Isolation And Identification Of Staphylococcus Aureus Phage Sau-p573 And Recombinant Expression And Functional Exploration Of LysZb48 Lysin

Staphylococcus aureus(S.aureus)is a Gram-positive coccus of the genus Staphylococcus,which has a wide range of transmission in mammals and can cause a range of diseases such as mastitis in dairy cows and sepsis.With the misuse of antibiotics,more and more drug-resistant bacteria have started to appear,and as a result,clinical treatment of mastitis in dairy cows has become increasingly difficult,so the development of new bactericidal biological agents has become extremely important.Bacteriophage are viruses that use bacteria as hosts and can bind to the cell wall of bacteria and then inject genetic material into the host and multiply inside the host bacteria,eventually killing the host bacteria and releasing the Offspring bacteriophage.Lysin is a class of enzymes expressed in the late stage of phage infection,which has the advantages of host specificity,trace amount efficiency,difficult to make the microflora resistant,relatively safe,etc.,and can be expressed in large quantities through prokaryotic or eukaryotic and mature technology.In this study,a strain of Staphylococcus aureus phage Sau-p573 was successfully isolated and extracted from a mixed sewage sample from a cattle farm using an enrichment method,and its head was observed by electron microscopy as a typical icosahedral head of a phage with a long non-contractible tail,which is a phage of the Siphoviridae.Its most efficient valence of 7×10¹⁰ PFU/m L was measured by the double-layer plate method,and the best multiplicity of infection was 10.The incubation period of Sau-p573 was known from the one-step growth curve to be 90min,and it reached the plateau at 3 h.Its lytic spectrum was determined using laboratory-preserved S.aureus and was found to be lytic for 7 strains of Staphylococcus aureus.The whole genome sequencing of Sau-p573 was performed,and the analysis of NR databases showed that ORF48 was tentatively identified as a lysin protein.In order to make the lysin Soluble expression,the MBP tag was inserted at the N-terminal end of the lysin sequence,and the expression vector p MBP2-Lys ZB48was constructed,and The product obtained after IPTG induced expression.SDS-PAGE analysis of the obtained product revealed that the lyase was soluble.The lysis effect of Lys ZB48 in the concentration range of 25-200?g/m L for 1h was measured by the turbidity drop test after the bacteria were lysed,and the lysis effect increased with the increase of concentration.Lys ZB48 at 200?g/m L at a host concentration of 3×10~8 decreased S.aureus 573 by 2.1 lg CFU/m L at 1 h as measured by plate counting.The lytic spectrum of Lys ZB48 was determined using 39 S.aureus and 7 E.coli strains kept in the laboratory,and the lysis effect of Lys ZB48 was found on 26 S.aureus and 4 E.coli strains.The effect of different conditions on the lysis of Lys ZB48 was tested,and the experimental results showed that Lys ZB48 was active from 4?-42?,p H 6-9,and the best lysis effect was in the range of 37?-42?,p H 8-9.The incubation of Lys ZB48 at37?for 3 h showed that almost all the lysis activity disappeared.The effect of bovine serum and different concentrations of milk powder also affected the lysis effect of Lys ZB48.Compared with the PBS control group in which Lys ZB48 decreased the host bacteria concentration by 2.2 lg CFU/m L,Lys ZB48 in bovine serum decreased the host bacteria concentration by 1.74 lg CFU/m L,and Lys ZB48 containing 4%milk powder decreased the host bacteria concentration by 1.7 lg CFU/m L,and the lysis effect gradually decreased with the increase of milk powder solution concentration,which proved that some components in serum and milk related to inhibit the lysis activity of lysin.In addition,the experimental results showed that Lys ZB48 had a scavenging effect on biofilm,so Lys ZB48 has greater potential for application.