| Objective:By measuring the frequency of integron capture resistance gene cassette in Escherichia coli BL21（DE3）under the influence of silver ion,the effect of silver ion on the integration frequency of type I integron in Escherichia coli BL21（DE3）host was studied,so as to provide ideas and ideas for solving the current problem of antibiotic resistance and the emergence of new drug-resistant strains.Methods:The integron and aminoglycoside adenosine transferase（aadA2）gene cassette were inserted into different positions of pACYC184 vector.The recombinant plasmid was named pACINAD.The recombinant plasmid pUCINT,with high expression of integrase was constructed by using pUC19 as vector.Two recombinant plasmids pUCINT and pACINAD were successively transformed into E.coli BL21（DE3）and named as HS2,.Only E.coli BL21（DE3）transformed by recombinant plasmid pACINAD was named HS1.80 HS2 strains were divided into 4 groups and cultured in liquid medium without silver ion,adding 0.3μg/ml silver ion,adding 0.6μg/ml silver ion and adding 0.8μg/ml silver ion LB liquid medium at 37℃for 24 hours,and another 20 HS1 strains were cultured in ordinary LB liquid medium at 37℃for 24 hours.Silver ions used in the experiment are provided by silver nitrate.After the strains were cultured,the integron copies and the total integron copies were determined by real-time fluorescence quantitative polymerase chain reaction（qPCR）,and the ratio of them was the integration frequency.At the same time,the changes of integration frequency were analyzed by three independent phenotypic screening methods and the protein expression of the strain was analyzed by mass spectrometry.Results:1.The integration frequency of HS1,HS2 without silver ion,0.3μg/ml silver ion group,0.6μg/ml silver ion groupand 0.8μg/ml silver ion groupwere 0.51×10-8(6.27×10-8),1.79×10-5(1.69×10-5),2.07×10-5(1.18×10-5),2.25×10-6(3.07×10-6),1.69×10-6(2.86×10-6)respectively.Statistical analysis of five groups of experimental data showed that there was no significant difference between HS2 strain without silver ion groupand0.3μg/ml silver ion group（P>0.05）,and between 0.6μg/ml silver ion groupand 0.8μg/ml silver ion group（P>0.05）.There were significant differences among the other groups（P<0.01）.2.The results of three times independent phenotypic screening showed that there were more colonies in HS2 without silver ion groupand 0.3μg/ml silver ion groupon LB plate containing streptomycin,while there were few colonies in HS1 group,0.6μg/ml silver ion groupand 0.8μg/ml silver ion group.3.Mass spectrometry analysis showed that there were differences in protein expression of the strains treated with silver ions.Conclusion:1.A certain concentration of silver ion can inhibit the frequency of drug resistance gene cassette captured by bacterial integron.2.Foreign substances in the environment can affect the integration frequency of integron.The new role of silver ions in reducing the integration frequency provides a new idea and direction for the research of bacterial antibiotic resistance.If we can deeply study the effect of silver ion on integron to reduce antibiotic resistance and assist clinical treatment of bacterial infection,it is of great significance to clinical work.3.Increasing the expression level of integrase can significantly increase the frequency of integration. |
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