采用长片段PCR、Western-blotting、质粒互补试验对大肠杆菌野生型和超突变子的MMR重要组分MutS进行了研究。为测定mutS可能的缺失，参照fhlA-mutS-rboS基因簇，在mutS的两侧及中间设计3对引物进行长片段PCR扩增，以K12为模式菌株，其mutS各片段与预期的大小相同，3条片段长度分别为12045，10668，8477 bp；超突变子CE3101和CE5116与 K12相比，3条片段均存在不同程度的缺失：第1条带分别缺失约3500 bp和7000 bp；第2条带分别缺失约3600 bp和7400 bp；第3条带分别缺失约2500 bp和7500 bp。采用Western-blotting方法对大肠杆菌K12、正常菌株、超突变子的MutS蛋白检测结果表明，大肠杆菌K12的条带最为清晰，MutS蛋白最为完整；其次为 CE1112、CE1305、CE2219、CE2307，条带较为清晰，MutS蛋白较为完整；CE5103、CE5120条带较为模糊，CE2205只有1条微弱的条带，MutS 蛋白不完全完整；而超突变子 CE3101、CE5116几乎没有条带，MutS蛋白不完整。以pBR322质粒对照，采用pBR322构建的mutS＋的pGW1811质粒进行质粒互补试验，CE1117、CE3101、CE1305、CE5116、CE6107、CE2313、CE7301、K12等8株菌株转化成功。转入pGW1811前后各株大肠杆菌对利福平（RIF）的突变频率及抑制率测定表明，菌株 CE1117、CE1305、CE6107、CE2313及K12在转入pGW1811前后对RIF的突变频率变化不大（40％）；而突变子CE3101、CE5116、CE7301在转入pGW1811前后对RIF的突变频率变化较大（＞95％）。以上试验结果表明，与野生型大肠杆菌相比，超突变子大肠杆菌的MutS均存在缺陷，说明大肠杆菌超突变子发生的分子基础是其MMR系统重要组分MutS存在缺陷。%The MutS (important constituent of MMR)of wild-type E .coli and hypermutators was studied using long PCR,Western blotting and plasmid complement essay .In order to determine possible deletion of mutS,three pairs of primers in two sides and middle of mutS were designed according to fhlA-mutS-rboS gene cluster and long PCR was carried out .K1 2 was model strain and each fragment of its mutS was identical with the anticipant fragment .Length of the three fragments was 1 2 045 bp,1 0 688 bp and 8 477 bp,respectively .Compared to K1 2,three fragment length of hypermutator CE31 01 and CE51 1 6 had different extent deletion:the first band had deletion of 3 500 bp and 7 000 bp,the second band had 3 600 bp and 7 400 bp and the third band had 2 500 bp and 7 500 bp,respectively .The MutS protein of K1 2,normal strains and hypermutators were determined by Western blotting .K1 2 had a very clear band and its MutS was the most complete .CE1 1 1 2,CE1 305,CE221 9 and CE307 had fairly band and their MutS were fairly complete,too .But CE51 03 and CE51 20 had vague band and CE2205 had only tenuous band,their MutS proteins were incompletely integrated .The hypermutator CE31 01 and CE51 1 6 nearly had no band and their MutS were not integrated .With pBR322 as a control,plasmid complement essay was carried out using pGW1 81 1 plasmid constructed from pBR322 .CE1 1 1 7,CE31 01 ,CE1 305, CE51 1 6,CE61 07,CE231 3,CE7301 and K1 2 were transformed successfully .Mutation frequencies of CE1 1 1 7,CE1 305,CE61 07,CE231 3 and K1 2 to RIF changed very little and their change rate was about 40%.But hypermutator CE31 01 ,CE51 1 6 and CE7301 mutation frequency changed drastically and their change rate was above 95%.These results suggested that the MutS of hypermutator E .coli had defection compared to wild-type E .coli .
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