C57BL／6小鼠ZFP580C端编码基因的克隆及序列分析

【目的】克隆C57BL／6小鼠ZFP580C端编码基因cDNA序列，并进行序列测定和分析。【方法】参照GENBANK公布的C57BL／6小鼠ZFP580eDNA序列（注册号为AK005257），根据ZFP580C端编码基因eDNA序列设计1对引物，采用RT-PCR技术，从C57BL／6小鼠的肾脏组织中扩增ZFP580C端编码基因eDNA序列，回收纯化后与T载体连接，构建重组子pMD18-T-ZFP580，转化大肠杆菌DH5a。阳性克隆以限制性酶切分析与PCR法鉴定后，测定序列并分析。【结果】自C57BL／6小鼠肾脏组织获得总RNA，扩增出255bp的基因片段，成功构建阳性克隆重组子pMD-T-ZFP580，经双酶切和PCR鉴定与预期结果一致。所测定的C57BL／6小鼠ZFP580C端基因eDNA序列与13本RIKEN基因组中心于GENBANK注册（注册号为AK005257）的完全一致，与人ZNF580C端编码基因eDNA序列比较，核苷酸序列同源性为90％；该eDNA序列所编码的氨基酸序列与人ZNT580基因C端比较，同源性为100％，均编码有3个串联重复的C2H2型锌指蛋白主构域。其氨基酸序列与大鼠、犬、牛比较，同源性分别为100％、100％、98％。【结论】成功克隆C57BL／6小鼠ZFP580C端编码基因，该基因在不同种属间高度保守。

Cloning and sequence analysis of ZFP580 gene fragment encoding carboxyl terminus in C57BL/6 mice

Objective ] To clone and analyze the cDNA sequence of C-terminal region of GP580 gene from mouse kidney. Methods] Total RNA was extracted from mouse kidney, and the C-terminal region of ZFP580 was obtained by RT-PCR with specific primers. The primers were designed according to the open reading frame of ZFP580 gene eDNA sequence reported by GENBANK （GENBANK Accession No. AK005257）. ZFP580 gene was cloned into pMD18-T-Easy vector and then transformed into E. coli DH5a. After the positive bacteria clones were cultured, pMD18-T-ZFP580 was extracted and identified by restriction enzymes digestion. Then pMD18-T-ZFT580 was sequenced and analyzed. Results] The 255bp eDNA sequence of C-terminal region of ZFP580 was obtained by RT-PCR. DNA sequencing result showed that ZFP580 gene was exactly consistent with the sequence reported by GENBANK. Homology analysis showed that mouse ZFP580 gene had a high similarity to human ZNF580 gene. The homology of nucleotide sequences was 90%, and the identity of the deduced amino acid sequences was 100% .The C terminns of ZFP580 and ZNP580 proteins contain 3 Cys2/His2 zinc fingers. The amino acid sequence had 100%, 100%, and 98% homology to the sequence of rat, canis, and bos taurus, respectively. Conclusions] The results showed that the cDNA sequence of C-terminal region of ZFP580 gene is successfully cloned and the sequence in differrent species is highly conserved.