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摘要：鸡传染性法氏囊病（Infectious bursal disease；IBD）会对养禽业造成巨大损失，是发病于雏鸡中的一种重要传染病。枯草芽孢杆菌是一种益生菌，作为黏膜免疫的抗原递送载体具有很多优势。本研究拟通过构建重组枯草芽孢杆菌，口服免疫雏鸡，以期通过益生菌枯草芽孢杆菌作为抗原递送载体表达鸡法氏囊病毒（Infectious bursal disease virus；IBDV）的抗原片段VP2，口服免疫雏鸡后评价其攻毒保护的效果，探索一种简便、高效的提高雏鸡黏膜免疫力，控制法氏囊病发生的免疫方法；本研究首先扩增（克隆）IBDV的VP2蛋白以及GFP蛋白基因，其次将克隆的基因通过Infusion克隆的方法直接克隆到枯草芽孢杆菌表达载体pIN-cotG中，构建重组表达VP2和GFP蛋白的枯草芽孢杆菌表面展示载体，使之成功表达IBDV表面的VP2蛋白，最后通过雏鸡免疫动物试验证实重组枯草芽孢杆菌的作用效果。本研究结果表明，应用枯草芽孢杆菌表达IBDV的VP2蛋白，有望成为一种简便、高效的控制法氏囊病发生的免疫方法；为新型口服黏膜免疫疫苗的开发奠定坚实基础。38756
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毕业论文关键词：法氏囊病毒；重组枯草芽孢杆菌；基因表达；口服黏膜免疫；雏鸡
Construction of Recombinant Bacillus Subtilis Which can Express Chicken VP2 Protein of Infectious Bursal Disease Virus and Study Its Immune Efficacy
Abstract：Infectious bursal disease (IBD), an important infectious disease in chicks, can cause great loss to the chicken industry. As a probiotic,Bacillus subtilis has many advantages in antigen delivery vector for mucosal immunity. In this study, we constructed the recombinant Bacillus subtilis and oral immunized chicks, in order to express the antigen fragment VP2 of Infectious bursal disease virus (IBDV) by probiotics Bacillus subtilis as antigen delivery vector, and the effect of the challenge was evaluated after oral administration of chicks, to explore a simple and efficient way to improve the immunity of chicks mucosa, control the occurrence of bursal disease immune method. In this study, the gene of VP2 protein and GFP protein of IBDV were amplified and cloned into the Bacillus subtilis expression vector pIN-cotG by Infusion cloning method,to construct recombinant expression vector expressing VP2 protein on Bacillus subtilis surface successfully.Finally,the effect of recombinant Bacillus subtilis was confirmed by immunized animal test. The results of this study show that the application of recombinant Bacillus subtilis which can express chicken VP2 protein of infectious bursal disease virus is expected to be a simple and efficient method to control the occurrence of bursal disease, and lay a solid foundation for the development of new oral mucosal immune vaccine.
Key words: Infectious bursal disease virus；Recombinant Bacillus subtilis；gene expression；Oral mucosal immunity；chicks
目 录
摘要3
关键词3
Abstract3
Key words3
引言4
1试验材料6
1.1实验材料 6
1.2实验试剂 6
1.3实验仪器 6
2试验方法6
2.1 整合型表达载体pIN-GFP-IBDV的构建6
2.1.1 IBDV病毒VP2蛋白基因的克隆6
2.1.2 GFP 绿色荧光蛋白基因的克隆6
2.1.3 连接片段与载体8
2.1.4 转化8
2.1.5菌落PCR8
2.1.6 提取去内毒素的质粒9
2.2化学转化感受态细胞构建重组整合型表达枯草芽孢杆菌10
2.3电击转化感受态细胞构建重组整合型表达枯草芽孢杆菌11
2.4雏鸡口服免疫重组枯草芽孢杆菌的研究12
3结果与分析 14
3.1枯草芽孢杆菌整合型表达载体pIN-GFP-IBDV的构建14
3.2化学转化构建重组整合型枯草芽孢杆菌16
3.3电击转化构建重组整合型枯草芽孢杆菌17
3.4 口服免疫雏鸡的结果测定17
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