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产肠毒素大肠杆菌（Enterotoxigenic Escherichia coli，ETEC）是一种重要的人-兽共患病原菌之一。仔猪感染后临床上多以排黄白色水样粪便及快速死亡为主要特征，常被称之为仔猪黄痢和仔猪白痢。仔猪黄白痢全年均可发生，并随着季节的更替表现出不同的发病率，秋冬季节感染尤为严重，给畜牧养殖行业带来了极大经济损失。传统的抗生素治疗方法加剧了细菌耐药性，同时还带来了药物残留等一系列问题。本研究以乳酸菌为递呈载体，扩增了鼠李糖乳杆菌（Lactobacillus rhamnosus GG）中编码亚油酸异构酶的mcra基因，将其与乳酸杆菌表达载体pPG612连接，构建工程菌益生菌LC-pPG-mcra。此外，以Escherichia coli Nissle 1917(EcN)为递呈载体，以双歧杆菌黏附性脂蛋白BOPA、K88菌毛蛋白FaeG、FaeC作为递呈抗原，构建携带双质粒系统的工程益生菌EcN-pPG-BOPA/pBbB8K-FaeGFaeC（EcN-BFF）。本研究以Caco-2细胞为细胞实验模型，检测野生株（Lactobacillus .cacei ATCC 393）、工程益生菌LC-pPG-mcra、EcN-BFF对肠上皮细胞的黏附与侵袭能力探究其特性，以便检测野生株及工程菌株对Caco-2细胞的黏附、侵袭能力，同时又将致病性大肠杆菌ETEC K88与两株工程益生菌共培养，检测工程益生菌竞争性抑制K88黏附侵袭细胞的能力。研究结果显示，两株工程益生菌通过基因、Western blot及间接免疫荧光试验证实成功构建，构建的两株工程益生菌均对Caco-2细胞均有较高的黏附与侵袭能力，能通过竞争性抑制K88菌株对Caco-2细胞，降低K88菌株对Caco-2细胞的侵袭感染。
Abstract：Enterotoxigenic Escherichia coli (ETEC) is one of the significant zoonotic pathogens. In piglets， clinical signs after ETEC infection often include diarrhea with yellowish-white watery stools and rapid death， commonly referred to as yellow scour and white scour in piglets. This condition is prevalent year-round， with a seasonal variation in incidence， particularly during autumn and winter， when the infection rate significantly rises， imposing a substantial economic burden on the livestock farming sector. Traditional antibiotic therapies have contributed to the exacerbation of bacterial resistance， concurrently posing challenges related to drug and other issues. This study employed Lactobacillus rhamnosus GG as a delivery vector to amplify and clone the mcra gene encoding the omega-3 isomerase， which was then integrated into the lactobacillus expression vector pPG612， resulting in the engineered probiotic strain LC-pPG-mcra. Additionally， using Escherichia coli Nissle 1917 as a carrier， and with the adhesion protein BOPA from Bifidobacterium， and the FaeG and FaeC flagellar proteins from Lactobacillus， the study constructs a dual-plasmid system in the engineered probiotic EcN-pPG-BOPA/pBbB8K-FaeGFaeC (EcN-BFF). This study employed the Caco-2 cell model to investigate the adhesion and invasive capabilities of the wild strain Lactobacillus cacei ATCC 393， engineered probiotic LC-pPG-mcra， and EcN-BFF. The objective was to assess the adhesion and invasive potential of these strains towards Caco-2 cells. Furthermore， a co-culture experiment was conducted with the pathogenic ETEC K88 strain and the two engineered probiotics to evaluate their competitive inhibition of K88's adherence and invasion. The study findings confirmed the successful construction of both engineered probiotics through genetic and biochemical validation， including Western blot and indirect immunofluorescence assays. Both engineered strains demonstrated enhanced adhesion and invasive capabilities towards Caco-2 cells， effectively competing with the K88 strain to inhibit its adherence and subsequent invasion of Caco-2 cells， thereby reducing the infection caused by K88.
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中图分类号:Q78;S852.61
引用信息:
[1]李惠,张婷婷,邵鑫,等.表达共轭亚油酸mcra基因与菌毛亚基工程益生菌的构建及特性研究[J].经济动物学报().
2024-10-22
2024-10-22
2024-10-22