抗菌药物浓度对整合子内耐药基因盒重组效率影响的研究  

Influence of antibiotic concentration on the integration frequency of resistance cassette in the integron

作　　者：杨泽华[1] 胡敏[1] 周双艳[1] 杜建明[1] 申彬 杜怡青 罗晓旭 梁转霞 Yang Zehua;Hu Min;Zhou Shuangyan;Du Jianmin;Shen Bin;Du Yiqing;Luo Xiaoxu;Liang Zhuanxia(Department of Laboratory Medicine,the First Hospital of Shanxi Medical University,Taiyuan 030001,China)

机构地区：[1]山西医科大学第一医院检验科,太原030001

出　　处：《中华检验医学杂志》2018年第7期531-535,共5页Chinese Journal of Laboratory Medicine

基　　金：山西省自然科学基金资助项目（2012011041-2）

摘　　要：目的研究在不同浓度链霉素下，aadA2基因盒在整合子attI位点的整合频率。方法将已知序列的1类整合子克隆到pACYC184质粒，该重组质粒命名为pACIDA；将该整合子上的整合酶基因克隆到pET28a质粒，该重组质粒命名为pETINT；这两重组质粒依次转化大肠杆菌BL21（DE3）。转化后的大肠杆菌在加不同浓度的链霉素的LB液体培养基中，37 ℃过夜培养。使用荧光定量PCR技术分别测定aadA2基因盒在attI位点发生整合作用的整合子的拷贝数和总整合子的拷贝数，前者除以后者即可获得整合频率。结果高表达整合酶的情况下，在加入的链霉素浓度分别为0、20、30、40、50 μg/ml时，aadA2基因盒在attI位点的整合频率依次为（1.97±0.24）×10－3、（3.23±1.77）×10－3、（3.27±0.67）×10－3、0.45±0.13、1.32±0.11。而在没有整合酶高表达的情况下背景频率低于（1.75±0.33）×10－7。结论高浓度的链霉素能够促进整合子中aadA2基因盒在整合子中attI位点的重组效率。（中华检验医学杂志，2018, 41：531－535）ObjectiveTo observe the integration frequency of aadA2 resistance cassette at attI site of the integron under different concentration of streptomycin. MethodsClass 1 integron with known gene sequence was cloned into plasmid pACYC184 to produce recombinant plasmid pACIDA, meanwhile the integrase gene was cloned into plasmid pET28a to construct recombinant plasmid pETINT. These two recombinant plasmids were consecutively transformed into E. coli BL（DE3）. These transformed bacteria was cultured in the LB medium at 37 ℃ overnight with addition of different concentration of streptomycin. The copy number of total integrons and the copy number of integrated aadA2 at attI site of integrons were determined by using real-time PCR. and the integration frequency is the result of the former divided by the latter. ResultsThe resulting frequencies were （1.97±0.24）×10-3, （3.23±1.77）×10-3, （3.27±0.67）×10-3, 0.45±0.13 and 1.32±0.11, with respective streptomycin concentrations of 0, 20, 30, 40 and 50 μg/ml. The background frequency of integration without integrase overexpression was less than （1.75±0.33）×10-7. ConclusionThese findings indicate that antibiotic concentration significantly increase recombination frequency of aadA2 resistance cassette at attI site of the integron, catalyzed by integron integrase.（Chin J Lab Med, 2018, 41： 531-535）

关 键 词：整合子类 链霉素 抗药性 微生物 质粒 实时聚合酶链反应 

分 类 号：R446.5[医药卫生—诊断学]