表达H9N2 AIV双脯氨酸突变HA蛋白的基因Ⅶ型重组NDV的构建及鉴定

Construction and identification of recombinant Newcastle disease virus type Ⅶ expressing H9N2 AIV double proline mutant HA protein

构建表达H9N2亚型禽流感病毒(H9N2 subtype avian influenzavirus, H9N2 AIV)双脯氨酸突变的HA蛋白的基因Ⅶ型重组新城疫病毒(Newcastle disease virus, NDV),为NDV和H9N2 AIV新型二联疫苗的研发提供前期基础。通过对H9N2亚型AIV的HA基因进行分析,将其编码区第403位氨基酸由I突变为P以及第411位氨基酸由V突变为P,并利用反向遗传操作系统,将突变前与双脯氨酸突变的HA插入到基因Ⅶ型NDV病毒弱毒株rDHN3-mF基因组P基因与M基因之间的非编码区,经病毒拯救后获得重组病毒(rDHN3mF-HA/HA-2P)。通过Western blot试验证明重组病毒rDHN3mF-HA-2P可在DF1细胞内表达特异性HA蛋白,且与rDHN3mF-HA相比,经双脯氨酸突变后的HA蛋白表达水平更高。重组病毒rDHN3mF-HA-2P可以在鸡胚中稳定传代,与亲本毒株具有相似的复制特性,保留了rDHN3-mF株在鸡胚中高滴度复制、低致病性等生物学特性。本试验成功构建表达H9N2亚型AIV的双脯氨酸突变HA基因的重组病毒rDHN3mF-HA-2P,将为研发同时防控H9N2亚型AIV毒株和基因Ⅶ型NDV感染的新型高效、廉价多联疫苗奠定基础。

The recombinant Newcastle disease virus(NDV)typeⅦexpressing double proline mutated HA protein of H9N2 subtype avian influenza virus(H9N2 AIV)was constructed to provide a preliminary basis for the research and development of new duplex vaccines of NDV and H9N2 AIV.Through the analysis of the HA gene of H9N2 AIV,the 403th amino acid in the coding region were mutated from I to P and the 411th amino acid was mutated from V to P.Using the reverse genetic operating system,the HA without mutation and HA with double proline mutation were inserted into the non-coding region between P gene and M gene of type Ⅶ rDHN3-mF attenuated NDV,and the recombinant virus rDHN3mF-HA/HA-2P was obtained after virus rescue.Western blot results showed that the recombinant virus rDHN3mF-HA-2P could express specific HA protein in DF1cells,and the expression level of HA protein mutated by double proline was higher than that of rDHN3mF-HA.The recombinant virus rDHN3mF-HA-2P could be propagated stably in chicken embryos,which had similar replication characteristics to its parent strain,and retained the biological characteristics such as high titer replication and low pathogenicity of rDHN3-mF strain in chicken embryos.In this study,a recombinant virus rDHN3mF-HA-2 Pexpressing double proline mutant HA of H9N2 AIV was successfully constructed,which will lay a foundation for the development of a new efficient and cheap multi-vaccine to prevent and control both H9N2 AIV strain and genotype Ⅶ NDV infection.