DEAE-Sepharose FF纯化脊髓灰质炎病毒Sabin株Ⅰ型稳定性研究
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摘要
目的观察离子交换纯化脊髓灰质炎病毒Ⅰ型Sabin株稳定性,以研究病毒纯化的回收率和质量控制。方法使用微载体培养Vero细胞和脊灰病毒Ⅰ型Sabin株,经过逐级过滤澄清和100K超滤膜浓缩,再经凝胶介质初步纯化后的病毒液,然后观察经过离子介质DEAE Sepharose FF层析纯化5批次的纯化效果。对每次纯化获得的病毒液测定D抗原、蛋白质含量、牛血清白蛋白含量、DNA残留量以评价其质量。结果凝胶纯化后的病毒液,经离子介质DEAE Sepharose FF纯化,其D抗原、蛋白质含量、牛血清白蛋白含量、DNA残留量和比活性的批间差异不显著(P>0.05)。结论离子介质DEAE Sepharose FF在一定批次内纯化脊灰病毒工艺稳定。
Objective To study the recovery efficiency and quality control system for poliovirus Type I Sabin strain, purified by ion exchange. Methods Vero cells were seeded on the microcarrier, infected by poliovirus type I Sabin strains. After harvesting of virus, the systematic purification process was taken, including multistage filtration clarification, 100 K ultrafiltration membrane and then gel medium. The purification efficiency of ion medium DEAE Sepharose FF chromatography was observed for 5 batches. The key quality control points including D antigen, protein content, bovine serum albumin content and DNA residue were monitored and evaluated for each times. Results After purified poliovirus Type I Sabin strain by DEAE Sepharose FF, there was no significant difference in D antigen, protein content, bovine serum albumin content, DNA residue and specific activity between each batch. Conclusion The purification method of DEAE Sepharose FF for poliovirus Type I Sabin strain was useful and stability.
Objective To study the recovery efficiency and quality control system for poliovirus Type I Sabin strain, purified by ion exchange. Methods Vero cells were seeded on the microcarrier, infected by poliovirus type I Sabin strains. After harvesting of virus, the systematic purification process was taken, including multistage filtration clarification, 100 K ultrafiltration membrane and then gel medium. The purification efficiency of ion medium DEAE Sepharose FF chromatography was observed for 5 batches. The key quality control points including D antigen, protein content, bovine serum albumin content and DNA residue were monitored and evaluated for each times. Results After purified poliovirus Type I Sabin strain by DEAE Sepharose FF, there was no significant difference in D antigen, protein content, bovine serum albumin content, DNA residue and specific activity between each batch. Conclusion The purification method of DEAE Sepharose FF for poliovirus Type I Sabin strain was useful and stability.
引文
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[9] Lifetime of chromatography medium and cleaning of chromatographic medium,GE Healthcare.
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[13] 甘淋,李娟,何涛,等.几种蛋白质含量测定方法的比较研究[J].泸州医学院学报,2004,6(27):500-502.
[14] 朱业培,王玮,吕青骎,等.动物过敏原牛血清白蛋白间接竞争ELISA检测方法的建立[J].南京农业大学学报,2016,2(39):305-311.
[15] 朱蓉,杜洪桥,谭洪兴,等.CHO宿主细胞DNA残留量检测方法的建立[J].中国生物制品学杂志,2010,11(23):1264-1266.
[16] 贾丽丽,周蕊葆,王辉,等.病毒类疫苗Vero细胞宿主蛋白残留量检测试剂盒的适用性验证[J].中国生物制品学杂志,2009,7(22):696-698.
[17] 司波,赵佳.离子交换层析技术在多糖分离纯化中的应用[J].科技风,2009,18:203.
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[19] 赵荣乐,郑光宇.离子交换层析和凝胶过滤纯化抗凝血多肽[J].北京师范大学学报(自然科学版),2002,4(38):544-548.
[20] 马晓轩,范代娣,王晓军,等.批量离子交换层析和凝胶过滤层析在类人胶原蛋白Ⅰ纯化中的应用[J].离子交换与吸附,2006,22(1):47-52.
[21] 张兴义.几种柱层析的特点和在纯化工艺中的选择[J].中国生物制品学杂志,1999,3(12):187-191.
[2] Heymann DI,Sulter RW,Aylwand RB,et al,A global call for new polio vaccines[J].Nature ,2005,434(7034):699-700.
[3] Liang X,Zhang Y,Xu W,et al.An outbreak of poliomyelitis caused by type Ⅰ vaccine-derived poliovirus in China[J].infect Dis,2006,194(5):545-551.
[4] 刘丹青,李黎,王斌冰,等.中国首例免疫缺陷疫苗衍生脊髓灰质炎病毒病例的流行病学调查分析及其控制对策[J].中国计划免疫.2006,12(6):459-463.
[5] Grnssly NC,Fraser C,Wenger J,et al.New strategies for the elimination of polio form India[J].Science,2006,314(5802):1150-1153.
[6] World Health,Organization Global polio eradication initiative strategic plan 2004-2008[J].Wkly Epidemiol Rec,2004,79(6):55-57.
[7] Trabesi K,Rourou S,Loukil H,et al.Optimiztion of virus yield as a strategy to improve rabies vaccine production by Vero cells in a bioreactor[J].Biotechnal,2006,121(2):261-271.
[8] 衡燮.Sabin株脊髓灰质炎灭活疫苗纯化工艺的建立[J].中国生物制品学杂志,2010,4(23):394-396.
[9] Lifetime of chromatography medium and cleaning of chromatographic medium,GE Healthcare.
[10] 杨红艳,隋礼丽.层析介质使用寿命缩小模型的建立和确认[J].中国生物制品学杂志,2013,6(26)6:894-896.
[11] 廖国阳,姜述德,郑从义,等.脊髓灰质炎减毒活疫苗的高效纯化及特性研究[J].中国病毒学杂志,2000,5(3):226-231.
[12] 杨怀德,姜述德,张新文,等.脊髓灰质炎病毒Sabin株D抗原定量及其中和抗体检测的研究[J].云南大学学报(自然科学版),2007,S3:373-381.
[13] 甘淋,李娟,何涛,等.几种蛋白质含量测定方法的比较研究[J].泸州医学院学报,2004,6(27):500-502.
[14] 朱业培,王玮,吕青骎,等.动物过敏原牛血清白蛋白间接竞争ELISA检测方法的建立[J].南京农业大学学报,2016,2(39):305-311.
[15] 朱蓉,杜洪桥,谭洪兴,等.CHO宿主细胞DNA残留量检测方法的建立[J].中国生物制品学杂志,2010,11(23):1264-1266.
[16] 贾丽丽,周蕊葆,王辉,等.病毒类疫苗Vero细胞宿主蛋白残留量检测试剂盒的适用性验证[J].中国生物制品学杂志,2009,7(22):696-698.
[17] 司波,赵佳.离子交换层析技术在多糖分离纯化中的应用[J].科技风,2009,18:203.
[18] 朱家文,武斌.离子交换层析分离纯化重组人血清白蛋白[J].华东理科大学学报,2002,1(28):341-345.
[19] 赵荣乐,郑光宇.离子交换层析和凝胶过滤纯化抗凝血多肽[J].北京师范大学学报(自然科学版),2002,4(38):544-548.
[20] 马晓轩,范代娣,王晓军,等.批量离子交换层析和凝胶过滤层析在类人胶原蛋白Ⅰ纯化中的应用[J].离子交换与吸附,2006,22(1):47-52.
[21] 张兴义.几种柱层析的特点和在纯化工艺中的选择[J].中国生物制品学杂志,1999,3(12):187-191.