犬瘟热病毒N蛋白基因真核表达质粒的构建与瞬时表达

【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,8 h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。

Construction and Transient Expression of Eukaryotic Expression Plasmid for N Protein Gene of Canine Distemper Virus(CDV)

【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.