7型腺病毒疫苗株载体的构建及β-半乳糖苷酶基因的表达

Construction of adenovirus type 7(vaccine strain) vector and expression of β galactosidase gene

目的构建Ｅ３区缺失的７型腺病毒疫苗株（Ａｄ７ｖ）载体并表达β半乳糖苷酶基因。方法从人二倍体细胞Ｗ１３８培养的Ａｄ７ｖ中分离病毒ＤＮＡ，利用Ａｄ７ｖＤＮＡ天然的酶切位点，经过多步亚克隆，克隆的同时将Ｅ３区７８８８７ｍｕ片段缺失，并将多克隆酶切位点带入Ａｄ７ｖ载体。为了验证载体的功能，将带有巨细胞病毒（ＣＭＶ）早期启动子β半乳糖苷酶基因插入缺失的Ｅ３区。将这一重组质粒和ＥｃｏＲⅠ酶切Ａｄ７ｖＤＮＡ共转染２９３细胞，获得表达β半乳糖苷酶重组病毒。结果构建了缺失部分Ｅ３区Ａｄ７ｖ载体，在ＣＭＶ启动子的作用下该载体能有效地表达外源基因。

Human adenovirus type 7 DNA was extracted from purified virus cultrured in WI 38 cells. The essential fragment (68-100mu) of Ad7 DNA was used for constructing a non defective Ad7 vector termed as pAd7△E3. The vector was characterized by a deletion at the E3 region that contains a multicloning site for the insertion of foreign genes. A helper independent adenovirus type 7 β galactosidase recombinant was established. The recombinant contains the β galactosidase gene flanked by CMV early promoter and SV40 poly A signal. The constructed recombinant viruse showed efficient capacity in expressing the foreign gene, β galactosidase.