表达鸡法氏囊病毒VP2蛋白重组枯草芽孢杆菌的构建及其免疫效力的研究
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