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目的比较两种不同的凝胶过滤介质对Sabin株脊髓灰质炎病毒的分离纯化效果。方法将Ⅰ、Ⅱ、Ⅲ型Sabin株脊髓灰质炎病毒收获液各3批经超滤浓缩后,分别采用Sepharose CL-6B和Sepharose 6FF填料进行凝胶过滤纯化,分段收集各纯化峰,检测D抗原及蛋白含量,计算比活性、蛋白去除率及抗原回收率,确定病毒峰收集范围。取抗原含量最高的病毒收获液,利用DEAE Sepharose FF离子交换填料进行纯化后,检测Vero细胞DNA残留量、Vero细胞蛋白残留量、牛血清白蛋白残留量、抗生素残留量。结果 Sepharose 6FF填料按6%柱体积上样进行凝胶纯化,纯化的Ⅰ、Ⅱ、Ⅲ型病毒收获液抗原回收率均值分别为78.69%、78.03%和78.37%,与Sepharose CL-6B填料按3%柱体积上样(77.29%、78.60%和77.36%)相比,差异无统计学意义(P>0.05)。两种填料纯化的各型收获液再经离子交换纯化后,蛋白去除率、比活性、Vero细胞DNA残留量、Vero细胞蛋白残留量、牛血清白蛋白残留量、抗生素残留量检测结果均符合《中国药典》三部(2015版)相关要求,两种填料纯化的各型收获液比活性差异无统计学意义(P>0.05)。结论使用Sepharose 6FF纯化的各型病毒纯化液抗原回收率和比活性与Sepharose CL-6B无明显差异,但可以减少纯化的上样次数,大幅提高纯化效率,可用Sepharose 6FF替换Sepharose CL-6B。
Objective To compare the isolation and purification of Sabin poliovirus by two different gel filtration media. Methods Three batches of Sabin poliovirus type Ⅰ, Ⅱ and Ⅲ poliovirus harvested liquid were concentrated by ultrafiltration and purified by gel filtration using Sepharose CL-6B and Sepharose 6FF respectively. Antigen and protein content, calculated specific activity, protein removal and antigen recovery rate, to determine the scope of the virus peak collection. The harvested virus solution with the highest antigen content was purified by DEAE Sepharose FF ion exchange packing. The DNA residues, Vero cell protein residues, bovine serum albumin residues and antibiotic residues in Vero cells were detected. Results Sepharose 6FF was purified by gel filtration on a 6% column. The recoveries of purified antigenic I, II and III viruses were 78.69%, 78.03% and 78.37% % Column volume (77.29%, 78.60% and 77.36%), the difference was not statistically significant (P> 0.05). After the two kinds of packing purified all kinds of harvested liquid were purified by ion exchange, the protein removal rate, specific activity, Vero cell DNA residue, Vero cell protein residue, bovine serum albumin residue, antibiotic residue test results are in line with “ Chinese Pharmacopoeia ”three (2015 version) of the relevant requirements, two kinds of filler purified various types of harvested fluid specific activity was no significant difference (P> 0.05). Conclusion The recovery rate and specific activity of Sepharose CL-6B purified by Sepharose 6FF were similar to those of Sepharose CL-6B. However, Sepharose CL-6B could be replaced by Sepharose 6FF by reducing the number of purified samples and greatly improving the purification efficiency.