脊髓灰质炎减毒活疫苗的高效纯化及特性研究
作者：郑从义 廖国阳 姜述德 李卫东 王明秀 陈光武 王燕
单位：郑从义(武汉大学中国典型培养物保藏中心，武汉 430072)；廖国阳 姜述德 李卫东 王明秀 陈光武 王燕(中国医学科学院 中国协和医科大学 医学生物学所，昆明 650107)
关键词：脊髓灰质炎减毒活疫苗； 凝胶柱层析； 高效纯化
中国病毒学000304摘要：应用Q-Sepharose Fast Flow对Vero细胞基质制备的I型口服脊髓灰质炎减毒活疫苗悬液进行纯化。病毒液经滤过澄清和超滤浓缩，获得85%的病毒感染性滴度回收率，而经Q-Sepharose F.F.纯化的病毒悬液，病毒感染性滴度回收率达100%。纯化后的病毒液用α-32P dATP标记DNA探针膜杂交法测定，宿主Vero细胞基质DNA残余含量远低于100 pg/剂量的标准；rct/40特征、病毒形态及病毒衣壳蛋白组份等生物学性状无显著变化。研究结果提示，Q-Sepharose F.F.是Vero细胞制备口服脊髓灰质炎减毒疫苗的理想纯化材料。
, 百拇医药
中图分类号：Q939.4，R512.4 文献标识码：A
文章编号：1003-5125(2000)03-0226-06
Studies on the Purification of OPV and its Characterization
LIAO Guo-yang，JIANG Shu-de，LI Wei-dong，WANG Ming-xiu，CHEN Guang-wu，WANG Yan
(Institute of Medical Biology, Chinese Academy of Medical Science & Peking Union of
Medical College, Kunming 650107, China)
, 百拇医药
ZHENG Cong-Yi
(China Center for Type Culture Collection, Wuhan University, Wuhan 430072, China)
Abstract: Q-Sepharose F.F. was adopted to purify the suspension of oral poliovaccine (OPV), which is prepared from Vero cells. After clarification and concentration by ultrafiltration, the recovery of virus infected titre may attain above 85%. Using column chromatography on Q-Sepharose F.F. to purify the concentrated virus suspension, the purified viruses attain 100% recovery of virus infectivity. Dot membrane hybridization was used to detect DNA with the probe of Vero cell genome DNA which was labed with α-32P dATP, the contents of residual substrate DNA was less than 100 pg/dose. The process of downstream had no significant influence on some biologiccal characters of purified viruses, such as virus morphology, tumorigenicity, rct/40 character and capsid protein. The results indicate that Q-Sepharose F.F. is an ideal chromatography material for purifying OPV prepared from Vero cells.
, http://www.100md.com
Key words: Oral polio vaccine (OPV); Q-Sepharose F.F.; Purification
作者简介：廖国阳(1960年-)，男，博士生
参考文献
[1] Hayflick L. History of the cell substrates used for human biologicals [J]. Develop Biol Standard, 1989,70:11-26
[2] Horaud F. Absence of viral sequences in the WHO Vero cell bank: A collaborative study [J]. Develop Biol Standard, 1992,76:43-46
, 百拇医药
[3] Nontagnon B. 1000 liter scale microcarrier culture of Vero cells for killed poliovirus vaccine-promising results [J]. Develop Biol Standard, 1983,55:37-42
[4] Florian H. Viral vaccines and residual cellular DNA [J]. Biologicals, 1995,23:225-228
[5] WHO. Requirements for Poliomyelitis vaccine (ORAL) [S]. WHO TRS, 1990,800:30-65
[6] Sepharose ion exchangers. Ion Exchange Chromatography: principles & methods [R]. Pharmacia, 1993,10-12
, 百拇医药
[7] James S R. A collaborative study on DNA quantitation in biological products [J]. Biologicals, 1995,23:199-205
[8] Floyd R, Sharp D G. Viral aggregation: Buffer effects in the aggregation of Poliovirus and reovirus at low and high pH [J]. Appl Environ Microbiol, 1979,38(3):395-401
[9] John C, Horaud F N. DNA, dragons and sanity [J]. Biologicals, 1995,23:233-238
[10] Chezzi C. Genetic stability of oral poliovaccine prepared on primary monkey cells or Vero cells-effects of passage in cell culture and the human gastrointestinal truct [J]. Vaccine, 1998,16:2031-2038
, http://www.100md.com
[11] Fleckenstein B, Muthian D, Ronald D H. Tumor induction with DNA of oncogenic primate herpes virus [J]. Nature, 1978,274:57-59
[12] Carcagne J. Evaluation of transforming activity of cellular DNAs from different origins by NIH 3T3 transfection test [J]. Biologicals, 1991,19:317-325
[13] Wierenga D E, Cogan J, Pettricciani J C. Administration of tumor cell chromatin to immunosuppressed and nonimmunosuppressed nonhuman primates [J]. Biologicals, 1995, 23:221-224
[14] Moray F, David J. Philp D M. Antigenic structure of poliovirus or IPV [J]. J Gen Virol, 1993,74:685-690
收稿日期：1999-04-21，修回日期：1999-10-21, http://www.100md.com
单位：郑从义(武汉大学中国典型培养物保藏中心，武汉 430072)；廖国阳 姜述德 李卫东 王明秀 陈光武 王燕(中国医学科学院 中国协和医科大学 医学生物学所，昆明 650107)
关键词：脊髓灰质炎减毒活疫苗； 凝胶柱层析； 高效纯化
中国病毒学000304摘要：应用Q-Sepharose Fast Flow对Vero细胞基质制备的I型口服脊髓灰质炎减毒活疫苗悬液进行纯化。病毒液经滤过澄清和超滤浓缩，获得85%的病毒感染性滴度回收率，而经Q-Sepharose F.F.纯化的病毒悬液，病毒感染性滴度回收率达100%。纯化后的病毒液用α-32P dATP标记DNA探针膜杂交法测定，宿主Vero细胞基质DNA残余含量远低于100 pg/剂量的标准；rct/40特征、病毒形态及病毒衣壳蛋白组份等生物学性状无显著变化。研究结果提示，Q-Sepharose F.F.是Vero细胞制备口服脊髓灰质炎减毒疫苗的理想纯化材料。
, 百拇医药
中图分类号：Q939.4，R512.4 文献标识码：A
文章编号：1003-5125(2000)03-0226-06
Studies on the Purification of OPV and its Characterization
LIAO Guo-yang，JIANG Shu-de，LI Wei-dong，WANG Ming-xiu，CHEN Guang-wu，WANG Yan
(Institute of Medical Biology, Chinese Academy of Medical Science & Peking Union of
Medical College, Kunming 650107, China)
, 百拇医药
ZHENG Cong-Yi
(China Center for Type Culture Collection, Wuhan University, Wuhan 430072, China)
Abstract: Q-Sepharose F.F. was adopted to purify the suspension of oral poliovaccine (OPV), which is prepared from Vero cells. After clarification and concentration by ultrafiltration, the recovery of virus infected titre may attain above 85%. Using column chromatography on Q-Sepharose F.F. to purify the concentrated virus suspension, the purified viruses attain 100% recovery of virus infectivity. Dot membrane hybridization was used to detect DNA with the probe of Vero cell genome DNA which was labed with α-32P dATP, the contents of residual substrate DNA was less than 100 pg/dose. The process of downstream had no significant influence on some biologiccal characters of purified viruses, such as virus morphology, tumorigenicity, rct/40 character and capsid protein. The results indicate that Q-Sepharose F.F. is an ideal chromatography material for purifying OPV prepared from Vero cells.
, http://www.100md.com
Key words: Oral polio vaccine (OPV); Q-Sepharose F.F.; Purification
作者简介：廖国阳(1960年-)，男，博士生
参考文献
[1] Hayflick L. History of the cell substrates used for human biologicals [J]. Develop Biol Standard, 1989,70:11-26
[2] Horaud F. Absence of viral sequences in the WHO Vero cell bank: A collaborative study [J]. Develop Biol Standard, 1992,76:43-46
, 百拇医药
[3] Nontagnon B. 1000 liter scale microcarrier culture of Vero cells for killed poliovirus vaccine-promising results [J]. Develop Biol Standard, 1983,55:37-42
[4] Florian H. Viral vaccines and residual cellular DNA [J]. Biologicals, 1995,23:225-228
[5] WHO. Requirements for Poliomyelitis vaccine (ORAL) [S]. WHO TRS, 1990,800:30-65
[6] Sepharose ion exchangers. Ion Exchange Chromatography: principles & methods [R]. Pharmacia, 1993,10-12
, 百拇医药
[7] James S R. A collaborative study on DNA quantitation in biological products [J]. Biologicals, 1995,23:199-205
[8] Floyd R, Sharp D G. Viral aggregation: Buffer effects in the aggregation of Poliovirus and reovirus at low and high pH [J]. Appl Environ Microbiol, 1979,38(3):395-401
[9] John C, Horaud F N. DNA, dragons and sanity [J]. Biologicals, 1995,23:233-238
[10] Chezzi C. Genetic stability of oral poliovaccine prepared on primary monkey cells or Vero cells-effects of passage in cell culture and the human gastrointestinal truct [J]. Vaccine, 1998,16:2031-2038
, http://www.100md.com
[11] Fleckenstein B, Muthian D, Ronald D H. Tumor induction with DNA of oncogenic primate herpes virus [J]. Nature, 1978,274:57-59
[12] Carcagne J. Evaluation of transforming activity of cellular DNAs from different origins by NIH 3T3 transfection test [J]. Biologicals, 1991,19:317-325
[13] Wierenga D E, Cogan J, Pettricciani J C. Administration of tumor cell chromatin to immunosuppressed and nonimmunosuppressed nonhuman primates [J]. Biologicals, 1995, 23:221-224
[14] Moray F, David J. Philp D M. Antigenic structure of poliovirus or IPV [J]. J Gen Virol, 1993,74:685-690
收稿日期：1999-04-21，修回日期：1999-10-21, http://www.100md.com