人转运蛋白SR2的原核表达及其与HIV-1整合酶体外相互作用的检测

Prokaryotic expression of tansportin-SR2 and its interaction with HIV-1 integrase in vitro

人转运蛋白SR2(TRN-SR2)与HIV-1整合酶(IN)相互作用是抗HIV治疗的有效靶点,寻找和设计该靶点的高效抑制剂具有重要的临床意义和广阔的应用前景。因此为了开展以TRN-SR2与HIV-1IN相互作用为靶点的抑制剂筛选研究,以pGEX-2T为载体的重组质粒pGST-TRN-SR2在大肠杆菌Rosetta(DE3)中进行原核表达,并利用亲和层析纯化得到浓度为0.80mg/mL的重组TRN-SR2蛋白;然后采用生物膜干涉技术(BLI)进行体外检测重组TRN-SR2蛋白与HIV-1IN的相互作用和结合情况。结果表明,重组TRN-SR2蛋白与HIV-1IN相互结合信号明显升高,且测得平衡解离常数KD值为45.2nmol/L。最后利用均相时间分辨荧光技术(HTRF)和交叉滴定实验确定了TRN-SR2与HIV-1IN在该靶点抑制剂筛选实验体系中的最适反应浓度分别为20和40nmol/L。以上研究结果说明原核表达的重组TRN-SR2蛋白的活性较好且与HIV-1IN体外相互作用明显,同时在筛选实验体系中TRN-SR2与HIV-1IN最适浓度的确定也为后续抑制剂的筛选提供了相应的研究基础。

The protein-protein interaction (PPI) between human transporter SR2 (TRN-SR2) and HIV-1 integrase (IN) is an effective target for anti-HIV therapy. It has important clinical significance and broad application prospects to find and design effective inhibitors of this PPI. In order to screen inhibitors targeting the PPI between TRN-SR2 and HIV-1 IN, the recombinant TRN-SR2 proteins were expressed in E. coli Rosetta (DE3) and purified by GST affinity chromatography column. The result showed that the recombinant TRN-SR2 protein was obtained with a concentration of 0.80 mg/mL. The interaction between TRN-SR2 and HIV-1 IN was confirmed in vitro by biofilm interferometry (BLI). The results showed that the binding signal increased significantly with TRN-SR2 binding to HIV-1 IN and the equilibrium dissociation constant (K D ) was determined as 45.2 nmol/L. The optimal reaction concentrations of TRN-SR2 and HIV-1 IN in the target inhibitor screening system were determined by homogeneous time-resolved fluorescence (HTRF) and cross-titration experiments, which were 20 and 40 nmol/L, respectively. All these results suggested that the recombinant TRN-SR2 protein was biologically active and could effectively interact with HIV-1 IN in vitro . The determination of the optimal concentration of TRN-SR2 and HIV-1IN in the screening system also provides a basis for the subsequent screening of inhibitors.