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目的 :应用酵母双杂交技术筛选和鉴定连接蛋白 2 6 (connexin 2 6 ,Cx2 6 )的相互作用蛋白质。方法 :以正常人DNA为模板 ,PCR扩增Cx2 6 (GJB2 )全长编码区作为诱饵 ,基因重组法定向克隆到第 3代MatchMakerGal4双杂交系统的 pGBKT7质粒 ,用构建的pGBKT7 Cx2 6质粒筛选人胎脑cDNA文库 ,获得的阳性克隆的插入子为Cx2 6的相互作用蛋白质 (猎物 ) ,将Cx2 6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 (neuroendocrinespecificprotein ,NSP)羧基端的 1 4 5个氨基酸残基序列一致 ,阅读框无移位。结论 :Cx2 6与NSP的羧基端具有相互作用 ,NSP可能在Cx2 6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
Objective: To screen and identify the interacting proteins of connexin 2 6 (Cx2 6) by yeast two-hybrid technique. Methods: The full length coding region of Cx2 6 (GJB2) was amplified by PCR using normal human DNA as a template. The recombinant plasmid was cloned into pGBKT7 plasmid of the 3rd generation MatchMakerGal4 two-hybrid system by gene recombination. The constructed plasmid pGBKT7 Cx2 6 Fetal brain cDNA library, positive clones obtained insert for the interaction protein Cx26 (prey), the Cx2 6 and the screening of the interacting proteins in response to a second yeast two-hybrid experiments to remove false positives. The DNA sequence of the positive cloned insert was sequenced and matched in GenBank and bioinformatics analysis. Results: The insert of one positive clone was consistent with the 1 45 amino acid residues of the carboxy terminal end of neuroendocrinespecific protein (NSP), with no shift in reading frame. CONCLUSION: Cx2 6 interacts with the carboxyl terminus of NSP. NSP may play a role in the physiological activities of Cx2 6 transport and assembly into gap junction or gap junction function