaccine,pooled and used as reference sera. The coupled antigen was added with the reference sera,incubated at 37 ℃,then added with goat F(ab′)2 against human IgG-Fc(PE)and further incubated at37 ℃,and subjected to multiplex detection by Luminex system. The concentration of coupled antigens,time for incubation and dilution of the second antibody were optimized,and the interference between five antigens was analyzed.The developed method was verified for specificity,accuracy and precision. Results The optimal concentrations of mHla,IsdB-N2,SpA5,mSEB and MntC for coupling were 30,150,7. 5,2. 5 and 7. 5 μg/2. 5 mil respectively,while both the optimal time for incubation of the first and the second antibodies were 30 min,and the optimal dilution of the second antibody was 1 ∶ 5 000. No significant cross epitopes were observed between the five antigens,while no significant interference during reaction with specific antibodies. The inhibitory rate of free recombinant protein to the reactions between coupled antigens and antibodies increased with the increasing concentration. The recovery rates of reference sera at three concentrations were 87. 0% ～ 120. 8%,of which the CVs in intra-and inter-assays were less than 10%.Conclusion A method for multiplex detection of specific antibodies against recombinant SA vaccine was successfully developed,which showed high specificity,accuracy and stability.

    2020 09 v.33 [Abstract][OnlineView][Download 449K]

  • Feasibility of BCA method for determination of protein content in intermediate product of component pertussis vaccine

    WANG Li;WANG Yin;HU Ye-qin;TIAN Cong;SHI Bin;CAO Jing;YANG Ming;Wuhan Institute of Biological Products Co.,Ltd.;

    Objective To evaluate and verify the feasibility of BCA method for determination of protein content in intermediate product of component pertussis vaccine. Methods By using the determination result by Lowry method 2 in Chinese Pharmacopoeia(Vol Ⅲ,2015 edition) as a control,the substances that interfered with the BCA method for determination of protein content in intermediate product of component pertussis vaccine were analyzed,based on which the limit of interfering substances was clarified. The BCA method was verified for specificity,linear range,reproducibility and accuracy,by which the determination result was compared with that by Lowry method 2. Results The 30% ammonium sulfate showed significant interference with the protein content determined by Lowry method 2 and BCA method,while the other buffer systems showed no effect. Ammonium sulfate at concentrations of not more than 25% showed no interference with the determination result by Lowry method 2,while that at concentrations of less than 5% could be eliminated by 1 mol/L hydrochloric acid thus did not affect the determination by BCA method. The determination result by Kjeldahl method showed that the concentration of ammonium sulfate was less than 5% in the intermediate product of component pertussis vaccine. The specificity,linear range,reproducibility and accuracy of BCA method met the requirements for verification. The determination results of intermediate product of component pertussis vaccine by Lowry method II and BCA method showed no significant difference(P > 0. 05). Conclusion BCA method was simple,rapid,accurate and with high-throughput,which might be used for the determination of protein content in intermediate product of component pertussis vaccine.

    2020 09 v.33 [Abstract][OnlineView][Download 318K]

  • Development of liquid chromatography tandem mass spectrometry for simultaneous determination of residual ADH and EDAC in polysaccharide conjugate vaccines

    LI Mao-guang;LONG Zhen;LI Ya-nan;WANG Chun-e;LI Yue-qi;MAO Qi-qi;YE Qiang;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 a liquid chromatography tandem mass spectrometry(LC-MS/MS) method for simultaneous determination of residual adipic acid dihydrazide(ADH)and N-(3-dimethylaminopropyl)-N′-ethylcarbo-diimide(EDAC) in polysaccharide conjugate vaccines including pneumococcal conjugate vaccine,meningococcal conjugate vaccine and Haemophilus influenzae type b conjugate vaccine. Methods The pretreatment condition for complete change of EDAC to EDU was determined by analysis of stability of EDAC in pure water,neutral aqueous solution(50 mmol/L phosphate buffer,pH 6. 8,200 mmol/L sodium chloride aqueous solution and medium)and 0. 1% FA aqueous solution.The retention of ADH,EDU and DEAC by various chromatographic columns,as well as influence of chromatographic condition such as product ions,fragmentor voltage,Q1 and Q3 voltages on the quantitative sensitivities of ADH and EDU,were investigated,based on which a LC-MS/MS method was developed by using C18 WCX column(2. 1 mm × 150 mm,5 μm)as stationary phase and the mixture of 0. 1% formic acid(FA)-water and 0. 1% FA-acetonitrile(pH 2 ～ 3)as mobile phase to achieve the highly sensitive quantitative analysis of ADH and EDAC(converted to EDU in practice). The developed method was verified for precision,accuracy,linear range limit of detection(LOD) and limit of quantitation(LOQ). Results ADH was stable in aqueous solution. EDAC was hydrolyzed into EDU starting with dissolution and was difficult to be converted completely,resulting in difficulty in determination,while was converted completely and rapidly after addition with FA into samples and standard. ADH,EDU and EDAC were not separated from vaccine matrix on C18 column due to very weak retention of these compounds on C18 column(eluted nearly dead time). However,A C18 WCX column was used as stationary phase to separate target compounds due to good retentions and peak shape. Precursor ions and product ions of target compounds were obtained by Q1 scan and product scan,and the product ions with high MS responses were used for the optimization of MRM parameters including Q1,CE and Q3 voltage. The ADH and EDAC contents determined by the developed method showed good linear relationship with the peak area,with R2 values of more than 0. 999. The LOD and LOQ of ADH were 3. 96 and 15. 63 μg/L,while those of EDAC were 0. 58 and 1. 17 μg/L,respectively,indicating a high sensitivity. The developed method showed a high precision(RSD% of peak area was less than2%) and a high accuracy(the recovery was 90% ～ 105%). Conclusion The LC-MS/MS method for highly sensitive determination of ADH and EDAC(quantified by determination of EDU) in polysaccharide conjugate vaccines was developed,which was more simple in pretreatment,more automatic and more sensitive as compared with the method in Chinese Pharmacopoeia(Volume Ⅲ,2015 edition).

    2020 09 v.33 [Abstract][OnlineView][Download 898K]

  • Determination of PEG modification degree of PEG-recombinant human granulocyte colony-stimulating factor by molecular exclusion chromatography with double detectors in series

    ZHANG Su;ZHANG Pei-biao;LIU Zhong;ZHANG Gui-min;Shandong Newtime Pharmaceutical Co.,Ltd.,Shandong Provincial Engineering Laboratory of Protein Pharmaceutical;

    Objective To establish and verify a method for determination of modification degree of PEG-recombinant human granulocyte colony-stimulating factor(PEG-rhG-CSF)by molecular exclusion chromatography with ultraviolet and refractive index detectors in series. Methods TSKgel G3000 SWXL(300 mm × 7. 8 mm) gel column was adopted using 0. 01 mol/L disodium hydrogen phosphate-sodium dihydrogen phosphate-0. 1 mol/L sodium chloride(pH 7. 0)as mo-bile phase at a flow rate of 0. 5 mL/min and a column temperature of 25 ℃. The refractive index detector temperature was 35 ℃,while the wavelength was 280 nm,and the injection volume was 20 μL. The modification degree was calculated by measuring the area of UV and RI peaks of rhG-CSF intermediates,the area of RI peaks of PEG,and the area of UV and RI peaks of bulk of PEG-rhG-CSF. The developed method was verified for specificity,linearity,precision and durability. Results The linear range of the developed method was 0. 2 ～ 1. 0 mg/mL(R2 = 0. 999). The RSDs in precision and durability tests were 1. 54% and 1. 77% respectively. The method also showed good specificity. Conclusion The developed method showed good specificity,linearity,precision and durability,and was simple to handle,which might be used for the determination of modification degree of PEG-rhG-CSF.

    2020 09 v.33 [Abstract][OnlineView][Download 390K]

  • Preliminary application of neutralizing antibody assay based on polio pseudoviruses

    JIANG Zheng;ZHU Xiu-juan;LIU Gui-xiu;LIU Ting;WANG Hui;LI Chang-gui;Department of Viral Vaccine,National Institutes for Food and Drug Control;

    Objective To preliminarily investigate the applicability of neutralizing antibody assay based on wild type polio pseudoviruses in clinical immunogenicity evaluation of poliomyelitis vaccines. Methods The cross neutralizing antibodies in 207 pairs of serum samples from phase Ⅱ clinical trial of sIPV,52 pairs from subjects in each of trial groups of sIPV at high,moderate and low dosages and 51 pairs from those in control group of wIPV,were determined by neutralization assay based on the wild-type polio pseudoviruses Mahoney,MEF-1 and Saukett strains established by the authors. The positive conversion rate and geomic mean titer(GMT) of neutralizing antibody in trial groups were calculated,and the results were compared with those determined in the same laboratory by conventional cytopathic inhibition method using SabinⅠ,ⅡandⅢstrains. Results The positive conversion rates in various trial groups determined by the two methods were identical,while the trends of determination results of high,moderate and low antibody titers were in agreement. The determination result by pseudo-Mahoney virus neutralizing antibody assay in sera after immunization with sIPV was about 4 times lower than that by cytopathic inhibition method using Sabin I strain in average,while those by pseudo-MEF-1 and pseudo-Saukett virus neutralizing antibody assays were about 2 and about 1. 5 times higher than those by cytopathic inhibition method using Sabin Ⅱ and Ⅲ strains in average respectively,which was significantly consistent with the trend of previous pseudo-assay validation. Conclusion The neutralizing antibody assay based on wild type polio pseudoviruses showed good suitability,high biosecurity and high sensitivity,which was an effective method for evaluation of the efficacy of polio vaccines.

    2020 09 v.33 [Abstract][OnlineView][Download 298K]

  • Development strategies and research progress of SARS-CoV-2 neutralizing antibody

    DU Jian-hui;PAN Yong-bing;YANG Xiao-ming;Wuhan Institute of Biological Products,National Engineering Technology Research Center for Combined Vaccines;

    Severe acute respiratory syndrome coronavirus 2(SARS-CoV-2) has evolved into a pandemic,which has imposed a burden on the global economy and public health,thus effective response measures are urgently needed.Antibodies have a good historic documented in the prevention and treatment of emerging infectious diseases. At present,hundreds of development projects for SARS-CoV-2 neutralizing antibodies around the world are ongoing by using different strategies,with some in clinical trials. This article reviews the current development strategies and research progress of SARS-CoV-2 neutralizing antibodies in the fields of target selection,antibody screening techinqne,functional evaluation and possible challenges.

    2020 09 v.33 [Abstract][OnlineView][Download 221K]

  • Progress in research on cell-based influenza vaccine and its production procedure

    GAO Ning;ZHAO Hui;JIANG Wei;LI Chang-gui;Changchun Institute of Biological Products;

    Influenza virus is one of the largest public health challenges in the world. WHO recommends annual influenza vaccination as the most effective way to prevent influenza. Influenza vaccine is generally produced by chicken embryos,which cannot meet the market needs during a pandemic. The production of novel influenza vaccines by microcarrier and suspension cultures using synthetic media is in study. An effective vaccine may be developed within a short period and used as a supplement to chicken embryo-based influenza vaccine. This paper reviews the progress in research on cellbased influenza vaccine and its production procedure.

    2020 09 v.33 [Abstract][OnlineView][Download 171K]


@2012 Editorial Department of "Chinese Journal of Biologicals"