基于ACSSH文库技术研究饲用蜡样芽孢杆菌对肉鸡免疫作用的分子机制

项目名称： 基于ACSSH文库技术研究饲用蜡样芽孢杆菌对肉鸡免疫作用的分子机制

项目编号： No.31472130

项目类型： 面上项目

立项/批准年度： 2015

项目学科： 畜牧学与草地科学

项目作者： 潘康成

作者单位： 四川农业大学

项目金额： 85万元

中文摘要： 芽孢杆菌制剂得到了广泛应用，具有促长、防病治病的作用。大量研究表明，饲用芽孢杆菌制剂对动物的特异性和非特异性免疫具有较好的促进作用，但对于免疫作用的分子机理，尚未见研究报道。抑制性消减杂交(SSH)是用于研究差异表达基因的技术，具有灵敏、稳定的特点，但此技术不仅程序复杂，且得到富集的cDNA 片段因克隆而出现假阳性，还有库容虚增等缺陷。本项目在前期研究已明确蜡样芽孢杆菌菌株具有促进动物免疫功能的基础上，拟采用DGGE优化SSH 技术(ACSSH)，并与传统SSH 法所构建的文库进行比对，研究饲用蜡样芽孢杆菌对肉鸡免疫器官差异表达基因，筛选免疫调节响应基因，用Real-time RT-PCR和Western blot 方法检测免疫调节新基因（X）的mRNA和蛋白水平的表达情况，应用RNAi技术研究X基因表达沉默对淋巴器官细胞相关功能，从而阐明蜡样芽孢杆菌的免疫促进分子调控机理。

中文关键词： 蜡样芽孢杆菌；免疫功能；抑制性消减杂交；差异表达；分子机理

英文摘要： Useful Bacillus has been used as probiotics, for its role in facilitating the growth of livestock, preventing and treating diseases. A lot of research show that Bacillus preparation can promote animal in specialficity and non-specialficity immune. However, the molecular mechanism of immunization haven't been reported. Suppression subtractive hybridization (SSH) is a technology for the study of differential gene expression, its characteristics are sensitive and stable, but the deformities of SSH such as the complicated procedure and some false and cDNA capacity increased is a major issue for urgent solution. The project is based on our former rearch which we have confirmed Bacillus cereus PAS38 owned the ability to promote animal immunity, we intend to adopt the new type of SSH optimized by DGGE (ACSSH) to study the differential gene expression in immune organs of broiler which are fed with Bacillus cereus PAS38, the traditional SSH mathod as control, quickly building the differential expressed gene library and screening immune response gene. Real-time RT-PCR and western blot to detect the expression of the X gene of interest mRNA and protein levels. Used the RNAi in X gene silencing technology, the function of lymphoid organs cell is detected by Real-time RT-PCR and western blot. The study elucidates the molecular regulation mechanism of Bacillus cereus promoting immunization, and provide the new techolonogy to research of differential gene expression and reveals the molecular mechanism of immune response.

英文关键词： Bacillus cereus;Immune function;Suppression subtractive hybridization;Differential expression;Molecular mechanism