紫外交联实验筛选HCV RNA结合蛋白的应用

Ultraviolet cross-linking assay in screening of cellular proteins binding to Hepatitis C virus RNA

摘要:
目的选择一种用于筛选与丙型肝炎病毒(HCV)RNA核心区结合的细胞蛋白质分子较佳方法。
方法用体外转录的方法制备地高辛标记(DIG)-HCV RNA; 从HepG2细胞中提取细胞蛋白质分子。分别用紫外交联实验(UV)和凝胶迁移实验(EMSA)筛选HepG2细胞中可与HCV RNA结合的蛋白质分子。
结果 2种实验方法均可检测到细胞蛋白质分子与HCV RNA结合, 但凝胶迁移实验结果所见DIG-RNA信号比较弥散, 而紫外交联实验的结果可见比较清晰的RNA-结合蛋白条带。
结论紫外交联实验是用于筛选HCV-RNA结合蛋白的首选方案。

Abstract:
Objective To select a better method for screening of cellular proteins binding to Hepatitis C virus(HCV)RNA.
Methods DIG-labeled RNA(DIG-RNA)transcripts were prepared by in vitro transcription.Cytoplasmic extracts were prepared from human hepatoma cells(HepG2).Ultraviolet(UV)cross-linking assay and electrophoretic mobility shift assay(EMSA)were used to screen the cellular proteins that would bind to HCV RNA.
Results Both UV cros-slinking assay and EMSA detected that there were proteins binding to HCV RNA.The signals of DIG-RNA detected by EMSA were dispersing and unclear.The results of UV cross-linking assay were clear RNA-binding protein bands.
Conclusion UV cross-linking assay is a preferred method for screening of cellular proteins binding to Hepatitis C virus RNA and other RNA molecules.