一种特异识别SV40启动子的人工转录因子的构建

Construction of a SV40 Promoter Specific Artificial Transcription Factor

转录因子是真核表达调控中非常重要的一类反式作用因子 ,通常由DNA结合域与效应域两部分组成 ,而锌指结构是DNA结合域的常见组成单元。人工转录因子就是基于转录因子的这种结构特点 ,人为地选择针对特定序列的DNA结合域与具有特定作用的效应域组合而成。利用噬菌体展示技术 ,筛选到与SV4 0启动子上 9bp序列特异结合的锌指结构 ,再连接KOX1的KRAB域构建了一种人工转录因子。转染实验表明它对SV4 0下游的报告基因的表达有很显著的抑制作用。

Transcriptions are regulated by transcription factors.Natural transcription factors usually consist of at least two functional domains:a DNA binding domain and an effector domain.According to this,novel artificial transcription factors are designed to up or down regulate transcription and expression of a target gene.The Cys2 His2 zinc finger domain is a DNA binding module that has been widely used as the DNA binding domain in artificial transcription factors.Each zinc finger domain,which comprises about 30 amino acids that adopt a compact structure by chelating a zinc ion,typically functions by binding 3 base pairs of DNA sequence.Several zinc fingers linked together would bind proportionally longer DNA sequences. According to the “bipartite complementary” library strategy, a pair of zinc finger phage display libraries were constructed.After construction of the libraries, a 9bp sequence (5′ GCAGAGGCC 3′) on the promoter of SV40 was chosen as a target for next step.After parallel selection,PCR amplification, desired fragments recovery,re ligation,and additional rounds of selection,phage enzyme linked ELISA experiments were performed to identify specific binding clones displaying the zinc fingers with predetermined sequence specificity to our target sequence.Then two clones with strong ELISA signals were chosen to be tested for binding both to its full target site (5′ GCAGAGGCC 3′) and to sites containing single transition mutations. The binding specificity of one of the two clones (clone 3) was shown to be fairly good. The three finger DNA binding domain targeted to SV40 promoter ,that is,zinc finger sequences on clone 3, was fused to KOX1 suppression domain KRAB and cloned into pcDNA3 1(+)(which expression product was artificial transcription factor).The zinc fingers(which expression product was the DNA binding domain of artificial transcription factor) and KRAB domain only(which expression product was effector domain of artificial transcription factor) were also cloned separately into the same expression vector.All constructs contained an N terminal nuclear localization signal.Every of the vectors (including pcDNA3 1(+) without inserting sequences)were cotransfected with pGL3 Control and pRL TK and the activity of luciferase was used to indicate the function of product from transfected expression vectors.Our artificial transcription factor was proved to repress the expression of reporter gene efficiently,while with only DNA binding domain or effector domain the repression was not remarkable. By adding different effector domains and changing the DNA binding domain,artificial transcription factor would have a wide range of potential applications.