唾液乳酸杆菌B1对仔猪肠道A型产气荚膜梭菌增殖及毒力基因表达调控的研究

Clostridium perfringenstypeA (CpA) widely exists in natural environment and intestinal tract of human beings and animals. CpA leads to many diseases, especially gastroentiritis. It is generally recognized that exotoxins produced by CpA are critical pathogenic factors of the disease. Of which, the β2 toxin, one of important exotoxins, is closely associated with the necrotizing enterocolitis (NEC)of neonatal piglets. However, the CpA harboring cpb2 gene could also be isolated from healthy piglets, andthe pathogenic mechanisms of CpA are roughly unknown yet. The occurring of NEC caused by cpb2-positive CpA perhaps associates with the micro-ecological environment of intestinal tract of piglets. The author observed that Lactobacillus could prevent the NEC of piglets in pilotstudy. In this study, we attempt to use a field isolated CpA-cpb2+strain as a backbone to construct a chimeric Luciferase-CpA-cpb2+which will be employed for a mouse model establishement by utilizing a Bioluminescence imaging (BLI) technology, and thus the proliferation of CpA-cpb2+ modulated by lactobacillus in intestinal tract of mouse will be investigated. Meanwhile，the expression of β2 gene and toxin are measured by real-time PCR and other conventional tests. In order to elucidatethe constitution and diversitiesof intestinal microbe-flora and hunt new probiotics against NEC, 16SrDNA-based metagenomics and other state-of the-art technologies will be used to analyse the difference in composition of gut flora between diseased and healthy piglets carrying CpA-cpb2+. The expected findings achieved from this study will provide insightinto the effects of Lactobacillus salivarius B1 on the proliferation and expression of virulence genes of CpA-cpb2+, the theoretical foundation for ecological prevention of NEC of piglets.

A型产气荚膜梭菌（CpA）广泛存在于自然环境和人畜肠道，有时能引起人类和动物疾病。外毒素是其致病的重要因子，其中β2毒素与新生仔猪坏死性肠炎（NEC）密切相关，然而，健康仔猪肠道也能分离到β2毒素阳性的CpA（CpA-cpb2+）。CpA致病机制目前还不明了。笔者在前期研究中已发现乳酸杆菌能预防NEC的发生，机理尚不明确。本项目拟从临床分离筛选CpA-cpb2+菌株，采用仔猪回肠结扎法和建立活体感染模型，通过生物发光图像（BLI）技术实时监测活体内乳酸杆菌对荧光素酶嵌合的CpA-cpb2+增殖影响；同时结合qRT-PCR和Western-blot技术检测β2毒素的表达，以揭示乳酸杆菌调控CpA机理；同时，采用细菌16SrDNA宏基因组高通量测序技术，研究携带CpA-cpb2+但临床上表现为NEC或健康状态的仔猪肠道菌群组成差异，解析肠道菌群对仔猪NEC的影响，为生态防御仔猪NEC提供理论基础。

为研究A型产气荚膜梭菌（CpA）及其产生的β２毒素对７日龄内仔猪腹泻的影响，本项目采用细菌16SrDNA V3-V4区高通量测序技术，对江西地区健康(H)、腹泻(D)仔猪肠道菌群分析，结果发现梭杆菌、拟杆菌、硬壁菌和变形杆菌门占总细菌的99%以上，梭菌分为两大类：腹泻组Clostridium Sensu Strito_2丰度偏高，在健康组丰度低，而Clostridium Sensu Stricto_1刚好相反；未分类的乳酸菌在种水平上两组有显著差异。Cp分离率统计结果为腹泻样品89.6 % (138/154)，健康样品为90.1 % (91/101)， 其中CpA 占分离菌株的89.5 % (205/229)，CpA 菌株的β2毒素携带率为99.5% (204/205)，表明江西地区腹泻与健康仔猪肠道CpA 携带率及CpA (β2+)比例均较高。依据小鼠毒性试验和兔回肠结扎结果选取来源于腹泻仔猪肠道的JXJA17（Genbank ID: CP028149.1）作为代表菌株，该菌株第三代全基因组测序结果：该菌株基因组全长1,302,938,004bp个碱基，共编码3396个基因，3092个CDSs,30个rRNA基因，95个tRNA基因和0个非编码RNA。菌株包含9个质粒，大小分别为3550 bp-62027 bp，β2毒素基因位于 58796 bp质粒上。通过酵母双杂交技术对IPEC-1 cDNA文库的筛选，得到了4个潜在与β2毒素相互作用蛋白。其中，LGALS1是一种可存在于细胞膜外侧的半乳糖凝集素。推测β2毒素主要通过与LGALS1蛋白C端结合侵入肠上皮细胞从而引起肠炎。最后以代表株为指示菌，从健康仔猪肠道筛选益生乳酸杆菌，经过体内、体外抑菌作用等生物学性能检测，筛选得到的益生乳酸杆菌研制成微生态制剂用于预防CpA所引起的仔猪肠炎，取得了良好效果。研究经费使用合理，公开发表论文5篇，申请和授权专利4项，会议论文7篇,培养了4位硕士研究生。