猪伪狂犬病毒gD蛋白抗原表位的克隆及表达

依据Gen Bank中猪伪狂犬病毒(PRV)BJ/YT株gD基因设计引物,扩增gD蛋白抗原表位,其设计扩增长度为702 bp,将扩增片段克隆到p MD19-T载体获得p MD19-T-gD。利用Bam HⅠ和HindⅢ限制性内切酶双酶切p MD19-T-gD质粒,进行琼脂糖凝胶电泳并回收酶切片段,将回收片段与原核表达载体p ET-28a连接,成功构建了p ET-28a-gD表达载体。将表达载体转化感受态细胞Rosetta,IPTG诱导表达后,进行SDS–PAGE电泳,结果显示,在25 ku处出现特异性蛋白质条带。Western blot分析结果表明,表达产物能够被PRV的阳性血清识别。综上,成功克隆并表达了gD蛋白抗原表位,且其表达产物具有较好的生物学活性。

Cloning and Expression of Epitopes of gD Protein of Pseudorabies Virus

According to the pseudorabies virus strain BJ/YT gD gene sequences in GenBank,a pair of primers were designed. The B-cell epitopes of gD was amplified,and the length of PCR product was 702 bp. The amplified fragment was cloned into the cloning vector pMD19-T successfully named pMD19-T-gD. The fragment was digested by BamH Ⅰ and Hind Ⅲ restriction enzymes and then cloned into the expression vector pET-28a named pET-28a-gD. The expression of protein was induced with IPTG,specific protein bands appeared at the site of 25 ku by SDS-PAGE electrophoresis. Western blot analysis showed that the expression product could be identified by pseudorabies virus positive serum. In this study, gD protein epitope was cloned and expressed successfully, and the expression product had good biological activity.