摘要
从北京医院临床分离的铜绿假单胞菌，经药敏试验确证对头孢唑林（ＣＥＺ）及氨苄西林（ＡＢＰＣ）耐药的菌株６９１中，提取总ＤＮＡ，分别用ＢａｍＨⅠ和Ｓａｕ３ＡⅠ部分酶切后，与ＢａｍＨⅠ酶切的载体ｐＡＣＹＣ１８４（ＣＰＲ，ＴＣＲ，４．２ｋｂ）连接，转化大肠杆菌ＨＢ１０１，在含氯霉素（ＣＰ）５０μｇ／ｍｌ和ＡＢＰＣ１００μｇ／ｍｌ的选择性ＬＢ琼脂平板上筛选，获得了稳定性耐ＡＢＰＣ的菌落，从中提取质粒ＤＮＡ，并经酶切分析，证明是重组质粒ｐＷＷ－１，但结构比较复杂，推测有可能铜绿假单胞菌的ＤＮＡ在大肠杆菌中发生了重排。将ｐＷＷ－１重新转化大肠杆菌ＨＢ１０１，以ＣＰ、ＡＢＰＣ筛选转化子，从中提取质粒ＤＮＡ，得到重组质粒ｐＷＷ－２，经酶切分析确定ｐＷＷ－２的分子量为５．１ｋｂ，外源片段约０．９ｋｂ，推测其中含编码β－内酰胺酶的基因。以ｐＡＣＹＣ１８４为探针进行Ｓｏｕｔｈｅｒｎ分子杂交，证明ｐＷＷ－２确实为来源于ｐＡＣＹＣ１８４的重组质粒。利用克隆的含有铜绿假单胞菌β－内酰胺酶基因的大肠杆菌ＰＳＡＥ作为指示菌，建立了微生物来源的β－内酰胺酶抑制剂抗菌物质筛选模型，利用该指示菌筛选的具抗菌活性的物质，大部分具抗铜绿假单胞菌的性能?
Total DNA was isolated from a clinical isolate, Pseudomonas aeruginosa 691, which was resistant to cefazolin (CEZ) and ampicillin (ABPC). After partial digestion with BamHⅠ or Sau3AⅠ, total DNA from P.aeruginosa 691 were ligated to plasmid DNA pACYC184 linearized by BamHⅠ and transformed into E.coli HB101. A stable resistant to ABPC colonies were selected on LB plates with ABPC (100μg/ml) and chloramphenicol (CP 50μg/ml). A recombinant plasmid pWW 1 with complicated structure was shown by endonucleases digestion analysis, indicated that a possible rearrangement of Pseudomonas aeruginosa DNA in E.coli cells. A recombinant plasmid pWW 2 was recovered by retransformation of pWW 1 DNA into E.coli HB101. Restriction analysis indicated that the molecular size of pWW 2 was about 5.1kb with 0.9kb of insert presumably containing β lactamase gene in it. Southern analysis using plasmid pACYC184 as a probe showed that pWW 2 was a recombinant plasmid originated from pACYC184. A E.coli PSAE strain containing cloned β lactamase gene from Pseudomonas aeruginosa as an indicator strain was applied for screening β lactamase inhibitor from microorganism. The substances which were active against indicator strain dominantly showed activity against Pseudomonas aeruginosa .
出处
《中国抗生素杂志》
CAS
CSCD
北大核心
1997年第3期161-165,169,共6页
Chinese Journal of Antibiotics
