利用表型筛选法测定整合子对耐药性基因盒的整合频率

Detection of integration frequency catalyzed by integrase using phenotypic screening method

目的建立一种利用表型筛选的方法来测定整合子对耐药性基因盒的整合频率。方法将整合子和aadA2耐药性基因盒克隆到同一质粒pACYC184的不同位点，该重组质粒和高表达整合酶的重组质粒分别转化大肠杆菌B121（DE3），挑选阳性克隆过夜培养后，取适量菌液涂布含有链霉素的LB琼脂平板，同时取适量菌液涂布不含链霉素的LB琼脂平板，过夜培养后计数菌落个数，用以计算整合频率。同时以链霉素平板上的阳性克隆为模板，进行PCR扩增。对扩增产物切胶回收纯化，然后进行测序，以确定aadA2耐药性基因盒整合位点。结果在大肠杆菌B121（DE3）宿主中，整合子对aadA2耐药性基因盒的整合频率为1．1×10^-3，主要的整合位点为attI。结论该系统可以用于整合子对基因盒捕获频率的测定。

Objective To establish a system for detecting integration frequency of antibiotic resistance integron. Methods We cloned integron and aadA2 gene cassette into different sites of plasmid pACYC 184, and the plasmid was transformed into E. coli BL21 （DE3） containing plasmid overexpressing integrase. The positive clone was cultured overnight and then was spread on LB agar plate with or without streptomycin respectively, and with appropriate amount of bacteria. Clones after cultured overnight were counted to detect the integration frequency. Meanwhile we used positive clones in LB agar plate containing streptomycin as templates to carry out PCR. The purified PCR products were sequenced to identify the integration sites. Results The integration frequency of integron capturing aadA2 gene cassette in BL21 （ DE3 ） host was 1.1 × 10^-3 mainly at attl site. Conclusion This system can be used to detect integration frequency.