| Due to the persistence and prophylactic or excessive use of antibiotics in animal husbandry and food production,bacteria continue to mutate in order to overcome the therapeutic effects of antibiotics,thereby causing bacterial resistance to form multi-drug resistant bacteria.The infection rate of various diseases caused by serious bacterial resistance is continuing to rise,so bacterial resistance has gradually become a serious threat to the health of biological groups around the world.In recent years,with the call for anti-aquaculture being recognized and responded by more and more people in the industry,as an effective alternative to antibiotics,the application of phage has gradually received attention.At the same time,the theory and technology of artificially modified phage are constantly being optimized and some progress has been made.The researchers have solved the problems of strong phage specificity,short half-life and release of endotoxin through genetic engineering technology,so that genetic engineering The transformation of phage has higher safety and application potential in clinical application.In this study,a strain of wild-type multi-drug resistant Escherichia coli was successfully isolated and purified by mitomycin-induced and double-layer plate plaque assay,and a ? mild phage was successfully isolated and named as PEA3 b.Study the characteristics and further perform genome-wide sequence determination and analysis.Based on this research,combined with BAC+Red ET reverse genetics operating system,? mild phage PEA3 b was ligated with CRISPR/Cas9 system which can specifically recognize NDM-1 drug resistance gene,and the construction of engineering mild phage was successfully completed.The obtained engineering phage was proliferated from C100 engineering bacteria and then acted on wild-type resistant host bacteria,and combined with fluorescence quantitative and wild-type resistant host bacterial resistance phenotype to evaluate the resistance of engineering mild phage to resistant bacteria.The drug gene performs directed shearing and elimination effects.The results showed that:(1)? mild phage PEA3 b could form a translucent,uniform circular plaque on the double-layer plate after two purifications;the phage had a size of 50 nm by projection electron microscopy using phosphotungstic acid negative staining method.The non-shrinking tail of the tail and the end has a small tail;its optimal multiplicity of infection is 10-1;its host spectrum is narrow,and it can only infect and lyse 3 strains of Escherichia coli isolated from the same batch(3/ 20).(2)High-throughput sequencing showed that the full-length genome of the phage was 52489 bp,and the average GC content was 49.40%.The search for the NDM-1 gene in the whole gene sequence revealed that the phage gene did not carry the NMD-1 resistance gene.(3)In this experiment,the p BAC11-15A-ccd B-HA plasmid with the phage at both ends as the homology arm was successfully constructed,and the plasmid and the C RISPR/cas9 plasmid and the phage whole gene were recombined in the Gbdir line,and the specific primers were used for PCR.After gel e lectrophoresis verification,the ligation of the engineered phage genome was completed,after which the genome was transformed into the competent C100 strain to obtain an assembled engineering phage.Combined with fluorescence quantitative and wild-type resistant host bacteria resistance phenotypic verification results,engineering phage can reduce the content of NDM-1 resistance gene in drug-resistant bacteria to some extent,and restore the sensitivity of some carbapenem antibiotics to bacteria..In summary,the engineering phage can eliminate the basis of the generation and spread of bacterial resistance to some extent.And lay a theoretical foundation and technical support for the engineering phage structure to eliminate other drug resistance genes in the future. |
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