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在大肠杆菌中,利用新构建的含T7g-10L RBS以及λ-PR启动子的新型原核表达载体,通过表达gag-pol基因片段,获得了具有天然序列的人类免疫缺陷病毒1型(HIV-1)核心蛋白p24的高效表达。克隆的gag-pol基因片段在其阅读框架移位区域插入了4bp碱基,其表达的病毒蛋白酶在阅读框架上与gag一致,从而实现了对gag-pol融合蛋白的有效加工,产生成熟的核心蛋白p24及其它产物。重组p24以可溶形式存在,可以被抗p24的单克隆抗体特异识别。测定的N端8个氨基酸序列与从病毒纯化的p24完全一致。在使用硫酸铵沉淀后,采用两步离子柱层析,可将重组蛋白纯化到95％以上的纯度。结果表明,纯化的p24可以作为特异性很强的试剂而用于HIV感染的诊断及病情的预后,并可用于p24的生化及结构分析。
In E. coli, a new type of prokaryotic expression vector containing T7g-10L RBS and λ-PR promoter was used to express human gag-pol gene fragment of human immunodeficiency virus type 1 (HIV-1 ) High expression of the core protein p24. The cloned gag-pol gene fragment inserted a 4 bp base in its reading frame shift region and the expressed viral protease was identical to the gag on the reading frame to allow efficient processing of the gag-pol fusion protein to produce a mature core Protein p24 and other products. Recombinant p24 exists in soluble form and can be specifically recognized by anti-p24 monoclonal antibodies. The N-terminal 8 amino acid sequence determined was completely consistent with the p24 purified from the virus. After precipitation with ammonium sulfate, the recombinant protein can be purified to a purity of 95% or more by two-stage ion-column chromatography. The results show that the purified p24 can be used as a highly specific reagent for the diagnosis of HIV infection and the prognosis of the disease, and can be used for biochemical and structural analysis of p24.