Preparation of porcine β-Defensin complex formulation and its synergistic antibacterial effects against enteropathogenic Escherichia coliin vitro and in vivo

<bold>Objective</bold> Defensins，as important members of antimicrobial peptides，are regarded as ideal alternatives to antibiotics.The purpose of this study is to investigate whether there is a synergistic antibacterial effect between porcine beta defensin 1（PBD1），porcine beta defensin 2（PBD2）and porcine beta defensin 114（PBD114），and an attempt was made to prepare a defensin compound preparation to achieve an antibacterial efficacy superior to that of a single defensin.By expressing three porcine β-defensins in vitro，we explored the optimal antibacterial ratio and the most susceptible pathogen，and evaluated the differences in antimicrobial efficacy between the composite preparation and single defensins through in vitro and in vivo experiments. <bold>Method</bold> The genetic sequence of PBD1，PBD2，PBD114 was obtained according to the GenBank database，and the codon was cloned to the pET-32a expression vector after optimization，and then transformed into BL21（DE3）PLysS competent cells for protein expression.The expressed proteins were identified by SDS-PAGE and Western Blot technology，and the target proteins were purified by magnetic bead method；by detecting the inhibitory effect of 3 defensins and their 7 combinations on 5 pathogens，the optimal inhibitory ratio and the pathogen with the best inhibitory effect were determined.The infection model of Bama pigs was established by intraperitoneal injection of <italic>Enteropathogenic Escherichia coli</italic>（EPEC）bacterial suspension.After intraperitoneal injection of the composite preparation for anti-pathogen infection intervention，clinical symptom assessment，histopathological examination，and determination and analysis of hematological and physical-chemical indicators，bacteriostatic titer，immune organ index，and intestinal bacterial load were performed on each test group respectively. <bold>Result</bold> （1）The pET-32a-PBD1，pET-32a-PBD2，and pET-32a-PBD114 expression vectors were successfully constructed.The induced proteins exhibited molecular weights of approximately 22.2 ku，22.1 ku，and 26.0 ku，respectively，and were primarily expressed in the supernatant.（2）The optimal ratio of the composite preparation was determined to be 1∶1∶1，which demonstrated significantly superior inhibitory effects against EPEC compared to single defensins，with a clear synergistic effect.In the EPEC infection model using Bama miniature pigs，piglets exhibited significant pathological changes post-infection，confirming the successful establishment of the model.（3）The clinical diagnosis results showed that，compared with the model control group，the piglets in the positive control group and the treatment group exhibited reduced diarrhea，normalized blood routine indicators，fewer intestinal hemorrhagic lesions，alleviated liver injury，and decreased bacterial load in the small intestine.Specifically，the single defensin showed better performance in terms of intestinal bacterial load and immune organ index，whereas the compound preparation demonstrated superior efficacy in protecting small intestinal villi and regulating blood routine indicators. <bold>Conclusion</bold> The porcine β-defensin composite preparation at a 1∶1∶1 ratio demonstrates significant synergistic inhibitory effects against EPEC.This formulation effectively reduces pathogen load in piglets and mitigated tissue damage，providing valuable experimental data for developing clinical therapeutics against EPEC infections.