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为了解人类肠道病毒68型3A蛋白可溶区的结构,本研究构建了EV-D68 3A蛋白可溶区的原核表达载体,在大肠杆菌中表达、纯化并对其结构进行了初步研究。首先,采用PCR的方法扩增EV-D68 3A(1~61)基因片段,克隆至原核表达载体pET-28a-His-SUMO中,转化进入BL21(DE3)感受态细胞,诱导表达出His-SUMO-3A(1~61)融合蛋白。经Ni-NTA树脂亲和层析初步纯化后得到大量融合蛋白,利用ULP酶酶切去除His-SUMO标签,通过Ni-NTA树脂亲和层析、阴离子交换层析柱、分子筛层析等方法分离标签并进一步纯化目的蛋白。最后通过化学交联反应检测目的蛋白的多聚化状态。结果显示,利用pET28a-His-SUMO-3A(1~61)原核表达载体能够在大肠杆菌中成功表达出大量可溶性的融合蛋白;经过多步纯化方法得到了大量高纯度的目的蛋白,平均每升大肠杆菌可得到约5mg目的蛋白,纯度达95%以上;通过化学交联反应证明了该蛋白的多聚化存在形式。本研究成功建立了EV-D68 3A蛋白可溶区的表达和纯化系统,为进一步获得3A蛋白晶体、研究3A蛋白功能以及设计以3A为靶点的抗病毒药物奠定了基础。
In order to understand the structure of the soluble region of human enterovirus 68 type 3A protein, a prokaryotic expression vector of EV-D68 3A soluble region was constructed and expressed in Escherichia coli. The structure of the fusion protein was also studied. First, the EV-D68 3A (1-161) gene fragment was amplified by PCR and cloned into the prokaryotic expression vector pET-28a-His-SUMO, which was then transformed into BL21 (DE3) competent cells to induce the expression of His-SUMO -3A (1 ~ 61) fusion protein. A large number of fusion proteins were obtained after preliminary purification by Ni-NTA resin affinity chromatography. The His-SUMO tag was digested by ULP enzyme and separated by Ni-NTA resin affinity chromatography, anion exchange chromatography and molecular sieve chromatography Label and further purify the protein of interest. Finally, the chemical cross-linking reaction to detect the target protein multi-state. The results showed that a large number of soluble fusion proteins were successfully expressed in E.coli using the prokaryotic expression vector pET28a-His-SUMO-3A (1 ~ 61). A large number of high-purity target proteins were obtained by multi-step purification method, Escherichia coli can obtain about 5mg of the target protein, with a purity of more than 95%; the chemical cross-linking reaction proves that the protein is in the form of multimerization. This study successfully established the expression and purification system of soluble region of EV-D68 3A protein, which lays the foundation for obtaining 3A protein crystal, studying the function of 3A protein and designing antiviral drug targeting 3A.