家蚕znfhit基因的克隆表达及序列分析  

Cloning and Sequence Analysis of the Gene znfhit in Bombyx Mori

作　　者：卜远虹[1] 张耀洲[1] 

机构地区：[1]浙江理工大学生命科学学院,杭州310018

出　　处：《浙江理工大学学报（自然科学版）》2009年第1期120-124,共5页Journal of Zhejiang Sci-Tech University(Natural Sciences)

基　　金：国家863项目(2005AA206120);国家973项目(2005CB121006)

摘　　要：在对家蚕蛹cDNA文库的测序中发现了znfhit(zinc finger HIT)的EST序列(GenBank登录号DY230769)。经过比对发现znfhit全长为1019bp,由297bp的5′端非编码区序列(5′UTR)、462bp的开放读码框(ORF)和260bp的3′端非编码区序列(3′UTR)组成。将该基因的cDNA和家蚕基因组序列比对,此基因由3个外显子和2个内含子组成。ZNFHIT蛋白的疏水性最大值为1.533,最小值为-3.267,且不含跨膜区域。用PCR方法扩增获得znfhit基因,将其克隆至质粒pET-28a上,构建重组质粒pET-znfhit,并在大肠杆菌中大量表达,经镍离子亲和层析纯化得到重组蛋白,质谱鉴定其分子量为21.11KD,为后续研究其功能提供了条件。The EST of znfhit（zinc finger HIT, GenBank accession no. DY230769）is found through searching cDNA bank of silkworm pupae. The full length of the gene is 1015 bp, which contains 5＇UTR 297bp, ORF 462 bp and 3＇UTR 260 bp. Alignment of the cDNA with the silkworm genomic sequences reveal that there are three extrons and two introns. The hydrophobicity of this ZNFHIT protein is predicted to be 1. 533 in maximum and -3. 267 in minimum. There is no transmembrane structure domain in ZNFHIT. The znfhit gene is amplified via PCR. The PCR product is cloned into the expression vector pET- 28a and successfully expressed in E. coli. The recombinant protein is obtained by Ni^2＋ affinity chromatography. Finally the molecular weight of the recombinant protein is 21. llKD, identified by mass-spectrum, supporting for functional studies of the gene.

关 键 词：锌指蛋白 克隆 表达 序列分析 

分 类 号：Q503[生物学—生物化学]