-
摘要：传染性法氏囊病（Infectious bursal disease, IBD）是危害世界养禽业的主要传染病之一。是由传染性法氏囊病病毒(Infectious bursal disease virus, IBDV)引起的一种急性、高度接触性传染病，传染性法氏囊病病毒(IBDV)属双股双节段RNA病毒，该病毒主要扩增部位在雏鸡的中枢免疫器官―法氏囊，因此会造成其免疫抑制，给养鸡业造成巨大的经济损失。本次试验采用载入VP3片段的pET28a重组质粒，将其转化到BL21(DE3)pLySS表达菌内，进行下一步纯化。此外本实验从时间以及IPTG浓度两个方面进行了蛋白诱导表达的条件进行摸索。同时，本实验还纯化了VP3蛋白以及其突变体：His-VP3(patch)，His-VP3(KR),His-VP3△16。经过初步表达后发现VP3呈上清融合表达和包涵体表达两种形式，因此在后面的实验中选择非变性表达来表达蛋白。经过对诱导条件的探索筛选得到最佳诱导条件，并在此条件下进一步大量表达得到纯度较高的VP3蛋白及VP3蛋白的突变体。28596
- 上一篇：过表达Circ-AKT2细胞系构建及Circ-AKT2功能初步研究
- 下一篇：产毒素性多杀性巴氏杆菌感染抗体间接ELISA方法的建立与优化
毕业论文关键词：传染性法氏囊病毒（IBDV）；VP3蛋白；蛋白纯化
Expression and purification of infectious bursal disease virus (IBDV) structural protein VP3 and its mutant
Abstract：Infectious bursal disease (IBD) is one of the major infectious diseases that endanger the world's poultry industry. It is an infectious, highly contagious infectious disease caused by infectious bursal disease virus (IBDV), which predominantly amplify in the central immune organs of the chicks —— bursa of Fabricius, resulting in immunosuppression.And caused huge economic losses to chicken industry. Infectious bursal disease virus (IBDV) is a double-stranded double-segment RNA virus that can damage chicks' bursal lymphocytes and cause severe immunosuppression and induce multiple diseases or immune to multiple vaccines.In this experiment pET28a recombinant plasmids which loaded with VP3 fragments were transformed into BL21 (DE3) pLySS expressing bacteria, and VP3 protein was purified by IPTG. In order to explore the optimal conditions of VP3 protein induction and expression VP3 protein was purified under different IPTG concentration gradient and different time gradient , and then the gradient was compared with each other. The optimal concentration gradient and time gradient were selected to explore the optimal VP3 purification conditions. At the same time, the VP3 protein and its three mutants were purified: His-VP3 (patch), His-VP3 (KR) and His-VP3 △ 16.After the initial expression, VP3 was found to be cleaved and expressed in two forms, so non-denatured expression was selected in later experiments. The optimal induction conditions were obtained by screening the induction conditions and the VP3 protein and VP3 protein mutations were further expressed under these conditions
Key words：IBDV ; VP3 protein ; Protein purification
目 录
摘要1
关键词1
Abstract1
Key words1
引言2
1材料与方法2
1.1材料 2
1.1.1重组质粒及重组表达菌 2
1.1.2表达菌株2
1.1.3 PCR菌液鉴定所用试剂及缓冲液2
1.1.4聚丙烯酰胺凝胶电泳（SDS-PAGE）试剂3
1.1.5蛋白质纯化试剂4
1.1.6 Western-blot试剂及抗体 .4
1.1.7主要实验材料生产厂家汇总4
1.2方法 7
1.2.1重组质粒转化7
1.2.2PCR鉴定重组质粒7
1.2.3蛋白的诱导表达 8
1.2.4蛋白纯化 8
1.2.5蛋白纯化鉴定9
2结果与分析11
2.1 pET28a-VP3重组质粒转化BL21（DE3）plySS感受态细胞及阳性单菌落鉴定11
2.2 His-VP3初步表达12
2.3 His-VP3包涵体表达分析13
2.4 His-VP3诱导表达条之IPTG浓度摸索14
2.5 His-VP3诱导表达条之时间梯度摸索.16
2.6 His-VP3大量诱导纯化.16
2.7 His-VP3(patch)，His-VP3(KR),His-VP3△16纯化实验 17