江西农业大学学报2025，Vol.47Issue(6)：1632-1645,14.DOI:10.3724/aauj.2025141
β防御素复合制剂的制备及其对肠致病性大肠埃希氏菌的体内外协同抗菌作用
Preparation of porcine β-Defensin complex formulation and its synergistic antibacterial effects against enteropathogenic Escherichia coli in vitro and in vivo
摘要
Abstract
[Objective]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.[Method]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 Enteropathogenic Escherichia coli(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.[Result](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.[Conclusion]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.关键词
猪β防御素1/猪β防御素2/猪β防御素114/肠道致病性大肠埃希氏菌/巴马香猪Key words
porcine beta defensin 1/porcine beta defensin 2/porcine beta defensin 114/intestinal pathogenic Escherichia coli/Parmesan pigs分类
农业科技引用本文复制引用
简硕果,祁克宗,宋祥军,王振宇,邵颖,涂健,崔珂玮,李哲,李皓男,崔桐彤,高佳音,缪周,张敏,柯航..β防御素复合制剂的制备及其对肠致病性大肠埃希氏菌的体内外协同抗菌作用[J].江西农业大学学报,2025,47(6):1632-1645,14.基金项目
国家重点研发计划项目(2023YFD1702102-11)Project supported by the National Key Research and Development Program of China(2023YFD1702102-11) 合肥市自然科学基金项目(HZR2427)和合肥综合性国家科学中心大健康研究院食品营养健康联合研究中心基金项目(2024SJY04,2023SJY01)同时对本研究给予了资助,谨致谢意! （2023YFD1702102-11）
