| Type Ⅵ secretion systems(T6SS)are bilayer transmembrane secretion systems that participate in different biological processes in bacteria and play an important role in interactions with competing bacteria,hosts and other environmental factors.The T6SS effector protein,Tae4,acts as a lytic enzyme mainly by breaking the peptidoglycan bonds in the bacterial cell wall,thus playing a role in killing bacteria.In recent years,the long-term use of antibiotics has accelerated the generation of pathogenic resistance in livestock and poultry,bringing certain hidden dangers and potential threats to human and animal food safety,so it is important to strengthen the study of Tae4 protein for subsequent antibiotic reduction and replacement.In this experiment,the recombinant plasmid pET-28a-Tae4 was first introduced into BL21(DE3)pLysS receptor cells,and the recombinant protein Tae4 was obtained by induced expression,followed by 3D structure modeling of Tae4 protein by PyMol and other software,and the storage performance and safety of the recombinant protein were tested by stability test and injection safety test.Finally,we evaluated the antibacterial effect of Tae4 on Staphylococcus aureus and Listeria monocytogenes,its MIC and the effect of combination with antibiotics.This study provides a new idea for antibiotic reduction substitution,and the main research contents and results are summarized as follows.1.Purification of Tae4 protein by prokaryotic expression and determination of stability and safetyThe recombinant plasmid pET-28a-Tae4 was introduced into BL21 receptor cells by chemo-transformation,and expression was induced in positive strains after PCR detection,and the expression of the target protein was examined by SDS polyacrylamide gel electrophoresis.The recombinant protein was purified using magnetic beads,and a single target protein with a molecular weight of about 21.5 kDa could be eluted using imidazole at a final concentration of 250 mmol/L.A 3D model of the Tae4 protein was successfully constructed using PyMol and other software,and subsequent stability tests showed that no significant degradation of the recombinant protein was observed within 5 d of treatment at4°C and 37°C.In order to evaluate the safety of Tae4,injection safety tests were performed on mice,and the results showed that the animals in each test group had good appetite and mental status,no mortality,no inflammation,necrosis and allergic reactions at the skin injection site,no abnormal reactions such as bleeding spots and fluid accumulation in all organs,and no obvious pathological changes in all tissues,which initially proved that Tae4 had no adverse effects on the mouse organism.2.Observation on the antibacterial effect of recombinant protein Tae4 on Staphylococcus aureusIn order to investigate the inhibitory effect of recombinant protein Tae4 on Staphylococcu saureus(S.aureus),it was firstly tested by agar well diffusion assay and the results showed that the recombinant protein had an inhibitory circle on three standard strains and three isolates.The results showed that the recombinant protein Tae4 had an MIC of 250 μg/mL against S.aureus.6 drug-resistant strains(S.aureus(ATCC 6538)、S.aureus 63、S.aureus67、S.aureus 76、S.aureus 92、S.aureus 93)were selected and evaluated the antibiotic substitution effect of Tae4 by combining recombinant protein with antibiotics.The results showed that the inhibition of S.aureus was enhanced to some extent when the combination was used,and the inhibition effect was enhanced to different degrees when the low concentration of protein was combined with low concentration of penicillin(recombinant protein concentration of 125 μg/mL and PG concentration of 200 U/mL),and the use of antibiotics was reduced.3.Observation of the inhibition effect of recombinant protein Tae4 on Staphylococcus aureusIn order to investigate the inhibitory effect of recombinant protein Tae4 on Listeria monocytogenes(L.monocytogenes),it was measured by agar diffusion assay,and the results showed that the recombinant protein had a certain inhibitory effect on all four isolates,and then the MIC of the recombinant protein was determined using the spot plate method and micro broth dilution method.The MIC of recombinant protein was determined by spot plate and micro broth dilution method,and the MIC of recombinant protein against Listeria monocytogenes was 125 μg/mL.Finally,the recombinant protein and PG concentrations were adjusted to 125 μg/mL and 200 U/mL,respectively,by recombinant protein and antibiotic combination test,and the results showed that the inhibition effect was improved.In summary,the T6SS effector protein Tae4 was successfully expressed and purified,and the 3D structure of the protein was constructed,and the protein was initially shown to have no adverse effects on mice and to be safe and non-toxic.It was subsequently determined that Tae4 had an inhibitory effect on Staphylococcus aureus and reduced the amount of antibiotics when combined with penicillin,and Tae4 also had an inhibitory effect on Listeria monocytogenes and reduced the amount of antibiotics when combined with penicillin,confirming its feasibility in antibiotic reduction.The results provide a theoretical basis and data support for the further application of Tae4 in follow-up. |
