4型腺病毒载体的构建和β－半乳糖苷酶基因的表达  

Construction of Type 4 Adenovirus Vector and Expression of Gene β-Galactosidase

作　　者：蒋国桥[1] 张炳华[1] 洪涛[1] 

机构地区：[1]北京中国预防医学科学院病毒学研究所

出　　处：《中华实验和临床病毒学杂志》1995年第1期1-6,共6页Chinese Journal of Experimental and Clinical Virology

基　　金：国家863高科技生物技术领域资助

摘　　要：以４型腺病毒疫苗株感染Ａ５４９细胞，提取病毒ＤＮＡ，将ＥｃｏＲＩ消化的Ｄ片段（７０．５－８３．０基因图谱单位）克隆入ｐＵＣ１８质粒，得ｐＡｄ４（７０．５－８３）质粒，质粒ｐＡｄ４（７０．５－８３）和ｐＡｄ４Ｃ１－２５经酶切、系列亚克隆、加接头等方法得到大部分和部分缺失Ｅ３区的重组质粒。聚合酶链反应（ＰＣＲ）法获得ｐｏｌｙ（Ａ），构建约８００ｂＰ腺病毒晚期表达盒，在所构建的腺病毒重组质粒Ｅ３缺失区或Ｅ４与ＩＴＲ间插入表达盒，得到可同时表达２个或２个以上外源基因和保留了Ｅ３区编码分子量为１９３００糖蛋白基因的３种Ａｄ４载体。将ｌａｃＺ基因插入载体表达区，与Ｂｅｌｌ消化的Ａｄ４ＤＮＡＡ片段共转染Ａ５４９细胞，ＯＮＰＧ法检测证实所构建的载体和表达盒功能良好。本项工作对于在国内研究口服活疫苗及开展基因治疗均具有重要意义。Type 4 human Adenovirus (Ad4) DNA was extracted and purified from A549 monolayer cell culture infected with vaccine strain of Ad4 (Wyeth,USA).EcoRI digested D fragment (70.5-83.0) was inserted into pUC18.and thus,a pAd4 (70.5-83.0) plasmid was constructed.A series of recombinant plasmids with partial or most part of E3 region deleted were obtained through digest ing,serially subcloning as well as linker treatment of the pAd4 (70.5-83.0) and pAd4 Cl-25.For obtaining the expressing vectors,an Ad4 late expressing cassette (about 800bp) was constructed and inserted into the deleted E3 region and in between the E4 and the inverted terminal repeat (ITR).The constructed Ad4 vectors can be used to express 2 or even more foreign genes.As the partially retaining of the E3 region,one of the vectors constructed here can express its glycoprotein (19300) simulteneously.The gene of β-Galactosidase was successfully expressed by co-transfecting A549 cells with Bcl I digested A fragment of Ad4 DNA to contest the validity of the vectors.

关 键 词：4型腺病毒 载体 Β-半乳糖苷酶 基因表达 疫苗 

分 类 号：R392－33[医药卫生—免疫学] R373.9[医药卫生—基础医学]