表达H5N1亚型禽流感病毒HA蛋白重组新城疫病毒的构建以及密码子优化的HA免疫增强作用

Construction of recombinants Newcastle disease virus expressing H5N1 subtypes avian influenza virus HA protein and the enhance protection by optimized-codons

为构建表达H5N1亚型禽流感病毒(AIV)HA蛋白重组新城疫病毒(NDV)及比较密码子优化的HA免疫增强作用,本研究利用NDV LaSota弱毒疫苗株的反向遗传操作系统,分别构建了表达野毒型H5N1亚型AIV HA蛋白和密码子优化的HA蛋白的重组NDV,并分别命名为rL-H5HA和rL-optiH5HA。对拯救的重组病毒进行RT-PCR、间接免疫荧光和western blot鉴定,结果表明,两种重组病毒均可以在BHK-21细胞中以及鸡胚成纤维细胞稳定地表达HA蛋白。此外,rL-optiH5HA在体外增殖能力及在体内刺激产生抗体均显著高于rL-H5HA。本研究表明,密码子优化的HA重组NDV具有明显的免疫增强作用。本研究为研制安全、有效的新型禽流感疫苗提供了实验依据。

In order to construct a recombinant Newcastle disease virus （NDV） expressing hemagglutinin （HA） of H5N1 subtype avian influenza virus （AIV） and evaluate the ability of enhancement for antibody product by HA with optimized-codons, we constructed and rescued the rL-H5HA and rL-optiH5HA of recombinant NDVs expressing the original HA protein and the codon-optimized H5HA （optiH5HA） protein of the H5N1 subtype AIV GD322 strain, respectively. The identification results showed that the HA proteins were stably expressed form the recombinant virus infected BHK-21 cell and CEF cell detected by RT-PCR, western blot, and indirect immunofluorescence assay. While, the HI titers in chickens immunized with rL-optiH5HA were significantly higher than those chickens immunized with rL-H5HA. These results demonstrated that the rL-optiH5HA had the potential of being used as a life NDV vector based A1V vaccine.