甲型流感病毒纯化及病毒生物学特征分析

Purification and biological characterization of influenza virus type A

目的评价蔗糖密度梯度离心纯化甲型流感病毒的方法,并对纯化的病毒颗粒特征和免疫原性进行分析。方法接种鸡胚培养病毒,收获尿囊液,超速离心浓缩,蔗糖密度梯度离心纯化病毒,用血凝试验、蛋白定量、透射电镜和SDS-PAGE鉴定病毒颗粒。取病毒纯化产物以0.03mg/只和0.005mg/只分组免疫小鼠,采集小鼠血清,利用血凝抑制试验测定血清血凝抑制滴度。结果成功利用蔗糖密度梯度离心法纯化出流感病毒,病毒原液经纯化后被分离成L、M、H三个病毒带,其中M病毒带的吸光度、蛋白浓度及血凝滴度最高,蛋白浓度可达1.2mg/mL,血凝滴度高达1×49。电镜观察显示,L区带为具有血凝活性的质膜和结构疏松的病毒组成,M区带病毒颗粒呈球形、丝状或棒状。同时,有大量空心病毒颗粒存在。H区带与M区带类似,但未发现空心病毒颗粒。纯化的病毒免疫小鼠后可产生具有较高血凝抑制活性的抗体。结论蔗糖密度梯度离心技术可用于流感病毒抗原的制备,且不影响病毒的免疫原性。因此,密度梯度离心法可为流感病毒的基础研究及疫苗开发提供技术支持。

Objective To evaluate the purification method of sucrose density gradient for influenza A virus and analyze the characterization and immunogenicity of influenza A virus. Methods Influenza virus was produced with allantoic fluid from fertilized eggs. Harvested allantoic fluid was concentrated by ultracentrifugation and the viral particles were purified utilizing sucrose density gradient ultracentrifugation. The purification of viral particles was analyzed by hemagglutination test, micro-BCA protein assay, transmission electron microscopy and SDS-PAGE. The mice were immunized with the purification of inactivated virus. The serum of the titer of hemagglutination inhibition was determined by hemagglutination inhibition test. Results Influenza viral particles were successfully purified utilizing sucrose gradient ultracentrifugation. The virus was found to be separated into three zones （zones L, M and H）. The optional density, protein concentration and HA titer of M zone were the highest. The protein concentration and HA titer were 1.2mg/mL and 1×49, respectively. TEM revealed that zone L contained viral particles and plasma membrane with HA activity. Viral particles of zone M were spherical or extended threadlike, with some hollow viral particles. H and M were the same; however, hollow viral particles were not found in zone H. The high HAI activity antibody responses were induced by the purified virus in mouse immunogenicity studies. Conclusion Sucrose density gradient ultracentrifugation is an effective method for preparation of influenza virus, which can ensure the immunogenicity of the virus. Therefore, the method of density gradient ultracentrifugation can provide technical support for viral fundamental study and the development of influenza vaccine.