不同致病性H7N9亚型流感病毒HA蛋白的比较分析

Comparative Analysis on HA Protein of H7N9 Subtype Influenza Virus with Different Pathogenicity

为明确高致病性和低致病性H7N9亚型流感病毒血凝素(HA)蛋白的血清学特性是否有差异,通过体外表达两种H7N9亚型流感病毒HA蛋白并进行分析。利用RT-PCR方法扩增出高、低致病性H7N9亚型流感病毒HA基因,测序,并克隆于真核表达载体pCAGGS-Flag中,构建两个重组表达质粒,命名为pCAF-H7HPHA和pCAF-H7LPHA,分别转染293T细胞,用间接免疫荧光试验和Western blot试验观察HA蛋白的表达情况。结果显示:与低致病H7N9亚型流感病毒比较,高致病性H7N9亚型流感病毒HA基因裂解位点有连续的碱性氨基酸插入,糖基化位点和受体结合位点没有差异。不同致病性毒株的HA蛋白均能表达良好,表达蛋白分子量约为70 ku;高致病性H7N9亚型流感病毒HA蛋白(或血清)可以与低致病H7N9流感血清(或蛋白)发生反应。结果表明:不同致病性H7N9亚型流感病毒HA蛋白在血清学上没有差异性,为H7N9亚型流感病毒HA蛋白的研究奠定基础。

In order to clear the difference of serological characteristics of hemagglutinin（HA） between low-pathogenicity and high-pathogenicity H7N9 subtype influenza virus, HA proteins of two influenza viruses were expressed in vitro, and analyzed. HA gene was amplified by RT-PCR, sequenced, and cloned into the eukaryotic expressive vector pCAGGS-Flag to construct two recombinant expression plasmids, pCAF-H7HPHA and pCAF-H7LPHA. Recombinant expression plasmids were transfected into 293T cell. The expression of HA proteins were measured by indirect immunofluorescence and western blot. The results showed that continuous amino acid insertion was only found at the cleavage site of HA gene of high pathogenicity H7N9 subtype influenza virus, but not in the low-pathogenicity H7N9 subtype influenza virus. There were no differences in glycosylation sites and receptor-binding sites between the two H7N9 by influenza viruses. HA proteins were stably expressed, and the molecular weight was 70 ku. The HA proteins（or serum） of high-pathogenicity H7N9 subtype influenza virus could react with low-pathogenicity H7N9 subtype influenza virus serum（proteins）. These results suggested that serological characteristics of HA protein from different pathogenic H7N9 subtype influenza virus were not different, which provided a basis for analysis on HA protein of H7N9 subtype influenza virus.