犬冠状病毒大熊猫株纤突蛋白全基因的克隆与序列分析(英文)

Cloning and SequenceAnalysis of Spike Protein Gene of Canine Coronavirus Giant Panda's Isolate

在国内首次对犬冠状病毒大熊猫野毒株(CCVDXMV)纤突蛋白基因进行了克隆和序列测定。该基因全长4362bp,编码1453个氨基酸,N端前18个氨基酸为推测的信号肽序列,后1435个氨基酸构成成熟蛋白。与GenBank中已发表的11个CCV毒株S基因相比,S基因核苷酸序列同源性在40.2%～99.5%之间;推导的氨基酸序列同源性在15.9%～99.0%之间。DXMV株S基因变异区主要集中在该基因前1/2处,其中350-370、439-478、1718-1818三个区域碱基变异较大,而1060-1700区却十分保守。基于S全基因及其蛋白的聚类分析表明,DXMV株与K378、NVSL和USpatent株亲源关系最近。推导的DXMV株S蛋白氨基酸序列潜在的N-联糖基化位点与CCV强毒V54相同,为34个,比Insavc-1弱毒多一个;其中第566-568位糖基化位点为多数强毒拥有而弱毒没有的。另外,DXMV株S蛋白疏水性及抗原表位与其它毒株有一定的差异,这些差异对DXMV株致病性和免疫原性等影响尚待进一步的研究。

Spike (S) gene of Canine coronavirus giant panda’s isolate (CCV strain DXMV) was amplified and cloned into pGEM-T for sequencing. The complete length of spike gene of strain DXMV was 4362 bp, which encoded 1453 amino acids. The initiative 18 amino acids were signal peptide. The homologies of nucleic acid and amino acid of spike among strains of CCV were 40.2-99.5% and 15.9-99.0%, respectively. The variation mainly exist in front half second region of S gene, especially in the regions of 350-370, 439-478 and 1718-1818 nucleotide acids, while the region 1060-1700 nucleotide acids shows high conservative. Phylogenetic tree based on S gene and deduced amino acid sequence of 12 different strains of CCV showed that strain DXMV had closer relationships with wild strain K378,NVSL and US patent. There are 34 N-glycosylation sites in deduced S protein of strain DXMV and V54, but 33 for strain Insavc-l. Noticeably, the potential glycosylation site 566-568 was owned by DXMV and most virulent strains but not for weak virulent strains. Moreover, there were some differences in the hydrophobicity and antigenic index between DXMV and other strains of CCV. The effects of the differences in S protein on pathogenicity and immunogenicity should be further studied.