摘要
设计表达了4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2个合适的9 bp长的序列(中间间隔6bp)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2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FokI的切割结构域分别与4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。
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. A firm foundation for further in vivo studies of gene thrapy with gene targeting will be laid. 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 pErT28a- ZFN. By transformation the recombinant plasmids into bacteria RossettaTM （DE3）, four zinc finger nuclease fusion proteins carrying His-tag were produced and purified.
出处
《中国生物工程杂志》
CAS
CSCD
北大核心
2008年第12期36-40,共5页
China Biotechnology
基金
国家自然科学基金(30671194)
广东省科技攻关计划(2006B20101012)资助项目
关键词
锌指核酸酶
设计
生物合成
表达
纯化
基因打靶
基因治疗
Zinc finger nucleases Design Biosynthesis Expression Purification Gene targeting Gene therapy
