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目的以重配技术制备H1N1流感病毒Vero细胞适应株,为以Vero细胞为基质生产流感疫苗奠定基础。方法以流感病毒Vero细胞适应株A/Yunnan/1/2005Va(H3N2)为母本株,与WHO推荐的2007～2008年度北半球流感疫苗生产用毒株A/Caledonia/20/99(H1N1)共同感染SPF鸡胚和Vero细胞,用抗A/Yunnan/1/2005Va(H3N2)抗体筛选重配病毒,在Vero细胞连续传12代,采用血凝抑制试验和RT-PCR鉴定病毒型别。将重配病毒经Vero细胞大量培养,重配前的病毒经鸡胚大量培养,分别经甲醛灭活,制备灭活疫苗,免疫ICR小鼠,检测其免疫原性。结果获得了1株Vero细胞适应的高产H1N1流感病毒株,连续传9代后,病毒血凝滴度维持在512,免疫原性与重配前的毒株相比,差异无统计学意义。结论通过流感病毒Vero细胞适应株与流行株的重配和抗体筛选,可以获得H1N1流感病毒Vero细胞适应株。
OBJECTIVE: To prepare Vero cell-adapted strains of H1N1 influenza virus by reassortment technology, which lays the foundation for the production of influenza vaccine based on Vero cells. Methods The influenza virus Vero cell adapted strain A / Yunnan / 1 / 2005Va (H3N2) was used as the female parent strain to co-infection with the WHO recommended strain A / Caledonia / 20/99 (H1N1) for influenza vaccine production in the northern hemisphere 2007-2008 SPF chicken embryos and Vero cells. The reassortant viruses were screened with anti-A / Yunnan / 1 / 2005Va (H3N2) antibody and continuously passaged in Vero cells for 12 passages. The virus type was identified by hemagglutination inhibition test and RT-PCR. The reassortant virus was extensively cultured in Vero cells. The virus before re-mating was extensively cultured in chicken embryos and inactivated by formaldehyde respectively to prepare an inactivated vaccine and to immunize ICR mice to test its immunogenicity. Results One Vero cell-adapted H1N1 influenza virus strain was obtained. After 9 consecutive passages, the virus titer remained at 512 and the immunogenicity was not significantly different from that before re-allocation. Conclusion The H1N1 influenza virus Vero cell adapted strain can be obtained through the reassortment and antibody screening of influenza virus Vero cell adapted strains and epidemic strains.