Production of antibodies to peptide sequences present in human O6-alkylguanine-DNA alkyltransferase and their use to detect this protein in cell extracts.
Production of antibodies to peptide sequences present in human O6-alkylguanine-DNA alkyltransferase and their use to detect this protein in cell extracts.
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针对人 O6-烷基鸟嘌呤-DNA 烷基转移酶中存在的肽序列的抗体的生产及其用于检测细胞提取物中的该蛋白质的用途。
DOI：
10.1093/carcin/12.9.1671
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发表时间：
1991
期刊：
影响因子：
4.7
通讯作者：
Dolan,ME
中科院分区：
文献类型：
--
作者：
Pegg,AE;Wiest,L;Mummert,C;Dolan,ME
兔抗血清中含有3个与人6-烷基鸟嘌呤- dna烷基转移酶氨基酸序列相对应的肽段（残基1-11、8-20和197-207）。这些抗血清在许多Mer+人肿瘤细胞系的Western blots蛋白上识别烷基转移酶蛋白，但在Mer -肿瘤细胞系中发现该蛋白不存在。这些抗血清也在人肝脏样品的一些提取物中检测到烷基转移酶蛋白，但其他具有高烷基转移酶活性的人肝脏提取物无法与针对该蛋白羧基末端的抗体发生反应，这表明该区域的一部分可以通过蛋白酶作用去除而不会失去活性，或者该区域存在遗传变异性。这一结果表明，研究与一些抗血清或抗体已知是针对一个基本的，不变的区域烷基转移酶将需要明确的检测烷基转移酶通过抗体筛选。在转染人DNA后，先前选择用于烷基转移酶表达的CHO细胞系中没有免疫反应性的人烷基转移酶蛋白。因此，仓鼠基因很可能在这些细胞中被重新激活。
Antisera were raised in rabbits to three peptides which correspond to sequences of amino acids present in humanO6-alkylguanine-DNA alkyltransferase (residues 1–11, 8–20 and 197–207). These antisera recognized the alkyltransferase protein on Western blots of proteins from a number of Mer+human tumor cell lines but this protein was found to be absent from Mer−tumor cell lines. The akyltransferase protein was also detected by these antisera in some extracts from human liver samples but other human liver extracts having a high alkyltransferase activity failed to react with antibodies directed towards the carboxyl terminus of the protein, suggesting that part of this region can be removed by protease action without loss of activity or that there is genetic variability in this region. This result indicates that studies with a number of antisera or with an antibody known to be directed towards an essential, invariant region of the alkyltransferase will be needed for unequivocal detection of the alkyltransferase by antibody screening. The immunoreactive human alkyltransferase protein was absent from CHO cell lines that had previously been selected for alkyltransferase expression after transfection with human DNA. It is therefore probable that the hamster gene has been reactivated in these cells.