重组戊型肝炎疫苗菌种库的建立及其稳定性
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摘要
目的建立重组戊型肝炎疫苗工程菌三级种子库,并分析其稳定性。方法将工程菌株HEV179/BL21(DE3)复苏活化,经全面检测合格后冻干保存即为原始种子库;原始种子库传代扩增,经全面检定合格后,冻干保存即为主种子库;主种子库经传代扩增和全面检定合格后,甘油冻存即为工作种子库。对三级种子库进行全面检定,并对工程菌进行冻干及甘油冻存稳定性、甘油冻存时间稳定性及质粒稳定性检测。结果建立的三级种子库经全面检定合格;菌种冻干稳定性、甘油冻存稳定性、甘油冻存时间稳定性及质粒稳定性指标均合格,菌种生长特性、遗传和表达稳定性均未发生变化。结论成功建立了HEV179/BL21(DE3)工程菌三级种子库,菌种稳定性良好,适用于重组戊型肝炎疫苗的工业化生产。
Objective To establish three-tier seed bank of recombinant hepatitis E vaccine and study its stability.Methods Recombinant E.coli HEV179 / BL21(DE3) was resuscitated,activated and subjected to overall control tests,and the qualified ones were lyophilized as primary seed bank.The seeds from primary seed bank were subcultured,propagated and subjected to overall control tests,while the qualified ones were lyophilized as master seed bank.The seeds from master seed bank were subcultured,propagated and subjected to overall tests,while the qualified ones were cryopreserved in glycerol and served as working seed bank.The three-tier seed bank was subjected to overall control tests,and the recombinant strain was evaluated for stabilities after lyophilization and cryopreservation in glycerol as well as the genetic stability.Results The established three-tier seed bank was qualified in overall control tests,of which the stabilities after lyophilization and cryopreservation in glycerol as well as the genetic stability met the relevant requirements.The growth characteristics as well as genetic and expression stabilities of recombinant strain showed no significant change.Conclusion Three-tier seed bank of HEV179 / BL21(DE3) was successfully established,and the seeds showed high stability and was suitable for industrial production of recombinant hepatitis E vaccine.
Objective To establish three-tier seed bank of recombinant hepatitis E vaccine and study its stability.Methods Recombinant E.coli HEV179 / BL21(DE3) was resuscitated,activated and subjected to overall control tests,and the qualified ones were lyophilized as primary seed bank.The seeds from primary seed bank were subcultured,propagated and subjected to overall control tests,while the qualified ones were lyophilized as master seed bank.The seeds from master seed bank were subcultured,propagated and subjected to overall tests,while the qualified ones were cryopreserved in glycerol and served as working seed bank.The three-tier seed bank was subjected to overall control tests,and the recombinant strain was evaluated for stabilities after lyophilization and cryopreservation in glycerol as well as the genetic stability.Results The established three-tier seed bank was qualified in overall control tests,of which the stabilities after lyophilization and cryopreservation in glycerol as well as the genetic stability met the relevant requirements.The growth characteristics as well as genetic and expression stabilities of recombinant strain showed no significant change.Conclusion Three-tier seed bank of HEV179 / BL21(DE3) was successfully established,and the seeds showed high stability and was suitable for industrial production of recombinant hepatitis E vaccine.
引文
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[9]贺石汉,李宝宗,郜金荣,等.重组pQE30tPAr质粒在JM109菌株中的稳定性研究[J].武汉大学学报:理学版,2003,49(4):497-500.
[2]耿彦生,王佑春.戊型肝炎病毒感染免疫研究进展[J].世界华人消化杂志,2010,18(9):897-901.
[3]Zhang H,Dai X,Shan X,et al.The Leu477 and Leu613 ofORF2-encoded protein are critical in forming neutralization anti-genic epitope of hepatitis E virus genotype 4[J].Cell Mol Im-munol,2008,5(6):447-456.
[4]Zhou EM,Guo H,Huang FF,et al.Identi?cation of two neutral-ization epitopes on the capsid protein of avian hepatitis E virus[J].J Gen Virol,2008,89(Pt 2):500-508.
[5]Meng JH,Dai X,Chang JC,et al.Identification and characteriza-tion of the neutralization epitope(s)of the hepatitis E virus[J].Virology,2001,288(2):203-211.
[6]孟继鸿,戴星,董晨.一种重组戊型肝炎病毒蛋白、其制备方法及用途[P]中国,200710021746.0.2007-12-26.
[7]王军志.生物技术药物研究开发和质量控制[M].北京:科学出版社,2007:390-392.
[8]国家食品药品监督管理药品审评中心.重组制品生产用哺乳动物细胞质量控制技术评价一般原则[S].2007-08-13.
[9]贺石汉,李宝宗,郜金荣,等.重组pQE30tPAr质粒在JM109菌株中的稳定性研究[J].武汉大学学报:理学版,2003,49(4):497-500.