丙型肝炎病毒核心蛋白DNA结合区的测定

目的 阐明丙型肝炎病毒（HCV）核心蛋白的DNA结合特性及其意义。方法 在大肠杆菌中表达谷胱甘肽转移酶（GST）融合HCV核心蛋白不同长度片段,并进行纯化。经聚丙烯酰胺凝胶电泳后,通过更换缓冲液的方法将这些蛋白片段相对固定在凝胶中,用γ－^32P[4667-0091]ATP标记人工合成的DNA进行2次电泳,通过放射自影检测核心蛋白的DNA结合区。结果 HCV核心蛋白N端第10－16位氨基酸残基和第46－70位氨基酸残基可独立地结合DNA,核心蛋白既能和单链DNA结合又能和双链DNA结合,对单双链DNA的结合域相同。对所结合的DNA序列无选择性。结论 HCV核心蛋白的N端含有两个DNA结合区,结合区序列与核内转移信号的部分重迭以及对结合靶DNA序列的非选择性可能是HCV核心蛋白具有多功能的基础。

Determination of DNA binding domains in hepatitis C virus core protein

Objective To determine the DNA binding domain in hepatitis C virus core protein and otelucidate the significance of binding. Methods Segments of hepatitis C virus core protein were expressed ill E.coli as fusion forms with glutathion S-transferase (GST). The core proteins were immobilized in SDS-PAGE gel after removing SDS from the gel by washing. 32P-ATP labeled oligonucleotides were electrophoresed through the gel in TAE buffer. The binding of DNA with core protein was detected by autoradiography. Results There were at least two DNA binding domains in the HCV core protein, the first locating at 10~16aa and the second at 46-70aa. The second region was divided into three adjacent parts, which could bind core protein independently. Core protein bound to single strand DNA as well as to double strand DNA without sequence specificity. Conclusions DNA binding regions of HCV core protein locate at its N-terminus. The binding regions of HCV core protein overlap its nucleus transfer signals and they bind to target DNA unselectively, suggesting a possible mechanism for its multifunction. The result provides basic data for understanding the biological function of HCV core protein.