猪繁殖与呼吸综合征病毒 FS 株 Nsp 9基因的克隆与序列分析

摘要

为了对猪繁殖与呼吸综合征病毒(PRRSV)Nsp 9基因的功能进行预测,利用 RT-PCR 法从细胞毒中扩增PRRSV FS 株的 Nsp 9基因,并将其克隆至 pMD18-T 载体上,测序得到 Nsp 9基因序列,利用多种生物信息学软件分析 Nsp9序列生物学信息。结果显示,Nsp 9基因全长1929 bp,目的基因的蛋白分子质量为70.5 ku,pI 为7.78,表明该蛋白不稳定；抗原性分析显示,Nsp 9基因存在多个抗原位点,柔韧性较好,多处位点的亲水性较强,适合作为单抗制备的表位；同种亚型不同毒株的 Nsp 9基因氨基酸同源性为96.9%~98.9%,这表明 Nsp 9基因高度保守,可进一步作为检测靶蛋白用于猪繁殖与呼吸综合征(PRRS)的诊断与疫苗免疫；亚细胞定位预测该基因可能定位于细胞质中,而不是细胞核内；同源建模预测三级结构结果显示,三维结构呈右手型,具有3个亚结构域,没有信号肽。此外,亲本株 Nsp 9基因与疫苗株 Nsp 9基因存在多个氨基酸的突变,这些氨基酸的插入与缺失是否与病毒的聚合酶活性和毒力有关还需要进一步试验验证。%In order to evaluate the function of Nsp 9 gene,the target gene of porcine reproductive and respiratory syndrome virus (PRRSV)FS strain was amplificated and sequenced after being cloned into the pMD18-T vector.The physical and chemical properties,homology,hydrophilicity, surface probability plot,antigenic index,secondary structure and subcellular localization were pre-dicted by various softwares.The results showed that the length of Nsp9 was 1 929 bp,its predic-ted molecular weight was 70.5 ku and pI was 7.78,and it was unstable protein.There were many antigen sites,and the flexibility and hydrophilicity of Nsp9 were ideal.The study showed that Nsp9 possessed potential antigenicity,and it fits for preparation of monoclonal antibodies.The re-sults of subcellular location showed that it might exist in the cytoplasm.Nsp9 of FS strain shared 96.9% to 98.9% amino acid homology with other strains of the some hypotype.Some amino acid mutations were found between the parent strains and vaccine strains,and insertions and deletions could be found among the European strains and the American strains.Whether these insertions and deletions correlate with the virulence and polymerase needed further research.