三株口蹄疫病毒的克隆和基因型鉴定

Cloning and Genotype Identification of Three Foot and Mouth Disease Virus Isolates

目的:将O、A、Asia-Ⅰ型等3株口蹄疫病毒(FMDV)VP3-VP1-2A区域的一个片段克隆到pMD18-T载体上,构建阳性重组质粒,并且鉴定3株病毒所属的基因型。方法:将从中国农业科学院兰州兽医研究所获得的O、A、Asia-Ⅰ型灭活FMDV提取RNA作为模板,采用RT-PCR技术扩增了VP3-VP1-2A区域的一个约1 070 bp的片段,包含了全部的VP1序列;将其克隆到pMD18-T载体上,鉴定后得到阳性重组质粒;将目的片段进行序列测定、分析、绘制系统发育树,进而确定各灭活病毒的基因型。结果与结论:经鉴定,O型灭活FMDV属Cathay基因型,它与该基因型3条参考毒株序列的相似性在87%以上;A型灭活FMDV与参考株的相似性差异较大,但在系统发育树上可以看出该毒株属于Asia基因型;Asia-Ⅰ型FMDV只有1个基因型,将测序结果在NCBI网站上BLAST,证实该灭活病毒为Asia-Ⅰ型。

Objective： VP3-VP1-2A fragments of serotype O, A and Asia-Ⅰ foot and mouth disease virus（FMDV） samples were cloned into pMD18-T vector in order to construct recombinant plasmid and identify the genotypes of these three strains. Methods： Serotype O, A and Asia-Ⅰ FMDV samples obtained from Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences were served as templates and a VP3-VPI-2A fragment was amplified by RT-PCR which was about 1 070 bp in length, including a complete VP1 sequence. The fragment was cloned into pMD18-T vector and positive recombinant plasmids were selected. The target segments were sequenced, analyzed and phylogenetic tree was drawn so that the genotype of each inactivated virus was determined. Results ＆ Conclusion： The results indicate that FMDV type O inactivated virus belongs to Cathay genotype. The VP1 similarity between this virus and the three reference viruses are over 87%. The similarity between type A inactivated virus and the three reference viruses are vary （85%, 72.6% and 76.9%, respectively）, howerver phylogenetic analysis proved the sample being Asia genotype. As FMDV type Asia-Ⅰ has only one genotype, the sequence of the cloned fragment was blasted and confirmed to be type Asia-Ⅰ.