EXPRESSION OF HIV ANTIGENS IN E COLI
EXPRESSION OF HIV ANTIGENS IN E COLI
批准号:
3792517
负责人:
C J MARCUS-SEKURA
金额:
$0.0万
依托单位:
--
依托单位国家:
美国
项目类别:
财政年份:
--
资助国家:
美国
项目状态:
未结题
起止时间:
至
关键词:
DNA binding protein DNA directed DNA polymerase Escherichia coli X ray crystallography antiserum antiviral agents chimeric proteins chromosomes drug design /synthesis /production enzyme activity enzyme linked immunosorbent assay enzyme structure gene expression human immunodeficiency virus 1 human immunodeficiency virus 2 hybridomas immunoglobulin structure laboratory rabbit molecular cloning monoclonal antibody protein purification southern blotting virus DNA virus antigen virus protein western blottings
中文摘要
HIV整合酶(IN)是病毒整合所必需的一种病毒酶
DNA进入宿主染色体。这种类型的集成是高度
针对逆转录病毒,因此HIV-1IN是
抗病毒治疗。要设计抗病毒药物,至关重要的是
描述一下In。我们已经在大肠杆菌中以融合形式表达了IN基因
蛋白。克隆的IN对HIV-1和HIV-2阳性均有反应
ELISA检测患者血清,兔抗重组蛋白血清
经Western印迹分析,只与HIV-1 IN反应,而不与HIV-2 IN反应。这些
数据已经在AIDS Res.Hum上发表。复古。杂交瘤已经被
用IN表达克隆制备的单抗与
Western印迹结果显示HIV-1不表达HIV-2。已经提交了一份发明报告
一份手稿正在准备中。表达N-的其他克隆
和C-末端的一半已被构建,它们的蛋白质
用西南印迹法检测Express结合DNA的能力
程序。完整的IN分子和C-末端蛋白
结合DNA;N-末端部分没有结合活性,
提示C-末端含有DNA结合位点。一个
手稿最近发表在艾滋病研究中心。复古。我们有
纯化了我们的全长重组IN,它在
含2个碱基对的酶活性的体外特异性测定
从与HIV-1 LTR对应的寡核苷酸中分离出来的。因为我们的IN是
产生的融合蛋白含有13个氨基酸的lambda CII at
它的氨基末端,检测我们的蛋白质的活性表明
活性不需要游离氨基末端。这可能是
与体内情况有关,因为IN最初合成为
一种在其氨基末端与RT相连的多蛋白。
为了更准确地定位C-末端内的DNA结合位点
分子的一半,我们已经构建了一系列额外的
亚克隆,我们目前正在提纯它们缺失的蛋白质
用于DNA结合分析的产品。我们最近建立了一个
与大卫·戴维斯在NIH的合作为他提供了
用于产生X射线FAB碎片的In MAb的量
IN的结晶学,这可能最终促进酶的设计
抑制剂。
英文摘要
HIV integrase (IN) is a viral enzyme required for integration of virus
DNA into the host chromosome. This type of integration is highly
specific for retroviruses, and HIV-1 IN is therefore a target for
antiviral therapy. To design antivirals, it is essential to
characterize IN. We have expressed the IN gene in E. coli as a fusion
protein. The cloned IN is reactive with both HIV-1 and HIV-2 positive
patient sera in ELISA, while rabbit antisera to the recombinant protein
are reactive only with HIV-1 IN but not HIV-2 IN by Western blot. These
data have been published in AIDS Res. Hum. Retro. Hybridomas have been
prepared using the IN expressing clone and the MAbs are reactive with
HIV-1 but not HIV-2 by Western blot. An invention report has been filed
and a manuscript is in preparation. Additional clones expressing the N-
and C-terminal halves of IN have been constructed and the proteins they
express examined for ability to bind DNA using a Southwestern blotting
procedure. The complete IN molecule as well as the C-terminal protein
bind DNA; the N-terminal portion exhibits no binding activity,
suggesting that the C-terminal region contains the DNA binding site. A
manuscript has recently been published in AIDS Res. Hum. Retro. We have
purified our full-length recombinant IN and it exhibits activity in a
specific in vitro assay for enzyme activity involving cleavage of 2 bp
from an oligonucleotide corresponding to the HIV-1 LTR. Since our IN is
produced as a fusion protein containing 13 amino acids of lambda cII at
its amino terminus, detection of activity with our protein indicates
that a free amino-terminus is not required for activity. This may be
relevant to the in vivo situation, since IN is initially synthesized as
a polyprotein linked to RT at its amino-terminus.
To more precisely localize the DNA binding site within the C-terminal
half of the molecule, we have constructed a series of additional
subclones and we are currently purifying the deleted proteins they
produce for analysis of DNA binding. We have recently established a
collaboration with David Davies at the NIH to provide him with large
quantities of IN MAb for generation of Fab fragments for X-ray
crystallography of IN, which may eventually facilitate design of enzyme
inhibitors.
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项目类别:
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资助金额:$0.0万
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财政年份:--
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依托单位:--
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资助金额:$0.0万
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批准号:3792518
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资助金额:$0.0万
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依托单位:--