抗辐射菌Deinococcus　radiodurans的DNA结合蛋白的检测及分析(英文)

Detection and analysis of DNA-binding proteins from the radioresistant bacterium Deinococcus radiodurans

抗辐射菌Ｄｅｉｎｏｃｏｃｃｕｓ ｒａｄｉｏｄｕｒａｎｓ是微小球菌属的一种，它对电离辐射、紫外线及其他的化学诱变剂所诱导的ＤＮＡ损伤都具有极强的修复能力，故倍受放射生物学界的关注。本研究旨在检测抗辐射菌的 ＤＮＡ结合蛋白（ＤＮＡ－Ｂｉｎｄｉｎｇ Ｐｒｏｔｅｉｎ； ＤＢＰ），并有选择地对其氨基酸序列加以分析，以进一步研究ＤＢＰ在抗辐射菌的辐射抗性中的作用和意义。主要研究方法：分离提取抗辐射苗染色体ＤＮＡ并用地高辛（ＤＩＧ）加以标记，作为探针；超声波破碎菌体，高速离心制备蛋白提取物， ＳＤＳ－ＰＡＧＥ用于蛋白质电泳， Ｓｏｕｔｈ－ｗｅｓｔｅｒｎ ｂｌｏｔｔｉｎｇ（碱性磷酸酶标记的酶联免疫技术） 检测 ＤＢＰ。结果表明，在野生型的抗辐射菌中，检测到了８种ＤＢＰ，分子量分别为１２２、９３、３３、２９、１６、１５、１４和１２ｋＤａ，其中９３ｋＤａ和 ３３ｋＤａ的两种 ＤＢＰ的表达水平随培养基和培养时间的变化而变化。 Ｎ－末端氨基酸序列测定表明，经 ＦＡＳＴＡ和 ＢＬＡＳＴ文库检索，这两种 ＤＢＰ和已知的其他蛋白质均没有同源性，即该两种 ＤＢＰ为新的蛋白质。由此得出结论，抗辐射菌的ＤＢＰ在数量上和氨基酸序列的结构上都与其他细菌不同。

Deinococcus radiodurans (D. radiodurans) possesses a prominent ability to repair DNA injury induced by various DNA-damaging agents including mitomycin C, ultraviolet light and ionizing radiation. In order to demonstrate the effect of DNA-binding proteins (DBPs) on the radioresistance of D. Radiodurans, the DBPs were detected from the bacteria and the amino-acid (aa) sequences of some DBPs were analysed. In this work, the chromosomal DNA labeled with Digoxin (DIG) was used as probe. The protein extract was prepared by sonicating the bacteria cells and centrifugting at 4'C. South-western blotting was employed for detecting DBPs. The results indicated that eight kinds of DBPs were detected from the wild type D. radiodurans, the molecular weights of them are 122, 93, 33, 29, 16, 15, 14 and 12 kDa respectively. The expression levels of 93kDa and 33kDa DBPs from wild type KR1 were changed with different culture media and different culture time. The results of N-terminal aa sequence analysis showed that these two DBPs were not homologous as compared with other known proteins according to FASTA and BLAST liberaries, i.e. they were two novel proteins. It could be concluded that DBPs and their aa sequences of D. radiodurans were very different from that of other bacteria. It was suggested that DBPs, especially 93kDa and 33kDa DBPs. might be closely related with the radioresistance of D. radiodurans.