Molecular Characteristics Analysis of D-strain Vaccine Virus of Muscovy Duck-origin Goose Parvovirus NS Protein Epitopes
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摘要: 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)。Abstract: To analyze the non-structural protein NS gene of vaccine muscovy duck (Cairina moschata) origin Goose parvovirus (GPV) D strain,apair 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 of MDGPV-D strain contained 1 884bps,encoding 627amino 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 11Bcell epitopes,13CD8 + CTL epitopes and 10CD4 + 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.
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Key words:
- MDGPV /
- NS protein /
- epitope /
- molecular characteristics
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