7株猪流行性腹泻病毒M基因的克隆与分析

Cloning and sequence analysis of M gene of seven strain porcine epidemic diarrhea viruses

采用RT-PCR方法对2012-2014年收集的7份四川规模化猪场送检猪流行性腹泻病毒（porcine epidemic diarrhea virus,PEDV）的阳性临床样品中病毒M基因进行扩增,纯化回收目的片段后连接到pMD19-T载体,经序列测定与GenBank收录的30条PEDV参考毒株的序列进行核苷酸和氨基酸同源性分析、构建系统进化树。结果显示,7株PEDV四川株间核苷酸以及氨基酸同源性分别高达98.4%~99.9%和97.4%~99.1%,与参考株之间的核苷酸以及氨基酸同源性分别是95.7%~99.9%和95.2%~99.6%,PEDV四川株与国内外近3年的PEDV毒株均在系统进化树上的同一分支,亲缘关系较近,而经典毒株CV777与国内早期PEDV株处于另外一个分支。表明目前流行的PEDV M基因与往年相比出现一定的变异,但是仍相对稳定。

To keep abreast of genetic variations of PEDV（porcine epidemic diarrhea virus） in China, this paper adopts RT-PCR to amplify gene M involved in 7 positive clinical samples of PEDV, which were collected from large-scale pig farms in Sichuan from 2012 to 2014. After purified frag- ments of recovery,connected to vector pMD19-T and sequenced. Then,we analyzed the homology in nucleotide and amino acid between the seven strains and 30 reference strains PEDV included in GenBank and constructed systematical tree of evolution. According to the consequence, the homol- ogy in nulcleotide and amino acid from 7 strain PEDV samples from Sichuan was from 98.4%to 99.9% and 97.4% to 99.1% separately,while they shared 95.7% to 99.9% and 95.2% to 99.6% homology with the reference strains. Thus, both the PEDV samples from Sichuan and domestic and abroad in recent 3 years are in the same branch of systematical evolution and are in closer rela- tionship. However, the classic strain 777 and domestic PEDV previously isolated are in another branch. It turns out that the epidemic gene PEDV M displayed a certain mutation in comparison to past years,but still remains steady.