犬Ⅱ型腺病毒弱毒DNA的酶切分析及分子克隆

RESTRICTION ENZYME ANALYSIS AND MOLECULAR CLONING OF MODIFIED CANINE ADENOVIRUS 2(CAV-2)DNA

犬Ⅱ型腺病毒(CAV-2)是犬喉气管炎的病原。CAV-2感染通常只引起轻微的呼吸道疾病,但在其它病毒或细菌继发感染下可诱发较严重的肺炎、支气管炎、扁桃体炎和咽炎。此病毒与引起传染性犬肝炎(ICH)的CAV-1有密切的抗原相关性。CAV-2弱毒株可产生抗CAV-1的保护性免疫,是目前用于预防ICH和狐狸脑炎的有效疫苗。由于CAV-2本身致病不严重,致弱的CAV-2更为安全。因此,用弱毒株构建成基因工程载体,把犬细小病毒或犬及狐狸的其它病毒的主要抗原基因重组到其中的非必需区,可获得安全、有效和多用的病毒载体疫苗。为此,我们首先分析了CAV-2弱毒DNA的酶切图谱,并克隆了全病毒DNA的76.5％。

Modified canine adenovirus 2 ( CAV-2 ) DNA was extracted direolly from infected MDCK cells using the procedure suggested by Carusi. The purified viral DNA was then analysed using restriction endonuclease Bam H I , EcoR I , Pst I , Sac I , Nsi I and Sph I , and the molecular weight of DNA fragments was measured in reference to 1kb ladder(BRL). Pst I digested CAV-2 DNA was recovered from agarose gel using DE-81 paper, and ligated into plasmids pBluescript SK and pBR322, followed by transforming DH5a ( E.coli) . Using photobiotin labeled CAV-2 DNA as probe, 6 recombinant plasmids were indicated containing virus specific fragments with different size ( they are 6.0, 4.6, 4.2, 3.9, 3.1, 3.0 and 0.2 kb) . These fragments cover 76.5% of the entire genome. Furthermore, the cloned fragments can be used for physical mapping, which is essential for constructing genetic engineering vector of CAV-2.