LEDGF／p75蛋白的可溶性表达、纯化及功能研究

Prokaryotic soluble expression, purification and function study of LEDGF/p75 protein

HIV-1整合酶(integrase,IN)是病毒复制过程中的一个关键酶,其与宿主晶状体上皮源性生长因子p75(lens epithelium-derived growth factor p75,LEDGF/p75)的蛋白-蛋白相互作用是筛选抗病毒药物的一个重要靶点。为开展以IN-LEDGF/p75相互作用为靶点的抑制剂研究,本研究构建了LEDGF/p75蛋白重组质粒,在原核细胞中进行了可溶性表达和功能研究。根据大肠杆菌密码子偏爱性,全合成高利用率密码子的LEDGF/p75基因序列,并克隆到表达载体pGEX-4T-1中构建重组质粒。在大肠杆菌中优化表达LEDGF/p75蛋白,经SDS-PAGE鉴定和亲和色谱纯化蛋白,采用酶联免疫吸附实验方法 (ELISA)测定了LEDGF/p75蛋白的生物学活性。结果显示,构建的重组质粒获得高效稳定的可溶性表达,ELISA证实体外表达的LEDGF/p75蛋白能够与HIV-1 IN相互作用,并促进IN链转移反应的完成。本研究为建立以LEDGF/p75-IN相互作用为靶点的抗HIV药物筛选平台打下了基础。

HIV-1 integrase （IN） is a key enzyme for the viral replication. The protein-protein interaction （PPI） between HIV-1 IN and a cellular cofactor lens epithelium-derived growth factor （LEDGF/p75） is a validated target for anti-HIV drug discovery. In order to build the platform for screening inhibitor against PPI between IN and LEDGF/p75, the vector containing the LEDGF/p75 protein cDNA was constructed and expressed in Escherichia coli and the function of the LEDGF/p75 protein was assayed. The LGDGF/p75 encoding gene optimized according to the preference codon usage of E. coli, was synthesized and cloned into the expression vector pGEX-4T-1 to form a recombined plasmid, then transformed into host cell E. coli BL21 （DE3）. The recombined clones were identified and confirmed by BamH I/Sal I digestion and sequencing, the successfully recombined plasmid in the host cell was induced by IPTG and the condition of the expression was optimized. The expressed protein was purified by the Ni2＋ affinity chromatography column and SDS-PAGE was used to analyze the molecular weight and specificity. In addition, ELISA assay was used to analyze thefunction of the recombinant protein. The recombinant LGDGF/p75 was soluble, and expressed highly and stably in E. coli. The protein was proved to enhance HIV-1 1N strand transfer activity in vitro by ELISA. It will be helpful to build the platform of screening inhibitors against PPI between 1N and LEDGF/p75.