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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?
From Pseudomonas aeruginosa isolated from Beijing Hospital, the total DNA was extracted from strain 691 which was resistant to cefazolin (CEZ) and ampicillin (ABPC) by drug susceptibility test and partially digested with BamHⅠand Sau3AⅠ , Ligated with BamHI-digested vector pACYC184 (CPR, TCR, 4.2 kb), transformed into E. coli HB101 and selected on a selective LB agar plate containing 50 μg / ml of chloramphenicol (CP) and 100 μg / ml of ABPC to obtain Stability of ABPC-resistant colonies from which plasmid DNA was extracted and analyzed by restriction enzyme analysis showed that the recombinant plasmid pWW-1, but the structure is more complex, speculated that Pseudomonas aeruginosa DNA rearrangement occurred in E. coli. The pWW-1 was transformed into E.coli HB101 again. The transformants were screened by CP and ABPC. The plasmid DNA was extracted from the recombinant plasmid pWW-2. The recombinant plasmid pWW-2 was confirmed by restriction analysis. The molecular weight of pWW- .9kb, presumably containing the gene encoding β-lactamase. Southern hybridization with pACYC184 probe proved that pWW-2 was indeed a recombinant plasmid derived from pACYC184. Using the cloned Escherichia coli PSAE containing Pseudomonas aeruginosa β-lactamase gene as an indicator bacteria, a screening model of microbial-derived β-lactamase inhibitor antibacterial substance was established, and the antibacterial activity Substance, most of the anti-Pseudomonas aeruginosa performance?