【摘要】 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。更多还原

【Abstract】 Capsid protein p24 (CA)of human immnodeficiency virus type 1 (HIV-1) was overproduced in Escherichia coli by using a novel expression vector to express gag-pot region which encodes p24,p15 and viral proteinase(PR). The expression vector utilized X PR promoter and T7 g-10L RBS for efficient translation initiation of the insert. Four base pairs were inserted at the frameshifting region of the gag-pol fragment. The expression plasmid constructed expressed viral proteinase, a pol gene fragment, in the gag reading frame .resulting efficient processing of mature CA protein and proteinase itself. The expressed CA was soluble, recognized by a monoclonal antibody directed a-gainst HIV CA. The N terminal sequences determined were identical to those of CA purified from HIV. A simple purification method was developed which could obtain milligrams of 95% pure CA protein based on two-step ion exchange chromtography following ammonium sulfate fractionation step. Our results show that the purified p24 may be of use as highly specific reagent for HIV-1 diagnosis. In addition .purified p24 can be used for stuctural and biochemical analysis.更多还原