摘要
参考GenBank上公布的猪传染性胃肠炎病毒(TGEV)S基因B、C抗原位点序列,应用Prim-er6.0设计一对含酶切位点的引物,用于RT-PCR扩增B、C抗原位点目的片段,将扩增产物连接于peasy-T克隆载体上,构建克隆载体,用EcoRⅠ和XhoⅠ对表达载体PET-32a(+)和重组质粒进行酶切,将酶切产物亚克隆至PET-32a(+)多克隆位点上,连接、转化至BL21(DE3),构建B、C位点的原核表达载体,并对阳性重组质粒进行酶切、PCR和测序鉴定。所扩增的目的片段的大小为357 bp,与原核表达载体连接后,经核苷酸及推导的氨基酸序列分析表明,该基因与其他猪传染性胃肠炎病毒相应基因具有很高的同源性,说明成功地构建了TGEV S基因B、C抗原位点的原核表达载体。TGEV S基因B、C抗原位点原核表达载体的成功构建,为TGEV诊断方法的建立提供良好的技术基础。
The target gene of the sites B／C of S gene was amplified by RT-PCR with a pair of primers, which was designed according to the published sequence of TGEV S gene with primer 6.0 software. The amplified gene was cloned into the vector peasy-T, then the gene and the vector PET-32a（＋）was digested by EcoR and Xho Ⅰ The recycled genes were inserted into the multiple cloning sites of the vector PET-32a （＋）. The recombinant plasmid was transformed into BL21 （DE3） strain and the positive plasmid was identified by restriction enzyme, PCR and sequencing. The results showed that the length of target gene was 357bp and the nucleotide had a relatively high homology with the corresponding gene from other TGEV strains according to the nucleotide and deduced amino acid sequences. The sites B／C gene was cloned into the vector peasy-T and the recombinant expression vectors were constructed successfully. The construction of recombinant expression vectors provided a technical basis for the diagnosis of TGEV.
出处
《动物医学进展》
CSCD
2008年第9期1-5,共5页
Progress In Veterinary Medicine
基金
农业应用新技术北京市重点实验室开放课题(KF2004-04)
北京市教委科研计划资助项目(KM200810020001)
