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目的:筛选间隙连接蛋白31.1(Cx31.1)的相互作用蛋白,进一步阐释Cx31.1在生殖健康方面可能的作用机制。方法:采用免疫共沉淀技术富集稳定表达Cx31.1-EGFP融合蛋白细胞中的Cx31.1结合蛋白,以稳定表达EGFP标签蛋白的细胞作为对照组。得到的蛋白复合物直接酶解后进行高效液相色谱-串级质谱(HPLC-MS/MS)分析,通过相应的肽段序列标签在Swiss Prot数据库中搜索蛋白质。结果:通过数据库搜索首次鉴定了5个与生殖相关的且与Cx31.1可能发生相互作用的蛋白,分别是HSPB1、HSPA2、HSPA5、BYP2和XAGE1A。其中HSPB1、HSPA2和HSPA5属于热休克蛋白家族,BYP2和XAGE1A蛋白在睾丸组织中特异表达。结论:Cx31.1可能与生殖相关蛋白具有相互作用。
AIM: To screen the interacting proteins of connexin 31.1 (Cx31.1) to further elucidate the possible mechanism of Cx31.1 in reproductive health. Methods: Cx31.1 binding protein was stably expressed in Cx31.1-EGFP fusion protein by co-immunoprecipitation. Cells stably expressing EGFP-tagged protein were used as control group. The resulting protein complexes were directly digested and subjected to high performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS) analysis to search for proteins in the Swiss Prot database by corresponding peptide sequence tags. Results: Five reproductive-related proteins that interacted with Cx31.1 were identified for the first time by database search, including HSPB1, HSPA2, HSPA5, BYP2 and XAGE1A. HSPB1, HSPA2 and HSPA5 belong to the heat shock protein family, and BYP2 and XAGE1A proteins are specifically expressed in testis tissue. Conclusion: Cx31.1 may interact with reproductive-related proteins.