摘要：杜兰病毒（Tulane Virus，TV）与人源诺如病毒（human norovirus，hNoV）同属于人类杯状病毒科，且杜兰病毒的基因组成和衣壳蛋白结构等几个重要特征与人源诺如病毒相同，所以可用杜兰病毒作为人源诺如病毒的替代物进行研究。本课题以重组大肠杆菌（Escherichia coli BL21/pET28a-InaQn-TB-P）为研究对象，对加入诱导剂IPTG浓度、诱导温度和诱导时间分别进行单因素优化实验和正交实验，通过SDS-PAGE凝胶电泳检测目的蛋白的表达量，先得出各单因素下的最优诱导条件，再进一步通过正交试验和极差分析初步获得TV衣壳蛋白P结构域表达效果最优条件。实验结果表明，当加入的IPTG终浓度为0.2mmol/L，诱导温度为30℃，诱导时间为18h时，目的蛋白的表达量达到最大为2061.12mg/L。该细菌细胞表面展示重组菌的构建为进一步表达TV衣壳蛋白P结构域奠定基础，为以TV为替代物研究hNoV与受体互作机制，了解hNoV分布及新受体的发掘提供了技术支撑。

关键词：杜兰病毒；人源诺如病毒；极差分析；蛋白质电泳

The IPTG-Induction Optimization of the Expression of P Domain from Tulane Virus Capsid Protein in Recombinant Escherichia Coli

Abstract: The Du Lan virus (Tulane Virus, TV) and the human norovirus (human norovirus, hNoV) belong to the human goblet family, and the gene composition of the Duran virus and the structure of the capsid protein are the same as those of the human norovirus, so the Duran virus can be used as a substitute for human nnovirus. In this study, the recombinant Escherichia coli (Escherichia coli BL21/pET28a-InaQn-TB-P) was used as the research object. The single factor optimization experiment and orthogonal experiment were carried out on the concentration of inducer IPTG, the induction temperature and the induction time respectively. The expression of the target protein was detected by SDS-PAGE gel electrophoresis, and the optimal lure under the single factors was first obtained. The optimal conditions for the expression of TV capsid protein P domain were preliminarily obtained through orthogonal test and range analysis. The experimental results showed that the maximum expression of the target protein was 2061.12mg/L when the final concentration of IPTG was 0.2mmol/L, the induction temperature was 30, and the induction time was 18h. The construction of recombinant bacteria on the surface of the bacterial cell lay the foundation for further expression of the TV P domain of capsid protein. It provides a technical support for the study of the interaction mechanism of hNoV and receptor with TV as a substitute, and to understand the distribution of hNoV and the discovery of new receptors.

Keywords: Duran virus; human Norovirus; Extreme analysis; Protein electrophoresis

目录

1.绪论 1

1.1杜兰病毒概述 1

1.2人源诺如病毒与杜兰病毒 1

1.2.1诺如病毒简介 1

1.2.2人源诺如病毒与杜兰病毒关系 2

1.3细胞表面展示体系 2

1.3.1细菌细胞表面展示技术概述 3

1.3.2细菌细胞表面展示体系的结构组成 3

1.4本课题的研究目的和意义 3

2.材料与方法 5

2.1实验材料 5

2.1.1菌株与重组质粒 5

2.1.2材料试剂 5

2.1.3主要仪器 5

2.1.4培养基及试剂的制备 6

2.2实验方法 7

2.2.1菌种的培养 7

2.2.2目的蛋白诱导表达条件优化 7

3.结果与分析 11

3.1目的蛋白诱导表达的单因素实验凝胶电泳结果 11

3.1.1以诱导剂IPTG终浓度为单一变量的SDS-PAGE凝胶电泳结果 11

3.1.2以温度为单一变量的SDS-PAGE凝胶电泳结果 12

3.1.3以时间为单一变量的SDS-PAGE凝胶电泳结果 13

3.2目的蛋白诱导表达正交实验结果 14

3.2.1正交实验凝胶电泳结果 14

3.2.2极差分析 17

4.结论与展望 19

4.1结论 19

4.2展望 19

致谢 20