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目的 研制出安全、有效、质量可控的SARS冠状病毒死活疫苗;同时,制备能用于SARS被动免疫和治疗用的异源高效价特异性抗体,用于SARS的免疫防治。方法 采用细胞培养法,从非典型肺炎患者标本中分离获得病毒株,通过电镜、血清学以及RT-PCR技术进行病毒鉴定。运用细胞规模化培养技术大量制备SARS冠状病毒。用温度灭活法和甲醛灭活法对SARS冠状病毒进行灭活处理,筛选灭活条件。将不同类型和不同剂量的佐剂与灭活病毒加以配伍,优化SARS灭活疫苗;同时,以小鼠、兔、猴等为实验对象,进行灭活疫苗的免疫原性评价。用灭活疫苗免疫马,获取异源(马)高免血清,并进行应用效果评价。结果 从分离得到的SARS冠状病毒中,优选出适合疫苗制作的SARS病毒株2株-F69和Y3,初步建立了大规模培养SARS病毒的技术体系,病毒TCID_(50)达到10~6以上。确定了SARS病毒的灭活方法,优选出灭活疫苗的佐剂,并已生产出一定量的SARS灭活疫苗。将灭活疫苗初步用于小鼠、兔等的免疫接种,对其体液免疫和细胞免疫效果进行了初步检测。结论 试验研制的SARS灭活疫苗具有较强的免疫原性,设计的灭活疫苗生产技术路线是可行的,进一步的研究工作结束后,可望进行规模化开发生产。
OBJECTIVE: To develop a safe, effective and quality controllable SARS coronavirus live and killed vaccine. At the same time, a heterologous high titer specific antibody that can be used for passive immunization and treatment of SARS was prepared for immune control of SARS. Methods The virus strains were isolated from the specimens of patients with atypical pneumonia by cell culture, and the viruses were identified by electron microscopy, serology and RT-PCR. Mass production of SARS coronavirus using cell-scale culture techniques. SARS coronavirus was inactivated by temperature inactivation method and formaldehyde inactivation method, and the inactivation conditions were screened. The different types and doses of adjuvants were combined with the inactivated virus to optimize SARS inactivated vaccine. Meanwhile, the immunogenicity of inactivated vaccine was evaluated in mice, rabbits and monkeys. Immunization of horses with inactivated vaccine, access to heterologous (horse) high serum, and evaluation of the application effect. Results From the isolated SARS coronavirus, two strains of SARS-F69 and Y3, which are suitable for vaccine production, were optimized. The technical system of large-scale cultivation of SARS virus was established. The TCID50 of virus was above 10-6. The method of inactivation of the SARS virus was identified, the adjuvant of the inactivated vaccine was preferred and a certain amount of SARS inactivated vaccine had been produced. The inactivated vaccine was initially used in immunization of mice and rabbits, and the humoral and cellular immune effects were preliminarily tested. Conclusion The SARS inactivated vaccine developed by this study is highly immunogenic. The technical route of inactivated vaccine production is viable. After further research is completed, it is expected to conduct large-scale development and production.