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目的筛选p53启动子结合蛋白,探讨p53对肝细胞调节的分子生物学机制。方法应用噬菌体表面展示技术,以p53作为固相筛选分子,对噬菌体人肝细胞cDNA文库进行5轮“吸附-洗脱-扩增”过程,经噬斑的PCR扩增后,构建克隆载体,最后对所筛选克隆进行DNA测序和生物信息学分析。结果噬菌体经5轮富集后,成功构建了噬菌体p53展示文库,该文库包含与p53结合的肝细胞蛋白有17种。其中2个克隆编码未知功能蛋白;其余的克隆分别编码人α2HS糖蛋白(AHSG)、人D4、锌指蛋白家族2(DPF2)、凋亡反应锌指基因(REQ)、Ⅰ型纤溶酶原激活物抑制因子结合蛋白、氨基甲酰磷酸合成酶1、磷酸化酶激酶、酪氨酸氨基转移酶、酪蛋白激酶细胞周期素激酶、谷胱甘肽过氧化物酶、组蛋白结合蛋白3、跨膜蛋白2、血清黏蛋白2、有核红细胞白血病病毒癌基因2、毛细管扩张失调症突变基因,涉及到细胞发育和代谢、细胞外基质、细胞周期调控、信号转导、细胞凋亡以及原癌基因。结论噬菌体表面展示技术成功筛选出了肝细胞p53启动子结合的多种类型和功能蛋白。
Objective To screen p53 promoter-binding proteins and investigate the molecular mechanism of p53 regulation on hepatocytes. Methods Phage display technique was used to screen the phage human hepatocyte cDNA library with 5 cycles of “adsorption-elution-amplification” using p53 as a solid-phase screening molecule. After plaque PCR amplification, a cloning vector Finally, the selected clones were subjected to DNA sequencing and bioinformatics analysis. Results After 5 rounds of phage enrichment, a phage p53 display library was successfully constructed, which contains 17 kinds of hepatocyte proteins that bind to p53. Two of the clones encoded unknown functional proteins. The remaining clones encoded human α2HS glycoprotein (AHSG), human D4, zinc finger protein family 2 (DPF2), apoptotic zinc finger (REQ), type I plasminogen, Activin inhibitor binding protein, carbamyl phosphate synthetase 1, phosphorylase kinase, tyrosine aminotransferase, casein kinase cyclin kinase, glutathione peroxidase, histone binding protein 3, Transmembrane protein 2, Serum mucin 2, Nucleocytopenic leukemia virus oncogene 2, Capillary dilatation disorder gene, related to cell development and metabolism, extracellular matrix, cell cycle regulation, signal transduction, apoptosis, Oncogene. Conclusion The phage display technique successfully screened many types and functional proteins of hepatocyte p53 promoter.