4型腺病毒疫苗株载体的构建及β—半乳糖苷酶基因的 …

目的 构建缺失Ｅ３区７８．９－８６ｍｕ的４型腺病毒疫苗株载体。方法 从４型腺病毒疫苗株感染的人胚肺二倍体细胞２ＢＳ中提取病毒ＤＮＡ，经过多卡亚克隆构建成功缺失７８．９￣８６ｍｕ的载体。将含有ＣＭＶ早期启动子的β－半乳糖苷酶基因插入缺失部位，将这一重组质粒与Ａｄ４Ｂｃｌ１大片段共转染２９３细胞，铺含有Ｘ－Ｇａｌ的营养琼脂，经蓝色斑斑纯化三代，ＯＮＰＧ法测定β－半乳糖苷酶基因的表达量。结果 所构建的载

Construction of adenovirus 4 vector with deletion of 78.9-86 mu fragment and express of beta-galactosidase gene]

OBJECTIVE: To construct a human adenovirus type 4(Ad4) vector with partial deletion at E3 region (78.9-86 mu). METHODS: Ad4 DNA was extracted from purified virus cultured in WI-38 cells. The essential fragment(71.3-100 mu) covering Ad4 E3 region was cloned and partial deletion of E3 region of this clone has been performed, generating plasmid pAd4 delta KS. Furthermore, a beta-galactosidase (beta-gal) gene flanked by CMV early promoter and SV40 poly A signal was inserted into pAd4 delta KS, resulting in pAd4c beta. This plasmid was cotransfected with Ad4 DNA, Bcl I A fragments into 293 cells, producing a non-defective recombinant Ad4 virus encoding B-gal. RESULT: The results showed that the constructed recombinant virus could efficiently express the foreign gene, beta-gal. CONCLUSION: Ad4 vector with a deletion of E3 region can be explored as a live vaccine for prevention of human infectious diseases.