CAJ | 학술논문

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:分离提取抗辐射菌染色体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-westernblotting(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,推测DBP特别是93kDa和33kDa两种新发现的DBP与抗辐射菌的辐射抗性有密切关系.
항복사균Deinococcus radiodurans시미소구균속적일충,타대전리복사、자외선급기타적화학유변제소유도적DNA손상도구유겁강적수복능력,고배수방사생물학계적관주.본연구지재검측항복사균적DNA결합단백(DNA --Binding Protein, DBP),병유선택지대기안기산서렬가이분석,이진일보연구DBP재항복사균적복사항성중적작용화의의.주요연구방법:분리제취항복사균염색체DNA병용지고신(DIG)가이표기,작위탐침;초성파파쇄균체,고속리심제비단백제취물,SDS-PAGE용우단백질전영,South-westernblotting(감성린산매표기적매련면역기술)검측DBP.결과표명,재야생형적항복사균중,검측도료8충DBP,분자량분별위122、93、33、29、16、15、14 화12kDa,기중93kDa화33kDa적량충DBP적표체수평수배양기화배양시간적변화이변화.N-말단안기산서렬측정표명,경FASTA화BLAST문고검색,저량충DBP화이지적기타단백질균몰유동원성,즉해량충DBP위신적단백질.유차득출결론,항복사균적DBP재수량상화안기산서렬적결구상도여기타세균불동,추측DBP특별시93kDa화33kDa량충신발현적DBP여항복사균적복사항성유밀절관계.
Deinococcus radiodurans (D. radiodurans) possesses a prominent ability to repair DNAinjury 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 theradioresistance of D. Radiodurans,the DBPs were detected from the bacteria and the amino-acid (aa) sequences of some DBPs were analysed. Inthis work, the chromosomal DNA labeled with Digoxin (DIG) was used as probe. The protein extract wasprepared by sonicating the bacteria cells and centrifugting at4C. South-western blotting was employed fordetecting DBPs. The results indicated that eight kinds of DBPs weredetected 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 of93kDa and 33kDa DBPs from wild type KR1 were changed with different culture media and different culturetime. The results of N-terminal aa sequence analysis showed thatthese two DBPs were not homologous ascompared with other known proteins according to FASTA and BLAST liberaries, i.e. they were two novelproteins. It could be concluded that DBPs and their aa sequences ofD. radiodurans were very differentfrom that of other bacteria. It was suggested that DBPs, especially 93kDa and 33kDa DBPs, might be closelyrelated with the radioresistance of D. radiodurans.