一些在组织和细胞分化中起重要作用的蛋白质包含锌指结构域.为了克隆分离和研究与造血细胞分化和发育成熟相关的蛋白基因,利用编码C2H2型锌指蛋白结构域中部分保守氨基酸序列设计简并引物,以骨髓cDNA为模板,进行PCR扩增,得到若干新的锌指蛋白基因EST.用其中一条为探针筛选人骨髓cDNA文库,获得了一个新的锌指蛋白基因全长cDNA,GenBank收录号为AF246126,长3 888 bp,包括一个完整阅读框,编码686个氨基酸,包括17个典型的和2个非典型的C2H2模体,命名为HZF2.RNA印迹、人多组织mRNA斑点杂交分析结果显示,其在T淋巴细胞发育和定居的器官组织胸腺、淋巴结中有较高表达,在脾脏、胎肝有中度表达,在B淋巴细胞发育的骨髓中表达很低,在外周血几个淋巴细胞系中仅有极微量的表达,提示HZF2可能对于T淋巴细胞发育和增殖有重要功能.该基因也在脑组织的若干部位、胎盘及肾上腺有较高表达,在多种其他组织细胞有微量表达,说明其可能对维持这些组织细胞的生理功能也起一定作用.将编码HZF2读框的DNA顺序克隆到pEGFP-N1载体中,转染3T3细胞,证明表达的HZE2-GFP融合蛋白定位于细胞核,这与根据HZF2蛋白结构推测其可能作为DNA结合蛋白行使调节基因转录的功能是一致的.%Some of proteins associated with differentiation and development of cells and tissues contain zinc finger domain.To clone and characterize proteins that are related to the hemopoietic cell differentiation and development. C2H2 zinc finger domains were amplified by RT-PCR with degenerated primers designed according to the conserved amino acids. Total RNA was extracted from human bone marrow. Some expressed sequence tags (ESTs) containing the zinc finger motif were acquired and one of them was used as probe to screen human bone marrow cDNA library. As a result, a new gene( GenBank accession number: AF246126) was cloned and designated HZF2. The full length of the cDNA is 3 888 bp and the open reading frame encodes a protein with 686 amino acid residues containing 17 typical and 2 atypical C2H2 zinc finger motifs. Northern blot and Human RNA Master Blot analysis suggested that HZF2 might play an important role in T cell development and amplification. The fragment encoding the complete HZF2 peptide was inserted into the eukaryotic expression vector pEGFP-N1 and introduced into 3T3 cells. The fusion protein was located in cell nuclei, which was a consistent with the conjecture that HZF2 may function as a DNA-binding protein to regulate gene transcription.
展开▼
