连接蛋白26与神经内分泌特异蛋白的羧基端相互作用

目的：应用酵母双杂交技术筛选和鉴定连接蛋白26（connexin 26, Cx26）的相互作用蛋白质。方法：以正常人DNA为模板,PCR扩增Cx26（GJB2）全长编码区作为诱饵,基因重组法定向克隆到第3代MatchMaker Gal4双杂交系统的pGBKT7质粒,用构建的pGBKT7-Cx26质粒筛选人胎脑cDNA文库,获得的阳性克隆的插入子为Cx26的相互作用蛋白质（猎物）,将Cx26和筛选到的相互作用蛋白再一对一回复进行酵母双杂交实验,去除假阳性。对阳性克隆插入子的DNA序列进行测序,在GenBank中作匹配及生物信息学分析。结果：1个阳性克隆的插入子与神经内分泌特异蛋白（neuroendocrine specific protein, NSP）羧基端的145个氨基酸残基序列一致,阅读框无移位。结论：Cx26与NSP的羧基端具有相互作用,NSP可能在Cx26蛋白的转运、装配成间隙连接或间隙连接功能等生理活动过程中起一定作用。

Interaction of connexin 26 with the C-terminal of neuroendocrine specific protein

OBJECTIVE: To screen and identify the interactive proteins with connexin 26 (Cx26) by the yeast two hybrid technique. METHODS: The whole coding region of Cx26 (GJB2) gene was amplified from normal human genomic DNA by polymerase chain reaction (PCR). The "bait" Cx26 was then subcloned into the vector pGBKT7 plasmid of the MatchMaker Gal4 Two-Hybrid System 3 as a target to screen its interactive proteins ("prey") from the human fetal brain eDNA library by the yeast two hybrid technique. The false positive clones were discarded from the preys by one to one yeast two hybrid method between Cx26 and the preys. The DNAs of the preys were sequenced and BLAST analyzed against the GenBank, and also underwent other bioinformatics analysis. RESULTS: The insert of one positive clone contained 145 amino acids residues that was identical to the C-terminal of the neuroendocrine specific protein (NSP) and the open reading frame of the insert was correct. CONCLUSION: Cx26 is interacted with the C-terminal of NSP. NSP may participate in the process of Cx26 transportation, assembling the connexon, or influencing the functions of its connexons.