心脏连接蛋白43羧基末端相互作用蛋白的酵母双杂交筛选

Identification of cardiac connexin 43 interacting protein using the yeast two-hybrid system

目的研究心脏连接蛋白43(Cx43)羧基末端与哪些心肌细胞内蛋白质存在相互作用。方法①通过PCR方法得到编码心脏Cx43羧基末端(AA235-382)的cDNA片段,并在其两端分别加上EcoRI和BamHI酶切位点,应用EcoRI/BamHI酶切PCR产物及pGBKT7空载体,凝胶分离后应用T4连接酶进行连接;②通过化学转化法将pG-BKT7-Cx43-CT转化酵母菌AH109;③通过“尿素/SDS”法从被转化的酵母菌中提取蛋白质;④应用抗C-myc抗体,通过Western blot方法检测”诱饵”蛋白的表达(即Gal4-BD-C-myc-Cx43-CT融合蛋白);⑤检测”诱饵”蛋白自我激活报告基因与否后,将已被“诱饵“质粒转化的AH109与人心脏cDNA文库进行杂交,筛选阳性克隆分离阳性克隆中的cDNA,并测序。结果①诱饵载体测序结果证明pGBKT7中的“Gal4 DNA binding domain-C-myc”与Cx43的羧基末端在同一读框中;②“诱饵”质粒转化AH109酵母菌成功率100%;③从被转化的AH109中能提取到浓度满意的总蛋白;④Western blot能检测到特异性条带,其位置与Gal4-BD-C-myc-Cx43-CT的分子量相当;⑤“诱饵”蛋白不能自激活报告基因Ade2和Mel1,但可自激活His3,5mmol/L的3-AT可有效抑制“诱饵”蛋白本身自激活报告基因His3,以利于下一步的杂交筛选,筛选得到10个准阳性克隆。结论心脏连接蛋白43羧基末端能与心肌细胞中的多种蛋白质存在相互作用,这些蛋白质可能参与对间隙连接通道的功能调控。

AIM To identify the connexin 43 （ Cx43 ） binding proteins in human heart by a yeast 2-hybrid screen. METHODS ① A eDNA cassette encoding the cardiac gap junctional connexin43 carboxylic terminal （AA235-382）introduced with EcoRI and BamHI restriction enzyme cutting sites was PCR amplified and pGBKT7-Cx43-CT was constructed by digesting the PCR product with EcoRI and BamHI restriction enzymes, followed by ligation to EcoRI/BamHI digested pGBKT7 plasmid using T4 DNA ligase; ② The pGBKTT-Cx43-CT was transformed into AH109 yeast cells using chemical method; ③ The extraction of protein from the transformed AH109 yeast cells was accomplished by the＂ Urea/SDS Method＂; ④ The expression of ＂bait＂ protein （Ga14-BD-C-myc-Cx43-CT fusion protein） was detected by Western blot using anti-C-myc antibody; ④After determining whether the bait itself auto-activated the reporter genes, the AH109 transformed with the pGBKTT-Cx43-CT mated with the human heart eDNA library and the positive clones were sequenced. RESULTS ① The ＂bait＂ plasmid was verified by se-quencing, ensuring that the Cx43-CT was inframed with the＂Ga14 DNA binding domain-C-myc＂ and the PCR mediated mutations were not inadvertently introduced; ② The ＂bait＂ plasmid was transformed into AH109 yeast cells with 100 percent success rate; ③ Total protein with approving concentration was extracted from transformed AH109 yeast cells; ④ The Western blot confirmed the stable expression of the Gal4-BD-C-myc-Cx43-CT fusion protein; ⑤ There was no auto-activation by the ＂bait＂ protein, but weak leaky His3 expression was found, which was inhibited by 5 mM/L 3-AT. After mating with a human heart cDNA library, 10 tentative positive clones were obtained. CONCLUSION Multiple proteins in cardiac myocytes interact with the cardiac gap junctional connexin43 carboxylic terminal and these proteins may be involved in the regulation of the GJ channel.