摘要
按常规方法培养大肾传代细胞(MDCK),待细胞长成单层后,以2%的量接种犬Ⅱ型腺病毒弱毒株(MV-CAV_2).当绝大多数细胞出现病变时(约35～40h),直接从感染细胞培养物中提取CAV_2 DNA.以该法提取CAV_2 DNA,不仅操作简便、耗费少,而且产量也比常规的先纯化病毒再提取DNA的方法高2～3倍.该法可普遍用于从感染细胞中提取与蛋白共价结合的所有病毒核酸.采用电泳洗脱法纯化上述CAV_2 DNA,以Eco RI、Bam HI、Pst I、Sac I、Nsi I和Sph I酶切,电泳分离,建立了该病毒DNA的6种限制性内切酶图谱.本实验结果为该病毒DNA的分子克隆奠定了基础.
The paper described a rapid technique of purifying DNA of canine adenovirustype 2 (CAV2) from infected cells by the modified Hirt method. This is based on the unique nature of CAV2 DNA that its 5' termini are linked covalently to the terminal proteins, so that the viral DNA-terminal protein complex extracted from infected cells is simply separated from RNA and fragmented cell DNA by phenol extraction, and the viral DNA was obtained within a culture area of 135 cm2. The DNA of the vaccine strain (MV-CAV2) was analysed by digestion with Eco RI, Bam HI, Pst I, Sac I ,Nsi I and Sph I. The restriction endonuclease mapping of the viral DNA was constructed by agarose gel electrophoresis. The experiment has laid a foundation for the molecular cloning of the viral DNA.
关键词
犬
Ⅱ型腺病毒
DNA
限制性酶切
canine adenovirus type 2
DNA
restriction enzyme analysis
