犬细小病毒VP2基因真核表达质粒的构建与瞬时表达

摘要

[Objiective] In order to set the base for studying vaccinum of nucleic acid, VP2 gene eukaryotic expression plasmid of canine parvovirus was constructed in this paper. [Method] The special primer was designed according to the VP2 gene sequence of CPV published in GenBank . VP2 gene was amplified from the stool samples in clinically infected dogs with CPV by PCR. The PCR product was cloned into eukaryotic expression vecyor pcDNA3 . 1( ＋ ) to construct a eukaryotic expression plasmid pcDNA3 . 1 - VP2 . After sequencing and identifying,the pcDNA3.1 - VP2 was injected into mice m vena caudal to induce the transient expression of VP2. The total RNA was extracted from murine livers at 8h after injection and the target strap was obtained by RT - PCR from the total RNA. [ Result]1 755 bp of VP2 gene fragment was obtained from the stool samples in clinically infected dogs by PCR . The eukaryotic expression Plasmid, pcDNA3 . 1 - VP2 was constracted and 1 755 bp of a bright stripe was found from total RNA of murine livers by RT - PCR . [ Conclusion ] The eukaryotic expression plasmid , pcDNA3 . 1 -VP2 was successfully constructed and expressed in murine bodies .%[目的]构建犬细小病毒(CPV)VP2基因真核表达质粒,为研究核酸疫苗奠定基础.[方法]根据CPVVP2基因序列设计特殊引物,采用PCR方法从疑似"犬细小病毒"的患犬粪便基因组中扩增VP2基因,将其克隆至真核表达载体pcDNA3.1(+),测序验证后,小白鼠尾静脉注射瞬时表达VP2基因,8 h后取小白鼠肝脏提取总RNA,进行RT-PCR扩增.[结果]在病犬粪便基因组中扩增得到1 755 bp的VP2基因片段,并构建了真核表达质粒pcDNA3.1(+)-VP2,从瞬时表达的小白鼠肝脏总RNA中可扩增到目的条带.[结论]成功构建了犬细小病毒VP2基因真核表达重组质粒,并在小白鼠体内进行了瞬时表达.