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[目的]筛选胎肾上腺cDNA文库中与α防御素HNP-1成熟肽具有相互作用的蛋白分子。[方法]通过聚合酶链反应(PCR)成功获得HNP-1成熟肽基因插入酵母表达载体pGBK-T7中构建诱饵质粒,同时提取胎肾上腺RNA,SMART技术制备人胎肾上腺cDNA文库,并采用Matchmaker LexA酵母双杂交系统从胎肾上腺cDNA文库中筛选与HNP-1成熟肽相互作用的蛋白。最后通过PCR筛选获得阳性克隆并测序,而后经回转实验,GST pull down以及免疫共沉淀再次验证两者的相互作用关系。[结果]成功构建诱饵质粒pGBKT7-HNP-1以及胎肾上腺cDNA文库并在酵母细胞中正确表达,筛选获得HNP-1成熟肽相互作用的蛋白-melanocortin 2 receptor (ACTH-R),CCAAT-enhancer-bind-ing proteins(C/EBP),Tramembrane trafficking protein(TMP21),low density lipoprotein receptor-related protein 6(LRP6)。最终选定促肾上腺皮质激素受体(ACTH-R)作为主要研究对象,且经GST pull down以及免疫共沉淀实验获得了证实两者间具有相互作用的相应的条带。[结论]从胎肾上腺cDNA文库中成功筛选得到α防御素HNP-1成熟肽相互作用蛋白——促肾上腺皮质激素受体。
[Objective] To screen the protein molecules that interacted with the mature peptide of α-defensin HNP-1 in fetal adrenal cDNA library. [Method] The mature peptide gene of HNP-1 gene was successfully inserted into the yeast expression vector pGBK-T7 by polymerase chain reaction (PCR) to construct the bait plasmid. Meanwhile fetus adrenal RNA was extracted and the fetal adrenal cDNA library was prepared by SMART technology. Yeast Two-Hybrid System Screens Fetal Adrenal cDNA Library for Proteins that Interact with HNP-1 Mature Peptides. Finally, the positive clones were screened by PCR and sequenced, and then the interaction between them was verified again by the rotation experiment, GST pull down and co-immunoprecipitation. [Result] The bait plasmid pGBKT7-HNP-1 and fetal adrenal cDNA library were successfully constructed and correctly expressed in yeast cells. The protein-melanocortin 2 receptor (ACTH-R), CCAAT-enhancer- bind-ing proteins (C / EBP), Tramembrane trafficking protein (TMP21), low density lipoprotein receptor-related protein 6 (LRP6). The adrenocorticotropic hormone receptor (ACTH-R) was finally selected as the main study object, and the corresponding bands confirmed by GST pull down and co-immunoprecipitation experiments confirmed their interaction. [Conclusion] The mature adrenocorticotropic hormone alpha-defensin HNP-1 interacting protein-adrenocorticotropic hormone receptor was successfully screened from fetal adrenal cDNA library.