痘苗病毒VV1原核增强子样序列的克隆及其功能和应用研究

Function and application study on prokaryotic enhancer-like element VV1 from vaccinia virus genome

目的 从痘苗病毒基因组中克隆原核增强子样序列VV1,并对其结构、功能和应用进行研究.方法 采用氯霉素乙酰转移酶基因（CAT）作为报告基因,从痘苗病毒基因组中筛选具有原核增强子样活性的序列,采用末端定向缺失试验对原核增强子样序列进行结构与功能研究,用携带增强子样序列表达载体表达干扰素基因.结果 从痘苗病毒天坛株DNA基因组中筛选到原核增强子样序列VV1,正反向分别可使lacZ基因活性提高10.9倍和3.8倍,末端定向缺失试验证实,5＆#39;末端20 bp的核苷酸序列和3＆#39;末端20 bp的核苷酸序列对于VV1的活性具有重要作用,因为缺失任意一个都会导致增强活性的大幅度下降.而5＆#39;末端30-50 bp的核苷酸序列对于保持VV1片段的基本活性非常重要,缺失它时VV1的活性完全消失.用携带VV1的表达载体表达的IFN-α2b型干扰素比原表达载体活性提高2.6倍.结论 从痘苗病毒天坛株DNA基因组中筛选到原核增强子样序列VV1,末端定向缺失试验证实VV1功能区,携带痘苗病毒增强子样序列VV1的表达载体可提高干扰素基因的表达水平.

Objective To clone enhancer-like sequences from vaccinia virus genome.Study on function and application of it.Methods Enhancer-like element from vaccinia virus genome was obtained by using the chloramphenicol acetyl-transferase（CAT）gene as reporter gene.Stepwise deletion experiment was used to identify the functional domain of VV1 element.Interferon was expressed by using an expression vector harboring VV1 sequence.Results An enhancer-like element VV1 of 283 bp from vaccinia virus genome DNA was obtained.Deletion of β-galactosidase activity showed that positive direction could increase the activity 10.9 times and negative direction could increase 3.8 times.Stepwise deletion experiment was used to identify the functional domain of VV1 element.The results suggested that the 20 bp at 5＆#39; terminal and 20 bp at 3＆#39; terminal were important to the activity of VV1 and its activity decreased greatly without them.Furthermore,the 30-50 bp at 5＆#39; terminal was essentiM to its activity and would lead to complete loss of its activity without it.The antiviral activity of interferon α-2b was increased by 2.6 times in comparison with the original expression plasmid.Conclusion VV1 enhancer-like sequences was obtained from vaccinia virus genome.Stepwise deletion experiment was identification of the functional domain of VV1 element.Interferon gene was highly expressed by using an expression vector harboring enhancer-like sequencesVV1.