灭活SARS病毒的纯化鉴定及初步应用

为制备纯化的灭活SARS病毒,取广东省CDC提供的灭活SARS-CoV病毒培养液进行超滤,收集相对分子质量介于100000至500000之间的组分,并浓缩。对纯化过程中各组分进行抗原活性鉴定和蛋白质含量测定,并加佐剂后免疫动物,测定抗体免疫应答。结果表明,超滤后活性抗原蛋白质回收率7.6%,纯化倍数为9,抗原活性达到1:480。免疫家兔获得的血清ELISA效价达到1:10000以上,中和抗体效价达到1:2000以上。表明该纯化工艺能获得较高纯度的灭活SARS-CoV病毒颗粒。

Purification,identification and preliminary application of inactivated SARS-CoV vaccine

To prepare the purified inactivated SARS-CoV vaccine, the SARS-CoV(F69) from CDC of Guangdong province was cultivated in Vero-E6 cells and the cultured cells were pooled and put on freezing under -20℃ and thawing for 3 times, the pooled cell suspension was centrifuged at 10 000 g for 10 minutes, and the supernatant fluid was inactivated with 0.4% formaldehyde for 4 hours. Then, samples were super-filtered with 500 Mr×103 permeable membrane for purification, and the components with molecular weight of 100-500 Mr×103 were collected and concentrated. The identification of antigenic activities and determination of protein contents were performed for each component collected. These purified product was used to immunize rabbits with adjuvant, and the specific IgG antibody responses were detected thereafter. It was found that the recovery rate of active proteins after super-filtration was 7.6% with a 9-fold purification of the active antigen and the antigenic activities reached up to 1:480. The specific IgG antibody titers assayed by ELISA were up to 1:10 000, and the neutralizing antibody titer of anliserum was up to 1:2 000. It concludes that a highly purified inactivated SARS-CoV virus prepa-ration was obtained by the method used in the present study.