犬2型腺病毒E3区缺失性表达载体的构建及外源基因表达

Construction of Deleted E3 Vector of Canine Adenovirus Type 2 Expressin g Foreign Genes

为构建可用于同源重组或体外重组的犬腺病毒 (canim e adenovirus,CAV) E3区缺失性表达载体 ,在克隆的 E3区两末端设计并合成了 2对突变引物 ,在引物中分别引入 1个 Bgl 酶切位点 ,以 p BE3质粒为模板 ,经 2次点突变后 ,得到含有 2个 Bgl 位点的重组质粒 p BE3M。该质粒经 Bgl 酶切后自连得到 E3区缺失的质粒 p BE3Δ。在 p BE3Δ质粒的 Bgl 位点加入接头后 ,产生含多个酶切位点的质粒 p BE3L,经相应的酶切鉴定 ,证明突变、缺失和接头连接正确后 ,利用 p BE3L 分别构建了带有和不带有外源性启动子的绿色荧光蛋白基因的重组表达质粒 ,并分别进行转染以检测其表达。结果显示 ,p BE3L CGFP质粒在转染 DK细胞后 ,于 36 h即可观察到荧光 ,72～ 96 h无明显差别 ,传 3代后仍可见表达荧光的细胞 ;而 p BE3L GFP质粒转染 DK细胞后经检测无表达。结果表明 ,构建的 E3区缺失性载体不能利用 E3区自身的启动子进行目的基因的表达 ,外源性启动子的加入是必要的。

To construct E3 expression vectors of canine ad enovirus for engineered vaccine recombination,2 pairs of primers each containing a BglⅡ site at the beginning and at the end of E3 were designed and synthesize d.PCR based quick mutations were performed,resulting in a recombinant plasmid pB E3 M.The region between the two BglⅡ sites was deleted,producing a plasmid pBE 3Δ.Multiple clonal sites were introduced into pBE3Δ at BglⅡ site,giving rise to a plasmid pBE3L.Using these base vectors,two candidate expression vectors of green fluorescent protein(GFP) with and without foreign promoters were construct ed,an d named as pBE3LGFP and pBE3LCGFP,respectively.Expression of the two candidate e xpression vectors was identified by transfecting them into DK cells.The results showed that pBE3LCGFP was expressive by fluorescent microscope observation.While pBE3LGFP could not be expressed.It is demonstrated that foreign promoter was ne cessary for gene expression in deleted E3 region of canine adenovirus type 2.