冰核基因2拷贝串联重复细胞表面展示体系的构建
The construction of cell surface display system using tandem repeats of Ice nucleation protein
Abstract: The ice nucleation protein (INP), a secretory protein from several Gram-negative bacteria, has drawn a wide intresting for development of the bacterial cell surface display system while used as a functional carrier protein. In this study, the N-domain of INP from Pseudomonas syringae was used as carrier protein and the green fluorescent protein (GFP) was used as passenger protein. Based on the previous work, a recombinant plasmid named pTrcHisC-(inpN)2-gfp, which contained two copies of inp-N gene, was constructed by PCR and restriction enzyme digest. Then the plamid was expressed in the Escherichia coli JM109. The recombinant plasmid was proved to be successfully constructed using the determination of fluorescence intensity. The construction of this recombinant plasmid can be used for the functional analysis of InpN as a carrier protein.
Keywords: ice nucleation gene, multi-copy, gene recombination.
目录
1前言 5
1.1冰核蛋白 5
1.1.1冰核蛋白的结构特性 5
1.1.2冰核蛋白的作用机制 7
1.2 细菌细胞表面展示技术概述 7
1.3 冰晶核蛋白的细胞表面展示研究进展 7
1.3.1 报告蛋白展示 8
1.3.2 工业酶展示 8
1.3.3抗原抗体展示 8
1.3.4多肽文库的细菌细胞表面展示 9
1.3.5真核基因展示 9
1.3.6重金属亲合蛋白/多肽的细菌细胞表面展示 9
1.4展望 10
2.本课题的研究目的和意义 10
3.材料与方法 10
3.1 材料 10
3.1.1 菌种与质粒 10
3.1.2 试剂 11
3.1.3 培养基和培养条件 11
3.2 实验方法 11
3.2.1质粒提取 11
3.2.2 引物设计 12
3.2.3 PCR扩增目的基因片段 12
3.2.4 PCR产物的连接与转化 13
3.2.5 两拷贝冰核基因表面展示重组质粒的构建与鉴定 14
4 结果分析 15
4.1 pTrcHisC-inpN-gfp质粒的提取 15
4.1 PCR扩增inpN基因片段 16
4.2 pMD19T-inpN重组质粒的构建以及验证 16
4.3 pTrcHisC-（inpN）2-gfp质粒的构建与验证 19
4.3.1 pTrcHisC-（inpN）2-gfp质粒构建流程图 19
4.3.2 pTrcHisC-(inpN)2-gfp质粒酶切验证图 21
4.4 重组菌的表达与荧光强度检测 21
5.讨论 22
5.1碱裂解法提取质粒不成功的原因分析 22
5.2荧光强度随拷贝数的增加而降低 23
1前言
1.1冰核蛋白
1974 年, 丁香假单胞菌Pseudomonas syringae从赤杨树的腐烂树叶中被分离出来，人们发现细菌可以在细胞外膜上诱导形成冰核类蛋白，从而催化过冷水( Super Cooled Water ) 形成冰晶，于是人们认识到冰¬¬核类细菌是诱发和加重植物霜害和冻害的重要因素。此后, 又相继在其它细菌和昆虫中发现了可以充当生物冰核剂的冰核蛋白[1]。