猪传染性胃肠炎病毒四川株的分离鉴定及其S基因的克隆和序列分析

Isolation and identification of transmissible gastroenteritis virus SC-A strain and cloning and sequencing of S gene of SC-A strain

通过核酸类型试验、脂溶性敏感试验,鉴定了1株分离自四川某猪场腹泻病猪粪便的猪传染性胃肠炎病毒(TGEV)。经电子显微镜观察,可以看到大小约90 nm的病毒粒子;通过中和试验,将病毒细胞液与TGEV超免疫血清进行中和反应,显示较高的中和抗体效价。根据GenBank中的TGEV序列,设计了1对包含TGEV S基因1 760 bp部分片段的引物,经RT-PCR扩增获得了1条约1.8 kb的条带,通过低熔点琼脂糖凝胶纯化回收该片段后,将其连接在pMD 18-T载体上,转化DH5α大肠埃希氏菌,用双酶切和PCR对重组质粒进行鉴定并测序,经用DNAStar软件进行同源性分析和多重序列比较,TGEV四川株(SC-A)与国外报道的部分TGEV分离株具有高达98.8%的同源性。

The transmissible gastroenteritis virus （TGEV） SC-A strain isolated from a pig farm in Sichuan was inoculated into ST cells, then pathological changes in the cells were observed by an electron microscope. A part fragment of S gene of the strain, about 1.80 kb in length, was amplified by RT-PCR with a pair of primers, whcih was designed according to sequences reported in GenBank. The fragment was cloned and sequenced. It was concluded from the sequenced that the S gene of the SC-A strain is composed of 1760 bp of nucleotides, encodes a polypeptide of 586 amino acids, and shares identity rates of 98.8%, 96.6%, 98.6% and 98.3% with that of Purdue, X53128, TH-98 and TO14 strains respectively.