猪细小病毒HNZM01株NS1基因的克隆及序列分析

Cloning and Sequence Analysis of NS1Gene of Porcine Parvovirus HNZM-01 Strain

为加强猪细小病毒病的监控,对分离的猪细小病毒(PPV)HNZM-01株NS1基因的序列进行了分析。设计一对特异性引物,以抽提的PPV HNZM-01株的DNA为模板,通过PCR反应扩增出大小约2.27kb的目的片段,并将其插入克隆载体pGEM-T Easy上,构建了重组质粒并测序。结果表明,NS1基因全长1 989bp,与预期目的片段大小一致。序列分析结果表明,PPV HNZM-01株的NS1基因与其他PPV毒株同源性在98.2%～99.8%,说明NS1基因具有高度保守性。同时应用ANTHEPROT软件分析了HNZM-01株NS1基因编码蛋白质的氨基酸组成和二级结构的含量、分布。结果表明,由于HNZM-01株中碱基的突变,造成其氨基酸序列与NADL-2株有所不同,形成的二级结构略有差异,但二级结构元件分布大致与NADL-2株相同。

In order to strengthen the monitoring PPV and get more analyse of the sequence of the NS1 gene,the special primer pair was designed and synthesized according to the NS1 gene sequence of PPV kress strain previously published in GenBank.After amplification from PPV HNZM-01-DNA,the PCR fragment in length of 2.27 kb was inserted in pGEM-T Easy vector.As expectedly,the cloned NS1 gene is 1 989 bp in length and encodes 662 aa.NS1 protein of HNZM-01 contains the conserved sequence GKRN.Compared to the corresponding region of other strains of PPV,the nucleotide sequence homology was between 98.2%-99.8%.The results showed that NS1 gene is highly conservative.The further analysis of the field strain was done by the phylogenetic tree.The amino acid composition and percentage of secondary structure were compared between HNZM-01 and NADL-2 isolated using the ANTHEPROT software.The results showed that for the changes of NS1 nucleotide sequence,the amino acid composition and secondary structure of HNZM-01 isolate had some difference compared with NADL-2 isolate,but the distribution of secondary structure unit changed not too much.