人类肠道病毒68型3A蛋白可溶区的表达及其结构初步研究

Expression and Preliminary Research on the Soluble Domain of EV-D68 3A Protein

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。

To understand the structure of the soluble region of Enterovirus 68 3Aprotein,we construct a prokaryotic expression vector expressing the soluble region of EV-D68 3Aprotein,and identify the forms of expression product after purification.The EV-D68 3A（1~61）gene was amplified by PCR and then cloned into the expression vector pET-28a-His-SUMO.The recombinant plasmid was transformed into Escherichia coli BL21 induced by IPTG to express the fusion protein His-SUMO-3A（1~61）.The recombinant protein was purified by Ni-NTA Agarose and cleaved by ULP Protease to remove His-SUMO tag.After that,the target protein 3A（1~61）was purified by a series of purification methods such as Ni-NTA,anion exchange chromatography and gel filtration chromato- graphy.Chemical cross-linking reaction assay was taken to determine the multiple polymerization state of the 3Asoluble region.A prokaryotic expression vector pET28a-His-SUMO-3A（1~61）expressing the solution region of EV-D68 3A was successfully constructed and plenty of highly pure target proteins were obtained by multiple purification steps.The total protein amount was about 5mg obtained from 1LEscherichia coli BL21 with purity95%.At the same time,those results determined the homomultimer form of soluble 3Aconstruct.These data demonstrated that the expression and purification system of the soluble region of 3Awere successfully set up and provide some basic konwledge for the research about 3Acrystal structure and the development of antiviral drugs targeted at 3Ato block viral replication.