猪传染性胃肠炎病毒SY株的分离鉴定及S基因的序列分析

Isolation and identification of transmissible gastroenteritis virus SY strain and phylogenetic analysis of S gene

取沈阳地区某猪场表现为腹泻症状的病死仔猪小肠病料,无菌处理后接种在PK-15细胞上分离病毒。通过无限稀释法进行病毒纯化、病毒中和试验和RT-PCR检测,证实其为传染性胃肠炎病毒（TGEV）,并命名为TGEV SY株。参照Gen Bank中TGEV序列设计引物,对SY株S基因进行RT-PCR扩增,克隆并测序,获得S基因组完全序列。将S基因序列和所编码的氨基酸序列与Gen Bank中登录的10个TGEV和1个PRCV S基因序列进行同源性和亲缘关系的比较分析。结果表明,SY株S基因核苷酸序列和编码的氨基酸序列与其他参考毒株的同源性分别为96.4%-99.2%和96.0%-97.9%;而与PRCV毒株的同源性分别为96.2%和96.6%。由系统进化树可知,SY株与国内的华毒株、TS株以及国外的Miller M60株位于进化树的同一分支,亲缘关系较近。总体来看,各毒株之间的S基因差异性不大,表明TGEV虽然处于不断遗传演化中,但不同毒株的S基因变异不是特别明显。

The intestinal materials of died piglets with clinical diarrhea symptom in a pig farm of Shenyang province were collected,inoculated and cultured on PK-I5 cells. Utilizing infinite dilution method to virus purification,virus neutralization test and RT-PCR,these results verified that the isolated virus was transmissible gastroenteritis virus,and named TGEV SY strain. The S gene was ampli- fied by RT-PCR,using the primers designed by referring to TGEV sequences in GenBank,then sequenced. Comparative analysis of gene homology and relationship of nucleotide and amino acid sequences was con- dusted with i0 TGEV strains and a PRCV strain published in GenBank. The result showed that nucleotide sequence homology was 96.4%-99.2% and amino acid homology was 96.0%-97.9%.The homology with PRCV strain was 96.2% and 96.6% ,respectively. The phylogenetic tree analysis showed that there was a close genetic relationship between SY strain and Hi6 strain,TS strain or Miller M60 strain isolated from China and America respectively. In general,the differences between S genes of various strains were not so obvious. It was indicated that,although TGEV mighe be constantly evolved,S gene mutation in dif-ferent strains was not particularly obvious.