猪细小病毒NS1基因和VP2基因的克隆与序列分析

Cloning and Sequence Analyzing of NS1 Gene and VP2 Gene of Porcine Parvovirus

根据GeneBank上发表的猪细小病毒（PPV）的基因组序列,分别设计扩增非结构蛋白NS1基因和结构蛋白VP2基因的特异性引物,采用PCR方法扩增PPV泰安株（PPV-TA）NS1基因和VP2基因。结果表明,PPV-TANS1基因长1989 bp,编码662个氨基酸,与参考PPVNS1基因核苷酸同源性在98.3%～99.9%之间。PPV-TA NS1蛋白有3个糖基化位点分别是356NIS、446NFS、513NLT,除PPV Tomau/1/02在356位缺失了一个糖基化位点之外,PPV-TA与其他PPV参考毒株一致;这表明不同毒株NS1蛋白调节功能可能有差异。PPV-TA NS1蛋白的潜在磷酸化位点是Thr435和Ser473,与PPV参考毒株相同。NS1基因系统发生分析表明,PPV-TA与PPV china亲缘关系最近。PPV-TAVP2基因长1740 bp,编码579个氨基酸,与PPV参考毒株VP2基因核苷酸同源性在98.3%～99.8%之间。PPV-TA VP2蛋白的378D、383H、436S氨基酸残基决定了PPV的组织嗜性,与PPV-NADL-2和PPV-china相同,这表明这3个毒株的组织嗜性是一致的。但与其他PPV参考毒株有差异,这表明不同毒株在动物体内的组织分布是有差别的。PPV-TA VP2蛋白有9个线性抗原位点,与PPV NADL-2和PPV china的线性抗原位点是一致的,这表明具有相同的抗原特性。VP2基因系统发生分析表明,PPV-TA与PPV NADL-2亲缘关系较近。

According to the sequences of porcine parvovirus genes in Gene Bank,the special primers for non-structural protein NS1gene and structural protein VP2 gene were designed,and the NS1 gene and VP2 gene of PPV-TA were amplified by PCR.As a result,the NS1 gene of PPV-TA include 1989 bp and encodes 662 amino acids,the nucleotide sequence identity is from 98.3 % to 99.9 % compared with PPV NS1 genes of the reference strains.PPV-TA NS1 protein has three glycosylation sites,which are 356NIS,446NFS,513NLT,and identical to the other reference strains except for PPV Tomau/1/02 that did not have a glycosylation site at site 356.It indicated that the NS1 protein function of different PPV strains might different.The phosphorylation sites of NS1 protein of PPV-TA are Thr435 and Ser473,which are identical to the reference PPV strains.The analysis of phylogenesis of NS1genes indicated that PPV-TA is the nearest with PPV-china.The VP2 gene of PPV-TA includes 1740 bp and encodes 579 amino acids,and the VP2 genes identity is from 98.3 % to 99.8 % between PPV-TA and the reference PPV strains.The 378D,383H,436S of VP2 protein of PPV-TA play a role on the tissue tropism,which are similar to PPV NADL-2 and PPV-china strains,and the tissue tropism of the three strains are coherent,but different from the PPV strains.It indicated the tissue tropism of different strains is differential in animals.PPV-TA VP2 protein has 9 linearity antigen sites,which are same to the PPV NADL-2 and PPV china strains.Three strains have the same antigen character.The analysis of VP2 gene phylogenesis indicated that PPV-TA has the nearest genetic relationship with PPV-NADL-2 strain.