基因Ⅶ型新城疫病毒HN蛋白茎区居间序列突变株的拯救及其生物学特性的分析

Recovery and biological properties analysis of genotype Ⅶ Newcastle disease virus with mutations in the HN intervening region

为了探究新城疫病毒(NDV)HN蛋白茎区居间序列氨基酸突变对其蛋白及病毒生物学特性的影响,利用overlapping PCR分别扩增包含E91A和S92A突变的HN基因,并将突变引入真核表达质粒pCAGGS及NDV全长cDNA中。通过红细胞吸附试验、神经氨酸酶试验等分析突变对蛋白功能的影响;并通过反向遗传操作技术拯救突变病毒,分析突变对病毒生物学特性的影响。结果显示,E91A突变能显著增强HN蛋白的红细胞吸附活性,同时显著降低神经氨酸酶活性;而S92A突变则导致蛋白的红细胞吸附活性及促融合功能显著下降;并且2个突变均会显著削弱病毒的复制能力,在一定程度上降低病毒毒力,影响其组织嗜性。研究结果表明,HN茎区居间序列的E91和S92氨基酸突变对蛋白及病毒的生物学特性具有重要影响,可作为减毒疫苗株改造及小分子药物设计的靶标位点。

To study how amino acid substitutions in the intervening region of Newcastle disease virus(NDV)HN stem influence both protein and viral biological properties,HN gene containing E91 Aand S92A mutations were cloned into pCAGGS vector respectively and into the whole NDV genomic cDNA for protein function study and viral recovery.In result,the HN-E91 Amutation increased significantly receptor binding activity but decreased markedly neuraminidase activity,while the HN-S92 Anotably impaired hemadsorpotion and fusion promotion functions.At the viral level,the two mutations reduced viral proliferation and resulted in attenuation to some extent,and the E91 Aand S92Arescued viruses caused a lighter tissue tropism compared to the parental virus.The result showed that the mutations(E91and S92)had great impacts on both protein and viral biological properties,which provided a theoretical basis for the development and design of attenuated vaccines and small-molecular drugs.