HCV NS3丝氨酸蛋白酶分子间及与其辅助因子NS4A分子间相互作用的研究

Studies on protein molecular interaction between NS3 and cofactor NS4A of HCV

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摘要丙型肝炎病毒（ｈｅｐａｔｉｔｉｓＣｖｉｒｕｓ，ＨＣＶ）非结构蛋白ＮＳ３丝氨酸蛋白酶位于ＮＳ３Ｎ端，对病毒前体蛋白的加工和病毒成熟具有重要作用。目的：用近年来发展起来的检测蛋白分子间相互作用的酵母双杂交系统，证实ＮＳ３分子间及与其辅助因子ＮＳ４Ａ分子间存在相互作用。为进一步建立酵母三杂交系统，用以检测针对ＮＳ３的寡肽小分子对ＮＳ３丝氨酸蛋白酶活性的竞争抑制作用奠定基础；并用计算机系统分析ＮＳ３及ＮＳ４Ａ参与分子间相互作用的可能区段，为抗ＨＣＶ的寡肽小分子药物的研究提供依据。方法：用异硫氰酸胍一步法提取病毒ＲＮＡ，经ＲＴ－ＰＣＲ扩增ＮＳ４Ａ基因片段。目的基因片段双酶切后定向克隆构建双杂交质粒ｐＧＰＴ９－ＮＳ３、ｐＧＡＤ４２４－ＮＳ３和ｐＧＢＴ９－ＮＳ４Ａ，然后转化酵母双杂交系统，分别以Ｘ－ｇａｌ和ＯＮＰＧ为底物对蛋白相互作用作定性和定量检测。蛋白疏水性和二级结构分析采用计算机软件进行。统计学处理采用ｔ检验。结果：ＮＳ３／ＮＳ３及ＮＳ３／ＮＳ４Ａ均出现蓝色反应，表明ＮＳ３／ＮＳ３分子间及ＮＳ３／ＮＳ４Ａ分子间的确存在相互作用，定量分析表明前者相互作用强于后者（Ｐ＜０．０５）。计算机分析结果显示，ＮＳ３参与分子间相? NS3 serine protease, located in the N-terminal domain of nonstructural protein NS3, is critical in processing HCV polyprotein. Objective: In order to evaluate the inhibitory effect of oligopeptides on NS3 serine protease, it is necessary to use yeast twohybrid system to prove the existence of interaction between NS3 and NS4A. Combination of the results of computer analysis of protein secondary structure and hydrophobicity would be useful for screening small hydrophobic molecules as anti-HCV drug, which disrupts NS3/NS3 or NS3/NS4A interaction. Methods: After isolation of viral total RNA with guanidinium method, the objective gene was amplified using RT-PCR. NS3, cloned in pGEMT vector, and the amplified NS4A were cleaved bimolecularly and subsequently cloned in twohybrid plasmids pGBT9 and pGAD424 respectively. The recombinant plasmids were transformed plasmids into yeast cell simultaneously and the interaction was assayed, using Xgal and ONPG as substrates. Results: Both NS3/NS3 Interaction and NS3/NS4A interactions showed blue color reaction, which indicated the existence of interaction between them, and the strength difference between NS3/NS3 and NS3/NS4A was significant(P<0.05). Computer analysis revealed that the interactions were hydrophobic ones, and the domains involved in the interactions were located in N terminal of NS3(1-30 amino acids) and NS4A(1-40 amino acids). Conclusion: The results of this study are consistent with those of other studies and provide the basis for developing NS3threehybrid system for further study of antiHCV drug of oligopeptides.