| 摘要: |
| 【目的】以猪基因组为模板，扩增天然锌指，以此建立人工锌指蛋白文库，为猪基因组的定点操作及基因表达研究奠定基础。【方法】利用质粒JMB378和JMB440构建锌指文库骨架载体JMB378-ZF1-MCS，然后根据天然锌指蛋白接头序列的保守性，设计5条简并引物扩增猪基因组中的天然单锌指结构域，将其与JMB378-ZF1-MCS载体上的人工锌指ZF1中识别GGC的锌指蛋白连接，构建二锌指蛋白文库，通过酶切方式再将天然单锌指结构域插入到二锌指蛋白文库中，构建三锌指蛋白文库。测定锌指库容量及阳性率，并通过酶切和测序检测锌指库的多样性。【结果】构建了基于猪天然锌指模块的包含ZF1的三锌指蛋白文库，库容量达到1.5×106 CFU。该文库中含有一个人工锌指蛋白ZF1和2个天然型锌指蛋白，能够对基因组中所有以GGC起始的9 bp靶序列进行筛选。【结论】构建了基于天然单锌指模块的三锌指蛋白文库，为构建新型锌指蛋白提供了一种可行的方法。 |
| 关键词: 锌指 天然锌指蛋白 文库构建 |
| DOI： |
| 分类号: |
| 基金项目:国家自然科学基金项目（31172186） |
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| Construction of artificial zinc finger protein libraries |
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ZHOU Gang,ZHANG Long,LIU Zhong-tian,et al
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| Abstract: |
| 【Objective】This study amplified the natrual zinc finger protein using murinegenome as the module to construct the libraries for artificial zinc finger protein.【Method】Plasmids JMB378 and JMB440 were used to construct backbone vector JMB378-ZF1-MCS for zinc finger library.Then,five degenerate primers were designed for swine natural zinc finger amplification base on the conservative adaptor sequences between zinc finger proteins.Amplified single zinc fingers were fused with GGC recognizing finger from Zif268 of JMB378-ZF1-MCS and the library of zinc finger dimer was constructed.And the amplified single zinc fingers were inserted into natural random zinc finger dimer library for the construction of trimer library.【Result】A natural random library for zinc finger protein trimer with a content of 1.5×106 was constructed.The constructed library contained an artificial zinc finger protein ZF1 and two natural zinc fingers,and could recognize any 9 bp sequence starting with GGC in genome.【Conclusion】A library of natural random zinc finger trimer was constructed using natural zinc finger modules.This study provides a feasible method for a novel zinc finger protein construction. |
| Key words: zinc finger nature zinc finger protein library construction |
