OVEREXPRESSION AND SIMPLE PURIFICATION OF HUMAN IMMUNODEFICIENCY VIRUS-1 GAG EPITOPE DERIVED FROM A RECOMBINANT ANTIGEN IN ESCHERICHIA-COLI AND ITS USE IN ELISA
OVEREXPRESSION AND SIMPLE PURIFICATION OF HUMAN IMMUNODEFICIENCY VIRUS-1 GAG EPITOPE DERIVED FROM A RECOMBINANT ANTIGEN IN ESCHERICHIA-COLI AND ITS USE IN ELISA
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DOI：
10.1016/0168-1656(94)90084-1
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发表时间：
1994-05-15
影响因子：
4.1
通讯作者：
LEE, YI
中科院分区：
文献类型：
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作者：
SOHN, MJ;CHONG, YH;LEE, YI
为了开发一种诊断人类免疫缺陷病毒-1 （HIV-1）暴露敏感性的检测方法，利用含有trp启动子的表达载体，克隆了gag基因的一部分并在大肠杆菌中表达。重组蛋白的免疫反应性采用HIV-1特异性抗体进行Western blot分析。重组质粒pYHCgag3将gag基因与羟胺（HA）切割识别序列上的trpE基因融合，诱导核心抗原（gag a.a 121-398）以不溶性包涵体的形式过表达。重组gag通过简单的单步纯化程序纯化。在包涵体部分纯化后，经过ha -裂解处理，用DEAE-Sepharose层析进一步纯化gag蛋白至均匀性。纯化的核心抗原在酶联免疫吸附试验（ELISA）中提供了可靠的结果，具有高灵敏度和特异性，可用于鉴定HIV-1抗体。这些结果表明，重组核心抗原的大规模生产将为HIV-1血清学诊断提供宝贵的资源，用于筛选大量献血者以预防HIV-1感染。
To develop a test for diagnosis of human immunodeficiency virus-1 (HIV-1) exposure sensitivity, a part of the gag gene was cloned and expressed in Escherichia coli, using expression vectors containing a trp promoter. The immunoreactivity of recombinant protein was determined using HIV-1 specific antibodies in a Western blot analysis. The recombinant plasmid, pYHCgag3, gag gene was fused to the trpE' gene linked to the hydroxylamine (HA) cleavage recognition sequence which was induced to overexpress a core antigen (gag a.a. 121-398 from plasmid BH10) as fusion protein in the form of insoluble inclusion body. Recombinant gag was purified by a simple single step purification procedure. After partial purification of inclusion bodies and subject to the HA-cleavage treatment, gag protein was further purified to homogeneity using DEAE-Sepharose chromatography. The purified core antigen offered reliable results with high sensitivity and specificity for identification of HIV-1 antibodies when tested in the enzyme-linked immunosorbent assay (ELISA). These results suggest that mass production of recombinant core antigen will provide a valuable resource to HIV-1 serodiagnostics for the screening of large groups of blood donors to prevent HIV-1 infection.