目的  构建表达甲型H5N1禽流感病毒（H5N1 avian influenza A virus，H5N1 AIAV）NIBRG14株结构蛋白的重组痘苗病毒，为研制新型人用流感疫苗奠定基础。方法  通过反转录PCR扩增H5N1 AIAV NIBRG14株的血凝素（hemagglutinin，HA）和神经氨酸酶（neuraminidase，NA）编码基因，并将改造的HA、NA基因克隆至痘苗病毒穿梭质粒 pSCCK。在重组质粒与痘苗病毒天坛株（vaccinia virus Tiantan，VTT）于Vero细胞中发生同源重组后，筛选同时表达HA和NA的重组痘苗病毒（rVTT-HA/NA），并对其进行鉴定。结果  反转录PCR扩增的HA和NA基因大小约分别为1 700和1 400 bp，与预期相同。DNA测序证实，改造的HA和NA序列正确。对获得的rVTT-HA/NA进行PCR及测序表明，HA和NA基因正确插入VTT。蛋白质印迹显示，rVTT-HA/NA感染的Vero细胞表达的HA和NA能与相应的抗体发生反应。表达的HA具有血凝活性，血凝滴度为1∶32。结论  重组痘苗病毒rVTT-HA/NA可稳定表达H5N1 AIAV NIBRG14株的HA和NA。

  Objective  To construct a recombinant vaccinia virus expressing the structural proteins of H5N1 avaian influenza A virus (AIAV) NIBRG14 strain, in order to lay the foundation for developing new influenza vaccine for human. Methods  Full length genes encoding hemagglutinin (HA) and neuramindase (NA) of H5N1 AIAV NIBRG14 strain were amplified with reverse transcription PCR (RT-PCR), and modified HA and NA genes were cloned into shuttle plasmid pSCCK of vaccinia virus. After the recombinant plasmid homologously recombined with the vaccinia virus Tiantan (VTT) in Vero cells, the recombinant vaccinia virus (rVTT-HA/NA) co-expressing HA and NA of H5N1 AIAV NIBRG14 strain was selected and validated. Results  The sizes of HA and NA genes amplified by RT-PCR were about 1 700 and 1 400 bp, respectively, and in line with expectations. The modified HA and NA sequences were correct according to DNA sequencing. The results of PCR and sequencing for the obtained rVTT-HA/NA showed that the HA and NA genes were inserted into VTT correctly. Western blotting confirmed that HA and NA expressed by rVTT-HA/NA infected Vero cells were able to react with corresponding antibodies. The expressed HA had hemagglutination activity, and its titer was 1∶32. Conclusion  The  recombinant vaccinia virus rVTT-HA/NA can stably express HA and NA of H5N1 AIAV NIBRG14 strain.