猪伪狂犬病毒gE基因的亚克隆及原核表达

Prokaryotic expression and subcloning of gE gene in porcine pseudorabies virus

为建立猪伪狂犬病毒gE-ELISA血清学检测方法,根据Genebank中发表的猪伪狂犬病毒gE基因序列,PCR扩增长度为750bp含gE基因的主要抗原区域,将其克隆到pMDTM19-T载体中,成功构建质粒pMD-gE。用BamHI和HindⅢ对重组质粒pMD-gE进行酶切、回收后与同样酶处理的原核表达载体pET32a连接,命名为pET32a-gE。经IPTG诱导后,SDS-PAGE电泳可检测到约为45ku的目的蛋白条带。经过Western blot分析,表达产物具有良好的反应原性。

To develop serological assay for PRV gE-ELISA. Using a pair of primers designed according to the revelant nucleotide sequence from GenBank, the specific genes encoding the main antigenic domains of PRV-gE were amplified. The PCR products of 750 bp were cloned into the pMD^TM19-T vectors. The plasmid pMD-gE＇ was constructed success-fully. The main antigen of PRV-gE was cut with BamH I and Hind Ⅲ ,and linked with prokary-otic expression vector pET32a which had been cut by the two enzymes before. The plasmid construced was named pET32a-gE＇. SDS-PAGE detected that the protein bands was about 45 ku, after the re-combinant plasmid was induced by IPTG. Western blot analysis showed that, the expression product has goodreactogenicity.