| 摘要: |
| 采用RT-PCR和重组PCR扩增猪传染性胃肠炎病毒(Transmissible gastroenteritis virus of swine,TGEV)TSX毒株纤突蛋白(spike protein,S)基因全长cDNA序列,分离纯化PCR产物,连接到pGEM-T载体,转化大肠杆菌DH5α,筛选阳性克隆pGEM-S,并对其进行酶切鉴定和测序,对测序结果及推导氨基酸序列进行同源性分析。结果表明,S基因全长为4 350 bp(GenBank登录号DQ001167),编码1 449个氨基酸,N端前16个氨基酸为推测的信号肽序列,其后1 433个氨基酸构成成熟蛋白;与GenBank中已发表的9个TGEV毒株S基因进行比较,核苷酸序列同源性为95%～98%,推导的氨基酸序列同源性为95%～98%。基于S基因及其推导氨基酸序列的聚类分析表明,TSX株与Miller、T014、TS和HN2002株亲缘关系较近。S基因变异是由于碱基突变造成10处氨基酸发生改变,但主要抗原部位未发生任何变异,为进一步研制猪传染性胃肠炎基因疫苗提供了良好的候选基因。 |
| 关键词: 猪传染性胃肠炎冠状病毒 纤突蛋白基因 克隆 序列分析 |
| DOI： |
| 分类号: |
| 基金项目:国家“863”高技术研究与发展计划项目（No.2004AA213072） |
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| Cloning and sequence analysis of spike protein gene of swine transmissible gastroenteritis coronavirus |
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| Abstract: |
| The spike (S) glycoprotein of transmissible gastroenteritis virus (TGEV) is the predominant inducer of neutralizing antibodies.S gene of TGEV TSX strain was amplified by RT-PCR and recombinant PCR.The amplified DNA fragments were separated and recovered from agrose gel,subsequently ligated into pGEM-T vector,and then transformed into E.coli DH5α.The positive clones named pGEM-S were screened and identified by restrict endonuclease digestion and then sequenced.The whole length of S gene of field TGEV strain was 4 350 bp,which encoded 1 449 amino acids.The initiative 16 amino acids were signal peptide.The homologies of nucleic acid and amino acid of spike among strains of TGEV were 95%-98% and 95%-98%,respectively. Phylogenetic tree based on S gene and deduced amino acid sequence of 10 different strains of TGEV showed that field TGEV had closer relationships with strain Miller,T014,TS,and HN2002.Moreover,there were some differences in the antigenic site between TSX and other strains of TGEV,but the main antigenic site didn’t change.Therefore,the study provides a good candidate gene for the research of gene engineering vaccine. |
| Key words: transmissible gastroenteritis coronavirus(TGEV),sipke protein,cloning,sequence analysis |
