番鸭小鹅瘟弱毒D株NS蛋白抗原位点特征分析

Molecular Characteristics Analysis of D-strain Vaccine Virus of Muscovy Duck-origin Goose Parvovirus NS Protein Epitopes

为获得番鸭小鹅瘟病毒弱毒株(MDGPV-D)非结构蛋白NS基因的相关信息,根据已发表的MDGPV-PT株全基因序列,应用DNAStar分子生物学软件设计1对引物,采用高保真PCR技术扩增MDGPV-D株NS全基因序列。对番鸭小鹅瘟病毒疫苗弱毒D株(MDGPV-D)NS基因进行克隆、测序,并利用生物信息学技术分析MDGPV-D株NS蛋白的同源性、遗传衍化、N-糖基化位点、磷酸化位点、B细胞抗原表位、T细胞抗原表位及其二级结构。结果表明,D株NS全基因大小为1 884bp,编码627个氨基酸(GenBank登录号:JF926696),与MDPV NS基因的亲缘性最近核苷酸及其推导氨基酸同源性分别为97.9%～98.6%,97.6%～98.2%;MDGPV-D株NS蛋白具有3个潜在的N-糖基化位点和27个磷酸化位点,可能存在11个B细胞抗原表位,13个CD8+CTL表位,10个CD4+Th抗原表位;二级结构分析显示,α螺旋和无规则卷曲含量高,分别为40.67%、41.15%,而β转角仅占4.63%。与亲本强毒PT株相比,弱毒D株的NS蛋白存在2个定点突变,分别位于核苷三磷酸区域(第338位氨基酸)与CD4+Th抗原表位(第225位氨基酸)。

To analyze the non-structural protein NS gene of vaccine muscovy duck （Cairina moschata） origin Goose parvovirus （GPV） D strain, a pair of primers were designed by DNAStar software based on the published sequences of MDGPV-PT strain from GenBank. We examed the homology, heredity evolution, N-glycosylation sites, phosphorylation sites, B cell epitopes, T cell epitopes and their secondary structure of NS protein with bioinformatics software. The full length NS gene was amplified by PCR and cloned into pMD18-T vector. Sequencing demonstrated that the NS gene ol MDGPV-D strain contained 1 884 bps, encoding 627 amino acids （GenBank number： JF926696）. The MDGPV-Dstrain NS gene was highly similar to that of MDPV （nt： 97.9%-- 98.6%, aa： 97.6%- 98.2%）. It was also shown that there were 3 potential N-glycosylation sites and 27 phosphorylation sites existed in MDGPV-D strain NS protein, and 11 B cell epitopes, 13 CD8＋ CTL epitopes and 10 CD4＋Th epitopes as well. Then the prediction of secondary structure of NS protein showed that alpha helix, random coil and beta angle account for 40.67%, 41.15% and 4.63% respectively. Comparison of the parental virulent strain PT with its attenuated vaccine strain D revealed two site-specific mutagenesis; one was in the nucleoside triphosphate （NTP） binding motif at amino acid 338, and the other in a CD4＋ Th epitope at amino acid 225. These studies has provided a molecular basis for investigating attenuation mechanisms of MDGPV, and benefitted for further researches on genetically engineered MDGPV vaccines.