本试验以犬2型腺病毒全基因组重组质粒pPolyⅡCAV 2及其E3 区重组质粒pVAX E3 为基础,通过DraⅢ和SspⅠ双酶切,缺失第25097bp 26141bp共1044bp的E3区片段,按与编码链相同转录方向插入由CMV启动子、狂犬病病毒SRV9 株糖蛋白基因、SV40 polyA基因构成的总长2424bp的表达盒,获得重组基因组质粒pPolyⅡCAV 2 CGS(34.7kb)。以AscⅠ和ClaⅠ双酶切,游离重组基因组(32.7kb),在脂质体LipofectamineTM 2000 介导下,转染MDCK细胞系,获得了E3 缺失区携带狂犬病病毒糖蛋白表达盒的重组犬2 型腺病毒CAV 2 CGS。Western印迹试验表明,CAV 2 CGS表达了狂犬病病毒糖蛋白。初步接种试验显示,重组病毒可以诱导犬产生狂犬病病毒特异性抗体。

To construct a new type of vaccine for dog Rabies prevention, Canine adenovirus type-2 was developed for Rabies virus glycoprotein expression. A 1044bp fragment of the E3 region of canine adenovirus type-2 in plasmid pVAX-E3(7.86kb) was deleted by Dra Ⅲ/SspⅠdouble digestion, and an expression cassette of 2424bp in length, composed of the cytomegalovirus immediately early promoter, Rabies virus strain SRV_9 glycoprotein cDNA and SV40 polyadenylation signal, was cloned into the deleted site. The NruⅠ-SalⅠfragment of pPolyⅡ-CAV-2 excluding the E3 region and the NruⅠ-SalⅠfragment of pVAX-E3 containing the expression cassette of the Rabies virus glycoprotein were recombined into pPolyⅡ-CAV2-CGS. The recombinant CAV-2 genome was released by AscⅠ/ClaⅠdigestion and was transfected into MDCK cells by Lipofectamine~(TM) 2000. Canine adenovirus typical CPE and the recombinant virus, CAV-2-CGS, were observed 8 d after transfection. Rabies virus glycoprotein expression was confirmed by Western blotting analysis. ˎ̥,Verdana,Arial; mso-font-kerning: 1.0pt; mso-bidi-font-family: 'Times New Roman'; mso-fareast-font-family: 宋体; mso-ansi-language: EN-US; mso-fareast-language: ZH-CN; mso-bidi-language: AR-SA">Preliminary immunization trial in dogs showed that the recombinant virus could stimulate specific immune response to both vector virus and Rabies virus .