摘要
目的采用复合模式介质Capto core 700纯化轮状病毒(rotavirus,RV),去除培养液中的残余细胞宿主蛋白和DNA,提高病毒收率。方法将RV LH9毒种接种Vero细胞,制备RV原液,澄清、超滤后,用Capto core 700纯化:样品LH9、LH9+150 mmol/L Na Cl[以缓冲液A(20 mmol/L PB+150 mmol/L Na Cl,p H 7.0)作为缓冲液]上样纯化分别标记为A、B组合,样品LH9+300 mmol/L Na Cl[以缓冲液B(20mmol/L PB+300 mmol/L Na Cl,p H 7.0)作为缓冲液]上样纯化标记为C组合,样品LH9+450 mmol/L Na Cl[以缓冲液C(20 mmol/L PB+450 mmol/L Na Cl,p H 7.0)作为缓冲液]上样纯化标记为D组合,洗脱,收集流穿峰,检测纯化后病毒的滴度和收率、RV RNA、RV抗原性、残余宿主细胞蛋白(HCP)和DNA;并用初步筛选出的纯化组合纯化3批RV超滤浓缩液,检测各项指标,进行进一步验证。结果用20 mmol/L PB+150 mmol/L Na CL缓冲体系和样品中加入150 mmol/L Na Cl的组合纯化RV,病毒收率在80%以上,病毒核酸完整,其抗原性未发生改变,可去除94%以上的HCP,残余DNA符合《中国药典》三部(2010版)标准。结论 Capto core 700纯化RV收率和残余蛋白去除效率均较高,组合B操作相对简便,工艺时间短,病毒收率高,更适于RV的纯化。
Objective To purify rotavirus （RV） with multimode media Capto core 700 so as to remove the residual host cell protein （HCP） and DNA in culture and increase the yield of virus. Methods The seeds of RV LH9 were inoculated to Vero cells, and the prepared bulk of RV was clarified, ultrafiltrated and purified by Capto core 700. LH9, LH9 ＋ 150 mmol/L NaCl [using buffer A （20 mmol/L PB ＋ 150 mmol/L NaCl, pH 7. 0）], LH9 ＋ 300 mmol/L NaCl [using buffer B （20 mmol/L PB ＋ 300 mmol/L NaC1, pH 7. 0）], LH9 ＋ 450 mmol/L NaCl [using buffer C （20 mmol/L PB ＋ 450 mmol/L NaCI, pH 7.0） ] were served as samples for purification in groups A, B, C and D, respectively. The flowthrough peaks of various groups were collected and determined for the titer and yield of purified RV, RV RNA, RV antigenicity, residual HCP and DNA. Three batches of RV concentrated by uhrafiltration were purified by the preliminarily screened procedure, of which various indexes were tested and further verified. Results The yield of RV purified by using 20 mmol/L PB ＋ 150 mmol/L NaCl as buffer and addition of 150 mmol/L NaCl into samples reached more than 80%, while the RV DNA was intact, the antigenicity showed no significant change, more than 94% of HCP was removed, and the residual DNA content met the requirements in Chinese Pharmacopoeia （Volume Ⅲ, 2010 edition ）. Conclusion Both the yield and removal rate of residual HCP of RV purified by Capto core 700 were high. However, the procedure in group B was relatively simple and time-saving, which was more suitable for the purification of RV.
出处
《中国生物制品学杂志》
CAS
CSCD
2015年第1期72-78,共7页
Chinese Journal of Biologicals
