犬瘟热病毒N蛋白基因真核表达质粒的构建与瞬时表达

摘要

[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]参考GenBank中发表的CDV N蛋白基因序列设计引物,采用RT - PCR方法从疑似犬瘟热病犬全血样品中扩增CDV N蛋白基因,将其克隆至真核表达载体pcDNA3.1(+).经测序验证后,小白鼠尾静脉注射瞬时表达CDV N蛋白基因,8h取其肝脏提取总RNA,进行RT - PCR方法扩增.[结果]在病犬的全血样品中扩增得到1 572 bp的CDV N蛋白基因片段,并构建了真核表达质粒pcDNA3.1(+)- CDV N,从瞬时表达的小白鼠肝脏总RNA中可扩增到目的条带.[结论]构建了犬瘟热病毒N蛋白基因真核表达质粒,并在小白鼠体内进行了瞬时表达.%[Objective] In order to further study the DNA vaccine against CDV, The eukaryotic expression plasmid, pcDNA3.1 - VP2 was constructed in this paper. [ Method ] One pair of the primers was designed according to the nucleocapsid (N) protein gene sequences of CDV published in GenBank. CDV N protein gene fragment was amplified from the total RNA template in the whole blood samples infected with CDV by RT - PCR. The cloned N protein gene of CDV was cloned into eukaryotic expression vector pcDNA3. 1 ( + ) to construct a eukaryotic expression plasmid pcDNA3. 1 - CDV N. After sequencing, identifying, the pcDNA3.1 ( + ) - CDV N was injected into mice in vena caudal to induce the transient expression of CDV N. The total RNA was abstracted from murine livers at 8 h after injection and the target strap was obtained by RT - PCR from the total RNA. [ Result] A 1 572 bp of CDV N gene fragment was obtained from blood samples in clinically infected dogs by RT - PCR. The eukaryotic expression Plasmid, pcDNA3. 1 - CDV N was constructed and 1 572 bp of a bright stripe was found from the total RNA of murine livers by RT - PCR. [ Conclusion] The eukaryotic expression plasmid, pcDNA3. 1 - CDV N was successfully constructed and expressed in murine bodies.