犬细小病毒南京株(CPV-GN)衣壳蛋白vp2基因的克隆与序列分析

Cloning and sequence analysis of the vp2 gene of canine parvovirus Nanjing isolate,CPV-GN

通过对结构蛋白vp2基因序列的分析可以对犬细小病毒进行分型的确定。本文以常规PCR技术扩增犬细小病毒南京株CPV-GN衣壳蛋白VP2基因,并将之插入到pMD 18-T中,构建克隆载体pMD-vp2。经测序,目的片段长1548 bp,编码516个氨基酸与CPV-d、CPV-15、CPV-N等其它标准株和参考株进行序列比较,CPV-GN株vp2基因第1276碱基处发生了A→G替换,使天冬酰氨426变为天门冬氨酸,从而确定CPV-GN株为CPV-2b亚型;与其它CPV参考株CPV-2b亚型进行序列比较,CPV-GN株vp2基因第18、354、405、1465和1543碱基处的突变是独有的,在这几处分别发生了T→A、A→G、G→A、G→A和T→A替换前三处替换并未改变所编码的氨基酸,而第1465位的G→A点突变使缬氨酸489变为异亮氨酸,第1543位的T→A点突变使丝氨酸515变为苏氨酸,推测这两处的突变很可能与该病毒株的毒力特性有关。

The vp2 gene of canine parvovirus CPV-GN strain isolated in Nanjing was amplified by PCR and cloned into pMD 18-T vector. The 1548-bp-long gene was sequenced and compared with American CPV reference strains CPV-d, CPV-15, and other standard strains. A nucleotide mutation was found at position 1276 with A→G change leading to asparagine 426 → aspardic substitution. Therefore the CPV-GN strain was classified as CPV type 2b （CPV-2b）. Comparing to other CPV type 2b strains, point mutations were found at positions 18, 354, 405, 1465, and 1543, with T→A, A→G, G→A, G→A, and T→A changes, respectively. The first three mutations resulted in no amino acid changes. However the G→A mutation at position 1465 and the T→A at position 1543 led to valine489→ileucine and sefine515→threonine substitutions, respectively. The implications of these substitutions were discussed.