痘苗病毒VV16原核增强子样序列的克隆及其结构与功能的研究

Functional and Structural Study of the Prokaryotic Enhancer-like Element VV16 from Vaccinia Virus Genome

利用带有氯霉素乙酰转移酶报道基因的检测载体 ,从痘苗病毒基因组DNA中筛选到一个增强子样片段VV16。序列分析表明 ,该片段长 112bp ,是痘苗病毒DNA依赖性RNA聚合酶、polyA聚合酶亚单位和DNA聚合酶基因RPO30的一部分 ,含有 4个AT丰富区。采用带有β 半乳糖苷酶报道基因的载体检测发现 ,该片段正向可以增强报道基因表达 9 0倍 ,反向可以增强报道基因表达 4 1倍。RNADotblotting实验证实 ,它对基因的增强活性表现在转录水平上。DNA删切实验证实 ,5′端 10bp及 3′端 12bp对其活性有重要调节作用 ,而该片段nt76～ 82的序列对增强子的活性至关重要。

An enhancer\|like element VV16 from Vaccinia virus genome DNA was obtained by using the plasmid with CAT reporter gene. Sequence analysis showed the element of 112bp is a part of the DNA\|dependent RNA polymerase,polyA polymerase and DNA polymerase (RPO30 gene). It contains 4 AT\|rich regions. Detection of β galactosidase acti vity showed that VV16 in the positive direction can increase the activity 9.0 times and VV16 in the negative direction can increase 4.1 times. The RNA dot blotting confirmed the enhancing activity of the element are on the transcription level. DNA deletion experiment indicated the sequences of 10bp at the 5′ end and 12bp at the 3′ end in the element are important to its function and the sequence from nt76 to nt82 is essential to its activity.