研究锌指蛋白与核酸作用的筛选模型的建立及应用

蛋白与核酸相互作用，是基因表达调节最常用最有效的一种方式，锌指是识别核酸最成功的一种结构元件。我们希望利用转录干扰、翻译抑制现象，建立体内遗传筛选模型，对经典锌指的核酸识别特异性进行研究，并进而设计序列特异的DNA、RNA结合蛋白，为进一步的基因治疗、疾病预防等研究提供新的工具和手段。

The study of protein-DNA interaction remains the central theme in the area of gene rgulation. Within the known classes of DNA binding proteins, the zinc-finger module remains to be the most successful structure owing to its prevalence in eukaryotic genome, its modularity and simplicity of structure and interaction with DNA. In this study, an in vivo transcription interference phenomenon was utilized to develop a genetic selection assay for investigating the DNA recognition properties of classical zinc-finger protein Zif268. By screening a library in which the eight amino acids of the first zinc-finger from Zif268 were randomized by overlap PCR, some functionally equivalent fingers which recognize the subtle sites such as 5ˊ-GCG-3ˊ，5ˊ-GCT-3ˊ, 5ˊ-GAT-3ˊetc. were obtained. The comparing of amino acid similarities of these mutants revealed the four primary positions for base contact and some other likely mode of DNA-zinc finger interaction. These results extend earlier insight into the DNA-protein interaction obtained by X-ray crystallography study. Using the selected zinc fingers and TATA-Box binding protein TBP as modular building blocks, several zinc-finger proteins (ZFPs) were constructed. After expression, purification, the in vitro DNA binding specificity of these ZFPs were determined by gel shift assay. The results showed that these ZFPs can bind to the congnate target sites with very high specificity and affinity. By fusion with the transactivation domain of VP16, several artificial transcription factors were also developed. The transient co-transfection assay indicated that these artificial transcription factors can regulate the report gene's expression with cognate tatget sequence in their promoter efficiently, thus demonstrating the potential of application for gene therapy.