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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?
Hepatitis C virus (HCV) nonstructural protein NS3 serine protease is located at the NS3 N-terminus and plays an important role in viral precursor protein processing and virus maturation. OBJECTIVE: To confirm the interaction between NS3 molecules and their cofactor NS4A molecules by the yeast two-hybrid system that was developed in recent years to detect the interaction between protein molecules. To further establish the yeast three-hybrid system for the detection of NS3 oligopeptide small molecule on the NS3 serine protease activity of competitive inhibition lay the foundation and the computer system analysis of NS3 and NS4A involved in the possible intermolecular interaction of the section for anti-HCV Oligopeptide small molecule drugs to provide the basis for the study. Methods: Virus RNA was extracted by one step method with guanidine isothiocyanate, and NS4A gene fragment was amplified by RT-PCR. Double-digested plasmid pGPT9-NS3, pGAD424-NS3 and pGBT9-NS4A were constructed by targeted digestion. The yeast two-hybrid system was then transformed into yeast two-hybrid system. The protein interactions were qualitatively and quantitatively determined by using X-gal and ONPG as substrates Detection. Protein hydrophobicity and secondary structure analysis were performed using computer software. Statistical analysis using t test. Results: The blue reaction of NS3 / NS3 and NS3 / NS4A showed that there was interaction between NS3 / NS3 and NS3 / NS4A. Quantitative analysis showed that the former interacted more strongly than the latter (P <0.05). Computer analysis showed that NS3 involved in intermolecular phase?