犬瘟热病毒核蛋白基因重组腺病毒的构建、鉴定与表达

核蛋白基因(N)位于犬瘟热病毒基因组的108—1679位置处,是保守性较强的免疫原性蛋白,因此选择N基因作为目的基因,利用酶切、连接等方法构建了含犬瘟热病毒核蛋白基因的穿梭质粒pVAX?E3LPN。以含CAV-2SY株全基因组的pPoly2-CAV-2为载体,构建了重组质粒pCAV-2-CDVLPN,利用脂质体介导法转染MDCK细胞,转染三次后,细胞出现了典型的腺病毒样病变。电镜负染、切片观察,酶切、PCR扩增及测序鉴定的结果表明表达犬瘟热核蛋白基因的重组犬2型腺病毒构建成功,表达的核蛋白分子量为58kDa。

Construction and Identification of a Recombinant Canine Adenovirus Expressing Nucleocapsid Protein of Canine Distemper Virus

The nucleocapsid protein gene(N)located 108 to 1679 situation of Canine distemper virus(CDV)genome and was immunogenicity protein with strong conservatism,so N gene was choosed as aim gene.A shuttle plasmid pVAX?E3LPN containing the gene encoding the nucleocapsid protein of CDV was constructed by restriction endonuclease digestion and ligation.A recombinant plasmid pCAV-2-CDVLPN was constructed with pPoly2-CAV-2 vector containing CAV-2 SY genome.The plasmid pCAV-2-CDVLPN was transfected into MDCK cells by Lipofectamine and after three transfections,a typical CAV-2 cytopathic effect(CPE)was observed.We demonstrated that a recombinant canine adenovirus type-2 expressing nucleocapsid protein of canine distemper virus was successfully constructed and observed by electron microscopy,enzyme digestion,PCR and sequencing.The molecular mass of nucleocapsid protein was 58 kDa.