人类免疫缺陷病毒1型(HIV-1)核心蛋白(p24)在大肠杆菌中的表达、纯化及鉴定

EXPRESSION OF CAPSID PROTEIN(P24) OF HUMAN IMMUNODEFICIENCY VIRUS TYPE1(HIV-1)IN ESCHERICHIA COLI AND ITS PURIFICATION AND CHARACTERIZATION

在大肠杆菌中，利用新构建的含Ｔ７噬菌体ｇ－１０核糖体结合位点（ＲＢＳ），以及λ噬菌体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（ＣＡ）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７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。ＥＬＩＳＡ分析表明，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。

Capsid protein p24(CA)of human immunodeficiency virus type 1(HIV-1)was over pro duced in Escherichia coli by using a novel expression vector to express gag-pol region which encodes p24,p15 and viral proteinase(PR).The expression vector utilized λ P R promoter and T7 g-10L RBS for efficient translation initiation of the insert.4 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 against HIV CA.The N terminal sequence determined was identi cal to that of CA purified from HIV.A simple purification method was developed which could obtain miligrams of 95% pure CA protein based on two-step ion exchange chromatography following ammonium sulfate fractionation step.Our results demonstrated that the purified p24 may be of use as highly specific reagent for HIV-1 diagnosis.In addition,purified p24 can be used for its structural and biochemical analysis.