兔出血症病毒JL株分离鉴定及VP60基因主要抗原表位分析

Isolation,identification and sequence analysis of VP60 gene of rabbit hemorrhagic disease virus JL strain

采用血凝试验、电镜观察、RT-PCR方法分离鉴定了1株JL株兔出血症病毒(RHDV),扩增衣壳蛋白VP60基因,将扩增片段克隆到pMD18-T载体上,经酶切鉴定后测序。结果显示,VP60基因全长1 740bp,编码580个氨基酸;JL株与其他RHDV分离株比较,核苷酸同源性为93.7%～99.2%,氨基酸同源性在97.3%～99.5%,在VP60 6个区中,A、B、D、F是稳定区,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;将JL株与国内外标准株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比较分析,预测RHDV VP60细胞表位。

A strain of RHDV named JL was isolated and identified by hemagglutination test ,electron microscope and RT-PCR in the research. Primers were designed referring to the genome sequences of RHDV published in GenBank. The VP60 gene was cloned into pMD18-T vector by RT-PCR and sequenced. The VP60 gene was l 740 nt in size and encoding 580aa. In comparison with sequences of 10 RHDV isolates in the world registered in GenBank, the JL strain shared 93.7 %-99.2 % nucleotide sequence homology,and 97.3 % -99.5 % amino acid sequence homology, respective- ly. 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 de- gree of variation in the regions C and E. The hydrophilicity plot, flexible regions, antigenic index and surface proba- bility plot of VP60 protein were also compared between RHDV JL strain and the international reference strain.