MAPREC技术检测Sabin株脊髓灰质炎病毒的基因突变率并评价疫苗神经毒力
|
摘要
目的:采用MAPREC技术检测Sabin株脊髓灰质炎灭活疫苗(s IPV)生产收获液中病毒神经毒力决定位点的基因突变并评估疫苗神经毒力。方法:采用MAPREC技术用CY5荧光标记引物替代同位素标记检测疫苗株SabinⅠ、Ⅱ型及PfizerⅢ型疫苗病毒收获液及毒种的神经毒力决定位点的突变率,并与猴体神经毒力比较;采用MAPREC技术检测在不同培养温度、传代的次数、降低病毒接种的感染复数(MOI)时病毒各位点突变率变化。结果:MAPREC方法检测显示,本研究收集的病毒收获液的突变率符合猴体神经毒力检测结果;当病毒培养条件发生改变导致突变率增加后,MAPREC检测出的神经毒力决定位点的突变率也同样增加。结论:MAPREC技术可用于检测Sabin株脊髓灰质炎病毒的基因突变率,并评价疫苗的神经毒力。
Objective: Adopting the MAPREC to test mutation rate of Sabin IPV and the neurovirulence. Methods: The isotope labeling of MAPREC technique was replaced by CY5 fluorescent labeled primer,and detect Sabin vaccine strain type Ⅰ,Ⅱ,and Pfizer Ⅲ vaccine virus harvest and nerve toxicity of the poison of decision locus mutation rate; to test whether the results consistent with the monkeys nerve toxicity test results. When the temperature of virus culture and the number of viral passages was increased,multiplicity of infection was decreased,the mutation of virus would be increased correspondingly. Results: The rate of mutation detected by the qualified vaccine MAPREC method was consistent with the results of monkeys nerve toxicity test. When the virus culture conditions changed and the mutation rate increased,the mutation rate of the nerve virulence localization sites detected by MAPREC also increased. Conclusion: MAPREC test can detect the gene mutation rate of SIPV,so as to determine the vaccine's neurovirulence.
Objective: Adopting the MAPREC to test mutation rate of Sabin IPV and the neurovirulence. Methods: The isotope labeling of MAPREC technique was replaced by CY5 fluorescent labeled primer,and detect Sabin vaccine strain type Ⅰ,Ⅱ,and Pfizer Ⅲ vaccine virus harvest and nerve toxicity of the poison of decision locus mutation rate; to test whether the results consistent with the monkeys nerve toxicity test results. When the temperature of virus culture and the number of viral passages was increased,multiplicity of infection was decreased,the mutation of virus would be increased correspondingly. Results: The rate of mutation detected by the qualified vaccine MAPREC method was consistent with the results of monkeys nerve toxicity test. When the virus culture conditions changed and the mutation rate increased,the mutation rate of the nerve virulence localization sites detected by MAPREC also increased. Conclusion: MAPREC test can detect the gene mutation rate of SIPV,so as to determine the vaccine's neurovirulence.
引文
[1]THOMPSON KM,DUINTJER TEBBENS RJ.The differential impact of oral poliovirus vaccine formulation choices on serotype-specific population immunity to poliovirus transmission[J].Bmc Infectious Diseases,2015(1):1-12.
[2]刘开民.脊髓灰质炎病毒抗原部位和疫苗[J].国外医学预防诊断治疗用生物制品分册,1985(1):37-39.
[3]SHIMIZU H.Development and introduction of inactivated poliovirus vaccines derived from Sabin strains in Japan[J].Vaccine,2016(16):1975-1985.
[4]MELNICK JL,姜述德.口服脊髓灰质炎活疫苗[J].国外医学预防诊断治疗用生物制品分册,1985(4):157-159.
[5]HAMIDI A,BAKKER WA.Innovative IPV from attenuated Sabin poliovirus or newly designed alternative seed strains[J].Pharmaceutical patent analyst,2012(5):589-599.
[6]车艳春,李琦涵.脊髓灰质炎灭活疫苗的研究进展与临床应用[J].中国新药杂志,2015(12):1364-1368.
[7]徐海明.脊髓灰质炎病毒Sabin株减毒的遗传学基础的研究进展[J].微生物学免疫学进展,1999(3):85-88.
[8]陈明文,李文.疫苗衍生脊髓灰质炎病毒相关病例的研究进展[J].疾病监测,2014(3):243-246.
[9]LU Z,DOUTHITT MP,TAFFS RE,et al.Quantitative aspects of the mutant analysis by PCR and restriction enzyme cleavage(MAPREC)[J].PCR methods and applications,1993(3):176-180.
[10]DUNN G,O’BRIEN J,HEATH A,et al.Report of the WHO collaborativestudy to establish a new International Standard and three new International Reference Preparations for the MAPREC test of poliovirus Type 1(Sabin)[J].World Health Organization,2009(09):2013
[11]马昌燐.放射性同位素标记核酸探针的方法[J].核科学与工程,1994(3):280-283.
[12]BELLA B,MAJID L,KONSTANTIN C.MAPREC assay for quantitation of mutants in a recombinant flavivirus vaccine strain using near-infrared fluorescent dyes[J].Journal of Virological Methods,2011(1):14-19.
[13]王潇潇,刘宇,温智恒,等.荧光定量PCR方法在脊髓灰质炎病毒突变分析中的应用[J].中国生物制品学杂志,2014(12):1579-1587,1594.
[14]邓燕.Sabin株脊髓灰质炎灭活疫苗毒种的遗传稳定性研究及Sabin株病毒深度测序方法的探索[J].北京协和医学院,2016(4):342-347.
[15]WORLD HEALTH O.WHO expert committee on biological standardization sixty-fifth report[J].World Health Organization technical report series,2015(993):261-262.
[16]吴照,温宁,罗会明,等.环境监测在疫苗衍生脊髓灰质炎病毒事件调查处理中的应用[J].中国疫苗和免疫,2012(1):67-69.
[2]刘开民.脊髓灰质炎病毒抗原部位和疫苗[J].国外医学预防诊断治疗用生物制品分册,1985(1):37-39.
[3]SHIMIZU H.Development and introduction of inactivated poliovirus vaccines derived from Sabin strains in Japan[J].Vaccine,2016(16):1975-1985.
[4]MELNICK JL,姜述德.口服脊髓灰质炎活疫苗[J].国外医学预防诊断治疗用生物制品分册,1985(4):157-159.
[5]HAMIDI A,BAKKER WA.Innovative IPV from attenuated Sabin poliovirus or newly designed alternative seed strains[J].Pharmaceutical patent analyst,2012(5):589-599.
[6]车艳春,李琦涵.脊髓灰质炎灭活疫苗的研究进展与临床应用[J].中国新药杂志,2015(12):1364-1368.
[7]徐海明.脊髓灰质炎病毒Sabin株减毒的遗传学基础的研究进展[J].微生物学免疫学进展,1999(3):85-88.
[8]陈明文,李文.疫苗衍生脊髓灰质炎病毒相关病例的研究进展[J].疾病监测,2014(3):243-246.
[9]LU Z,DOUTHITT MP,TAFFS RE,et al.Quantitative aspects of the mutant analysis by PCR and restriction enzyme cleavage(MAPREC)[J].PCR methods and applications,1993(3):176-180.
[10]DUNN G,O’BRIEN J,HEATH A,et al.Report of the WHO collaborativestudy to establish a new International Standard and three new International Reference Preparations for the MAPREC test of poliovirus Type 1(Sabin)[J].World Health Organization,2009(09):2013
[11]马昌燐.放射性同位素标记核酸探针的方法[J].核科学与工程,1994(3):280-283.
[12]BELLA B,MAJID L,KONSTANTIN C.MAPREC assay for quantitation of mutants in a recombinant flavivirus vaccine strain using near-infrared fluorescent dyes[J].Journal of Virological Methods,2011(1):14-19.
[13]王潇潇,刘宇,温智恒,等.荧光定量PCR方法在脊髓灰质炎病毒突变分析中的应用[J].中国生物制品学杂志,2014(12):1579-1587,1594.
[14]邓燕.Sabin株脊髓灰质炎灭活疫苗毒种的遗传稳定性研究及Sabin株病毒深度测序方法的探索[J].北京协和医学院,2016(4):342-347.
[15]WORLD HEALTH O.WHO expert committee on biological standardization sixty-fifth report[J].World Health Organization technical report series,2015(993):261-262.
[16]吴照,温宁,罗会明,等.环境监测在疫苗衍生脊髓灰质炎病毒事件调查处理中的应用[J].中国疫苗和免疫,2012(1):67-69.