猪细小病毒疫苗株NS1基因克隆及生物信息学分析

为进一步开展猪细小病毒（porcine parvovirus，PPV）NS1蛋白功能研究，根据GenBank登录的PPV参考株NADL-2（登录号：NC001718）的NS1基因序列设计1对特异性引物，采用PCR技术扩增PPV疫苗株NS1基因，将PCR扩增产物克隆入pTA2载体构建重组质粒pTA2-NS1，并进行测序鉴定。测序结果显示，构建的pTA2-NS1质粒含有一个开放阅读框（ORF），全长1989 bp，共编码662个氨基酸，与GenBank登录的PPV参考株NADL-2、Kresse株的NS1基因的核苷酸同源性分别为99.85%和99.65%，氨基酸同源性分别为99.70%和99.70%。上述结果表明试验成功构建了含有PPV NS1基因的重组质粒pTA2-NS1。应用DNAStar软件及在线ProtScale、SignalP 4.1、TMHMM、Scansite、PSORTⅡ等生物信息学软件对NS1基因及其编码蛋白进行生物信息学分析，结果显示NS1蛋白分子质量为75.7 ku，等电点PI为6.82，是弱酸性蛋白；NS1蛋白不含信号肽序列，无跨膜区，为可溶性蛋白，主要定位于细胞核内，T199、Y309、T338、T512、S631、T635为其潜在磷酸化位点。以上研究为进行PPV NS1蛋白表达，进一步开展NS1蛋白功能研究奠定了基础。

Cloning and Bioinformatics Analysis of NS1 Gene of Porcine Parvovirus Vaccine Strain

In order to study the NS1 protein founction of porcine parvovirus(PPV) further, a pair of primers was designed according to PPV strain NADL-2 NS1 gene. The whole NS1 gene was obtained by PCR, and cloned into pTA2 to sequence. Sequence analysis result indicated that the recombinant plasmid(pTA2-NS1) contained one open reading frame with 1989 bp encoding 662 aa. Compared with PPV strains NADL-2 and Kresse, nucleotide homologies were 99.85% and 99.65%, putative amino acid homologies were 99.70% and 99.70%, respectively. Bioinformatics software(e.g. DNAStar) and net servers were applied to predict and analyze the structures and biochemical properties of NS1 protein. The results showed that the molecular weight of the protein NS1 was predicted as 75.7 ku, the isoelectric point PI was 6.82. It was a soluble protein, mainly located in the nucleus, containing neither signal peptide nor transmembrane region, with T199, Y309, T338, T512, S631, T635 being the potential phosphorylation sites. The construction of pTA2-NS1 and bioinformatics analysis laid the foundation for further functional research.