Production of Polyclonal Antisera to Choline Acetyltransferase Using a Fusion Protein Produced by a cDNA Clone Victor
Production of Polyclonal Antisera to Choline Acetyltransferase Using a Fusion Protein Produced by a cDNA Clone Victor
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使用 cDNA 克隆 Victor 产生的融合蛋白生产抗胆碱乙酰转移酶的多克隆抗血清
DOI：
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发表时间：
1988
影响因子：
4.7
通讯作者：
P. Salvaterra
中科院分区：
文献类型：
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作者：
J. Muñoz‐Maines;J. Slemmon;M. Panicker;N. Neighbor;P. Salvaterra
摘要：从大肠杆菌λ gtll裂解物中纯化了含有果蝇胆碱乙酰转移酶（Drosophila choline acetyltransferase, ChAT） cDNA片段的融合蛋白。该cDNA插入物含有728个氨基酸的ChAT编码区，用于免疫家兔。制备了三种不同的抗血清，可以识别低效价的原生果蝇ChAT。此外，所有三种抗血清都使用Western blot技术以高滴度染色酶多肽。部分纯化酶的Western blot结果显示，抗血清可识别分子量为67和54千道尔顿的ChAT多肽；这些多肽先前已使用单克隆抗ChAT抗体鉴定，并且是完全纯化酶的主要成分。令人惊讶的是，当这些抗血清用于果蝇头部匀浆的Western blots染色时，主要免疫反应带的分子质量为75千道尔顿。利用快速高效液相色谱凝胶过滤层析对新鲜蝇头匀浆进行分离，研究了该75千吨多肽与ChAT活性的关系。酶活性和免疫反应性多肽的柱分分析表明，75千道尔顿和67千道尔顿的多肽可以被分离，并且都具有酶活性。此外，当新鲜蝇头匀浆的上清液在37°C下自溶时，观察到75 -和67 -千道带的相对免疫染色强度与ChAT活性之间存在相关性。我们的研究结果表明，ChAT主要作为一种分子质量为75千道尔顿的活性酶存在于新鲜的果蝇头部中。先前报道的原生果蝇ChAT和部分纯化酶制剂中ChAT多肽的分子质量为67千道尔顿，这可能是由于酶的75千道尔顿形式的蛋白质水解所致。重组DNA技术提供了一种很好的方法来培养高滴度、序列导向的ChAT多克隆抗体，而不需要直接从果蝇中纯化酶。
Abstract: A fusion protein containing a Drosophila choline acetyltransferase (ChAT) cDNA insert was purified from a λ gtll lysate of Escherichia coli. The cDNA insert, which contained a 728‐amino acid coding region for ChAT, was used for immunizing rabbits. Three different antisera were produced that could recognize native Drosophila ChAT with low titer. In addition, all three antisera stained enzyme polypeptides using the Western blot technique at high titers. The antisera recognized ChAT polypeptides with molecular masses of 67 and 54 kilodaltons in Western blots of partially purified enzyme; these polypeptides had previously been identified using monoclonal anti‐ChAT antibodies and are the major components of completely purified enzyme. It was surprising that when these antisera were used to stain Western blots of Drosophila head homogenates, the major immunoreactive band had a molecular mass of 75 kilodaltons. The relationship of this 75‐kilodal‐ton polypeptide to ChAT activity was investigated by fractionating fresh fly head homogenates using rapid HPLC gel filtration chromatography. Analysis of column fractions for enzyme activity and immunoreactive polypeptides indicated that the 75‐ and 67‐kilodalton polypeptides can be resolved and are both enzymatically active. In addition, a correlation was observed between the relative immunostaining intensities of both the 75‐ and 67‐kilodalton bands and ChAT activity when supernatants from fresh fly head homogenates were autolyzed at 37°C. Our results indicate that ChAT is present in fresh Drosophila heads primarily as an active enzyme with a molecular mass of 75 kilodaltons. The previously reported molecular mass of 67 kilodaltons for native Drosophila ChAT as well as ChAT polypeptides in partially purified enzyme preparations may have resulted from proteolysis of the 75‐kilodalton form of the enzyme. Recombinant DNA technology provides a good approach to raise high‐titered, sequence‐directed polyclonal antibodies to ChAT without the difficulties encountered in purifying the enzyme directly from Drosophila.
胆碱乙酰转移酶的多种等电点和分子量变体。
DOI：
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发表时间：
1984
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
Hersh,LB;Wainer,BH;Andrews,LP
通讯作者：
Andrews,LP
DOI：
10.1073/pnas.80.5.1194
发表时间：
1983-01-01
期刊：
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA-BIOLOGICAL SCIENCES
影响因子：
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作者：
YOUNG, RA;DAVIS, RW
通讯作者：
DAVIS, RW