表达犬瘟热病毒融合蛋白基因重组腺病毒的构建与鉴定

利用pVAX1载体的表达元件CMV启动子和poly(A)多聚腺苷酸信号构建了含犬瘟热病毒融合蛋白基因(F)的重组犬腺病毒。首先,利用酶切、连接等实验方法构建了含CDV启动子F蛋白基因表达盒的转移质粒pVAXΔE3LPF。再以含CAV-2SY株全基因组的pPoly2-CAV-2为载体,构建重组质粒pCAV-2-pVAXLPF,利用脂质体介导方法转染MDCK细胞,转染3次后,细胞出现了典型的犬腺病毒感染样病变。电镜负染和切片观察、酶切、PCR扩增及测序鉴定的结果表明,成功构建了含犬瘟热病毒融合蛋白基因的重组犬腺病毒,表达的融合蛋白分子质量为72ku。在MDCK细胞上连续传30代试验表明重组病毒CAV-2-pVAXLPF具有良好的遗传稳定性。

Construction and identification of recombinant canine adenovirus expressing fusion protein gene of canine distemper virus

Recombinantcanine adenovirus containing the fusion protein gene ofcanine distempervirus was constructed on the basis of pVAX1 vector containing CMV promoter and poly (A).Firstly,the shuttling plasmid pVAXΔE3LPF containing F protein gene expressing cassette of CDV was constructed by restriction endonuclease digestion and ligation.Then,The recombinant plasmid pCAV-2-pVAXLPF was constructed with pPoly2-CAV-2 vector containing CAV-2 SY genome.The plasmid pCAV-2-pVAXLPF was transfected into MDCKcells by Lipofectamine.Afterthree transfections,the typical CAV-2 cytopathic effect (CPE) was observed by microscope.It was proved that the recombinant canine adenovirus type-2 expression fusion protein gene of CDV was successfully constructed by electron microscope observation,enzyme digestion,PCR and sequencing identification.The molecule mass of fusion protein was 72 ku.The genetic characteristics of recombinant virus CAV-2-pVAXLPF was also good in stability proved by the serial passage experiment.