感染性犬2型腺病毒全基因组克隆及鉴定  

Cloning and Identification of the Full Length Infectious Genome of Canine Adenovirus Type-2

作　　者：张守峰[1] 扈荣良[1] 牛建强[1] 夏咸柱[1] 

机构地区：[1]解放军军需大学军事兽医研究所,吉林长春130062

出　　处：《中国兽医学报》2002年第6期533-535,共3页Chinese Journal of Veterinary Science

基　　金："十五"军队医药卫生重点项目 ( Z2 0 0 10 95 ) ;国家自然科学基金资助项目 ( 30 170 70 4)

摘　　要：以犬 2型腺病毒 (canine adenovirus type- 2 ,CAV- 2 )自然弱毒株 YCA18基因组 DNA为模板 ,经 PCR扩增分别获得其双链两末端 10 13bp(U)和 972 bp(D)的 DNA序列 ,以首尾相接方式克隆入载体 p Poly2 ,获得重组质粒p Poly2 - UD。以 Hind 和 Bst B 双酶切 p Poly2 - UD,回收含载体和部分 U、D的大片段并与 CAV- 2 DNA共转化感受态 E.coli Sure TM,经菌体内同源重组 ,获得含有 CAV- 2全基因组序列的重组质粒 p Poly2 - CAV- 2。后者以 Cla 和 Asc 双酶切 ,释放 CAV- 2全基因组 ,通过脂质体介导转染 MDCK细胞 ,盲传 3代后重复转染 1次 ,4d后 ,出现腺病毒典型病变。细胞上清血凝效价为 1∶ 6 40 ,并可被 CAV- 2抗血清有效抑制。以上结果表明 ,本研究获得了具有感染性的CAV- 2全基因组重组质粒 ,为以 CAV- 2作为重组疫苗载体的系列研究奠定了重要基础。A 1 013 bp fragment (U) at the left end and a 972 bp fragment(D) at the right end of the genome of Canine Adenovirus type-2(CAV-2) strain YCA18 were separately amplified by PCR.They were then cloned into plamid pPoly2 with direction from left fragment to right fragment,resulting in a rescue plasmid pPoly2-UD.The pPoly2-UD was linearized by HindⅢ and BstBⅠ digestion and was cotransformed with the purified CAV-2 genome into competent E.coli Sure TM.Recombinant plasmids harboring the full CAV-2 genome were obtained after bacterial intermolecular homologous recombination.One of the recombinant plasmids,pPoly2-CAV-2,was further identified by enzyme digestion analysis and sequencing.The cloned CAV-2 genome was amplified in plasmid form,released by ClaⅠ/AscⅠ double digestion,and transfected into MDCK cells by Lipofectamine mediated transformation.Retransfection of the transfected MDCK cells was performed after three passages.Typical cytopathogenic effect was observed 4 days after retransfection.The virus Heamagglutination to human red blood cell can be inhibited by anti-CAV-2 sera.All above demonstrated that an infectious CAV-2 genome in plasmid form was gained and this laid a solid foundation for further manipulating the genome as a live vaccine vector.

关 键 词：鉴定 犬2型腺病毒 同源重组 感染性全基因组 基因克隆 重组疫苗载体 

分 类 号：S852.659.1[农业科学—基础兽医学]