兔出血症病毒Yaan株衣壳蛋白VP60基因的克隆和序列分析

Cloning and sequence analysis of VP60 gene of RHDV Yaan strain

采用RT-PCR方法扩增兔出血症病毒(RHDV)Yaan株衣壳蛋白VP60基因,将扩增片段克隆到pMD 18-T载体上,经酶切及PCR鉴定后测序.结果显示,VP60基因全长1 740 bp,编码579个氨基酸.将Yaan株与其他RHDV分离株进行比较,结果显示,核苷酸同源性为90.5%～97.5%,氨基酸同源性为94.3%～99.5%,氨基酸变异多发生在衣壳蛋白C、E区,表明毒株具有高度保守性;同时对Yaan株与国外标准株AST/89的VP60蛋白氨基酸变异及其亲水性、柔性区、抗原区和表面结构进行了比较分析.

Particles of rabbit hemorrhagic disease virus（RHDV）Yaan strain were extracted from liver of experimentally-infected rabbits. The VP60 gene was cloned into pMD 18 T vector by RT-PCR and sequenced. The VP60 gene was 1740 nt in size and encoding 579aa. In comparison with sequences of 14 RHDV isolates in the world registered in GenBank the Yaan strain shared 90. 5%- 97. 5% nucleotide sequence homology,and 94.3%- 99.5 % amino acid sequence homology respectively. The results indicated that the sequences of VP60 gene among different RHDV isolates were highly homologous. In all the six regions of VP60 gene, low degree of variation was found in the regions A,B,D,F, and relative high degree of variation in the regions C and E. The hydrophilicity plot, flexible regions,antigenic index and surface probability plot of VP60 protein were also compared between RHDV Yaan strain and the international reference strain AST/89.