人工锌指核酸酶的设计与表达纯化

设计表达了四个锌指核酸酶，用于切断人基因组中的rRNA基因家族的内部转录间隔序列，造成双链断裂，以此提高针对多位点基因打靶的效率，为后续基因打靶应用于基因治疗研究奠定基础。首先，在人rRNA基因家族ITS1序列中找到两个合适的9 bp长的序列(中间间隔6 bp)为锌指蛋白识别位点，根据识别位点序列每个位点分别设计两个三锌指蛋白。通过设计引物进行重叠延伸PCR得到全长编码锌指蛋白的DNA，分别克隆到表达载体pET-28a（+），构建重组质粒pET28a-ZFP，转化大肠杆菌RossettaTM（DE3），实现带组氨酸标签的锌指融合蛋白的表达与纯化。同时，将限制性内切酶Fok I的切割结构域分别与四个锌指蛋白序列采用PCR拼接后克隆到表达载体pET-28a（+），构建重组质粒pET28a-ZFN，转化到大肠杆菌RossettaTM（DE3），实现带组氨酸标签的锌指核酸酶融合蛋白的表达并纯化。

Design, expression and purification of specific DNA cleavage proteins:zinc finger nucleases

Four engineered zinc finger nucleases were produced for cleaving the internal transcribed spacers(ITS) of the human rRNA gene family to generate double-strand breaks, thereby increasing the efficiency of gene targeting. This study will lay a firm foundation for further in vivo studies of gene thrapy with gene targeting. First, two 9 bp-long appropriate sequences (with an internal sequence of 6 bp) within the ITS1 of the human rRNA gene family were selected as the recognition loci of zinc finger proteins. Based on the sequences of the recognition loci, two tri-zinc finger proteins were designed for each locus. The full-length DNA encoding zinc finger proteins were obtained through the gene splicing by overlap extension PCR (SOE PCR) method, then they were inserted into the expression vector pET-28a(+) to generate recombinant plasmid pET28a-ZFP. The zinc finger fusion proteins carrying a His-tag were produced by transformation of the recombinant plasmids into bacteria RossettaTM（DE3）. At the same time, the coding sequences of four zinc finger proteins were linked with the sequences encoding the DNA-cutting domain of Fok I, respectively, by SOE PCR. The resulting sequences encoding four zinc finger nucleases were then inserted into the expression vector pET-28a(+) to construct recombinant plasmid pET28a-ZFN. By transformation the recombinant plasmids into bacteria RossettaTM（DE3）, four zinc finger nuclease fusion proteins carrying His-tag were produced and purified.