摘要
目的筛选和鉴定连接蛋白26(connexin 26 ,CX26)的相互作用蛋白质,并分析其在小鼠耳蜗的表达,探讨CX26在胞内核糖体合成后的运输、装配和在胞膜形成间隙连接的生理过程中可能相关的蛋白质。方法应用酵母双杂交技术筛选CX26的相互作用蛋白质。聚合酶链反应(polymerase chain reaction,PCR)法扩增正常人GJB2 (CX26)全长编码区,基因重组的方法定向克隆于第3代酵母双杂交系统的诱饵质粒pGBKT7 ,用构建的诱饵pGBKT7/CX26筛选人胎脑cDNA文库,获得CX26的相互作用蛋白质的初步阳性克隆,再用这些克隆分别与CX26酵母双杂交去除假阳性。对阳性克隆的插入子DNA进行测序、生物信息学分析。用逆转录-聚合酶链反应(reverse transcription-polymerase chain reaction, RT-PCR)方法分析相互作用蛋白质编码基因的mRNA在小鼠内耳的表达。结果 1个阳性克隆的插入子DNA全长867 bp,前525 b为编码区。DNA序列及编码读框均与Nogo蛋白的编码基因RTN4的编码区终止密码子前525 bp(包括终止密码子)及终止密码子后非翻译区238 bp完全相同,编码Nogo蛋白的3个异构体Nogo-A、Nogo-B和Nogo-C的羧基端的174个氨基酸残基。RT-PCR分析示RTN4的mRNA在小鼠内耳表达。结论 Nogo蛋白的羧基端与CX26相互作用,Nogo蛋白在内耳表达。Nogo可能参与了CX26蛋白在内耳细胞的转运或间隙连接功能的生理过程。
Objective To screen and identify the proteins that interact with connexin 26 （CX26） and to analyze the expressions of these proteins in cochlea so as to explore the proteins that relate to the trafficking, assembly, localizing and gap junction functions of CX26. Methods The whole coding region of GJB2 （CX26） gene was amplified from norreal haman genomic DNA by polymerase chain reaction （PCR） and then directionally subcloned into the vector pGBKT7 plasmid of the Match Maker Gal4 Two-Hybrid System 3 as a target to screen the interactive proteins of CX26 from the human fetal brain cDNA library by the yeast two hybrid technique. The false positive clones were discarded from the preys by repeated yeast two hybrid method between CX26 and everyone of the preys respectively. The DNAs of the insert of the identified positive clone were sequenced and BLAST analyzed against the GenBank. Lastly, the mRNA of the gene encoding the identified protein was analyzed in the murine inner ear by reverse transcription-polymerase chain reaction （RT- PCR）. Results The insert of one positive clone contained 867 bp with the former 525 bp being coding region. The DNA sequence and the open reading frame of the insert were identical to the 525 bp before the stop codes （ including the stop codes） and the 238 bp after the stop codes ofRTN4 gene which encoded Nogo protein. And the 174 amino acid residues encoded by the insert were those of the C-terminal of Nogo protein- Nogo-A, Nogo-B and Nogo-C. RTN4 mRNA expressed in the murine inner ear was confirmed by RT-PCR method. Condusion The C-terminal of Nogo protein interacts with CX26. Nogo protein expresses in the inner ear and may take part in the trafficking of CX26 or CX26 gap junction function.
出处
《中华医学遗传学杂志》
CAS
CSCD
北大核心
2006年第5期492-496,共5页
Chinese Journal of Medical Genetics
基金
国家自然科学基金(30000094
30572021)~~
