鸡痘病毒载体复制非必需片段优化及强启动子的筛选  

Optimization and Selection of Nonessential Fragments and Strong Promoter for Fowlpox Virus Vector

作　　者：彭大新[1] 刘秀梵[1] 吴艳涛 高崧 朱爱华 张如宽 

机构地区：[1]扬州大学畜禽传染病农业部重点开放实验室,扬州225009

出　　处：《农业生物技术学报》2000年第2期129-132,共4页Journal of Agricultural Biotechnology

基　　金：国家863计划项目!960332025;江苏省应用基础研究!BJ95107;江苏省自然科学基金!BK9706;江苏省教委基金

摘　　要：以痘苗病毒启动子P7．5调控的β-半乳糖苷酶（LacZ）基因为检测基因，分别插入三个复制非必需片段构成重组质粒，脂质体介导转染鸡痘病毒中国疫苗株282E4感染原代鸡胚成纤维细胞（CEF）单层，蓝斑筛选纯化病毒；将重组病毒感染次代CEF收获细胞后制备提取物，检测其中的β-半乳糖苷酶的活性，由此得到一个基因表达效率高的复制非必需片段pFPV7s。根据痘苗病毒天然启动子P7．5的结构，人工合成4条寡核苷酸，形成一个早期和一个晚期启动子，将合成的启动子进行不同组合，最终形成10个较有代表性的启动子组合；以P7．5为对照，在其下游分别插入大肠杆菌β-半乳精苷酶基因LacZ，并进一步插入pFPV7s构建成鸡痘病毒转移载体，以同样的方法检测β-半乳糖苷酶的活性，得到了表达活性约为p7．5的3．5倍的LLEE启动子组合。The β-galactosidase (LacZ) report gene controlled by vaccinia virus (VV) promoter P7.5 was inserted into three replicate nonessential fragments. DOSPER liposome-mediated transfection with these recombinant plasmids was performed on chick embryo fibroblast (CEF) monolnyers infected with FPV Chinese vaccine strain 282E4 3 hours earlier to produce recombinant viruses.Recombinant FPVs with blue plaque were selected and purified on secondary CEF overlaid with agarose containing X-gal. The level of theβ-galactosidase expressed by these recombinant viruses with LacZ gene was assayed. The result of this study suggested that pFPV7s was the best nonessential fragment. Four olignucleotides were synthesized according to the essential structure of the VV promoter P7.5 and were used for generating early and late promoters. Ten artificial promoters designated PS10 were constructed with different combinations ofearly and late promoters, which were known by restriction endonuclease analysis and sequence analysis. LacZ gene transfer vectors of FPV with these promoters and P7.5 were developed by a series of molecular biology manipulation. The expressing activity of the β-galactosidase controlled by various synthetic promoters and P7.5 was assayed and compared. The LLEE combination was the strongest synthetic promoter which expression of β-galactosidase three more times higher than that of the P7.5.

关 键 词：鸡痘病毒 复制非必需片段 启动子 活性 

分 类 号：S858.312.6[农业科学—临床兽医学] S852.65[农业科学—兽医学]