The Research Of Excision And Integration Mechamism About Antibiotic Resistance Gene Cassettes For Functional Class 2 Integron In Bacteria

Objective:To verify and compare the activity of all natural functional class 2 integrase found so far,and to determine the key amino acid sites of class 2 integrase to catalyze gene cassette excision and integration.To purify class 2 integrase protein by affinity chromatography and provides the basis for further experiments.Finally,to provide clues for clarifying the response and regulatory mechanism of the integron captures resistance gene cassette.Methods:Taking class 2 integrons as the research object,the data about class 2 integrons are screened through the online database INTEGRALL(http://integrall.bio.ua.pt).Natural functional class 2 integron genes which found in INTEGRALL database were obtained by site-directed mutagenesis to construct plasmids capable of high expression of class 2 integrase protein under IPTG induction.Recombinant plasmid pACIa was constructed which containing gene cassette sat2 and attI2-dfrA14 fragments.Quantitative real-time PCR system was established to detect the natural functional class 2 integrase mediated gene cassette sat2 excision and integration efficiency.Quantitative real-time PCR was used to detect and compare the recombination activity of natural functional class 2 integrons which found in INTEGRALL database.Using the Proteus 47437 genome as a template,a class 2 integrase mutation library was created by random mutated the functional class 2 integrase gene.Quantitative real-time PCR was used to detect changes in mutated class 2 integrase mediated gene cassette excision and integration efficiency,and determines the key sites for class 2 integrase to catalyze gene cassette excision and integration Finally,by constructed,expressed and purified the maltose and functional class 2 integrase fusion protein,to provide the experimental basis for further research.Results:A total of 236 pieces of data on class 2 integrons were screened from the online database INTEGRALL.Through sorting and analysis,a total of 5 different natural functional class 2 integrons were found.Successfully constructed plasmids pINTI2P(IntI2A176_V256),pINTI2E(IntI2),pINTI2F(IntI2A176),pINTI2V(IntI2S175_A176)and pINTI2W(IntI2C172_T174_A176)which can provide expression of 5 natural functional class 2 integrase proteins,and plasmid pACIa which provide the gene cassette sat2 and the integration site attI2.Quantitative real-time PCR system was successfully constructed to detect the class 2 integrase mediated gene cassette sat2 excision and integration efficiency.The recombinant activities of 5 natural functional class 2 integrases were detected and compared by quantitative real-time PCR.The results showed that there was no significant difference in the activity.The highest activity IntI2A176 was about twice as high as the lowest one IntI2s175.Using the Proteus 47437 genome as a template,a library of 2000 clones was created by random mutated the functional class 2 integrase gene.Quantitative real-time PCR was used to detect changes in mutated class 2 integrase mediated gene cassette excision and integration efficiency,results showed that changes in T23N,F34S,L73I,V111E,L129P,L134S,R135H,I154N,F272L,T274R,R284S,G291W and Y301C sites can cause loss of class 2 integrase activity.Recombinant plasmids pMAL-IntI2,pMAL-IntI2T274R,pMAL-IntI2T297P,pMAL-IntI2V144D,pMAL-IntI2Y208F were constructed by gene cloning,and proteins MBP-IntI2,MBP-IntI2T274R,MBP-IntI2T297P,MBP-IntI2Y208F were successfully purified.Conclusions:IntI2 encoding amino acid sequence polymorphisms are limited to amino acids 172,174,175,176,and 256,suggesting that functional class 2 integrons have lower genetic polymorphisms.By verifying,identified T23,F34,L73,V111,L129,L134,R135,I154,F272,T274,R284,G291 and Y301 are the key sites for class 2 integrase to catalyze the excision and integration of gene cassettes.The successfully purified functional class 2 integrase protein provides the basis for further research.Finally,it will provide a theoretical basis for clarifying the catalyzed mechanism of IntI2.Figure[20]table[5]reference[45]...