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摘要：利用冰晶核蛋白（Ice nucleation protein, INP）的N末端作运载蛋白就能高效展示外源蛋白于细菌细胞表面，但展示体系的稳定性能及展示效率的提高还需进一步探讨。用MB 108中提取出的质粒pMB 102转化大肠杆菌JM 109，得到相应重组菌株。通过酶切验证，结果表明质粒成功转化进入大肠杆菌JM 109。本实验基于以INP的N末端为载体蛋白，绿色荧光蛋白GFP为报告蛋白构建大肠杆菌细胞表面展示体系，通过调节可能影响融合蛋白表达及表面固着稳定性的因素，如IPTG浓度、诱导时间、诱导温度等，优化培养条件和诱导条件，最终获得稳定展示GFP的大肠杆菌细胞表面展示条件。经荧光分光光度计检测，结果显示在0.1 mM IPTG室温下(28℃以下)诱导24 h时大肠杆菌细胞表面展示效率达到最优。
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关键词 大肠杆菌；细胞表面展示；绿色荧光蛋白
Preliminary optimization of Escherichia coli cell surface display conditions
Abstract: The N-domain of Ice nucleation protein (INP) can be used as a carrier protein to display foreign proteins on the bacterial surface. However, the stability and efficiency of this surface display system require further study. In this research, a recombinant plasmid named pMB102 was extracted from the recombinant strain MB108, transformed successfully into the Escherichia coli JM109 and tested by restriction enzyme digestion. Then, the recombinant strain, harboring N-domain of INP as carrier protein and green fluorescent protein GFP as a reporter, was used to determine factors affecting the stability and efficiency of surface display system. Finally, the cultural and inducing conditions for the surface display system were figured out by testing the concentration of IPTG, inducing time, inducing temperature and so on. According to the results of fluorescent intensities, the optimal efficiency of this surface display system was showed under room temperature (under 28℃) using 0.1 mM IPTG for 24 h.
Key words Escherichia coli; cell surface display; green fluorescent protein
目录
1前言 1
1.1细胞表面展示技术简介 1
1.2国内外研究状况 2
1.3存在的问题及发展 4
1.4本课题的研究目的和意义 4
2材料与方法 6
2.1实验材料 6
2.1.1菌株与质粒 6
2.1.2培养基配方及培养条件 6
2.1.3试剂 6
2.1.3.1主要分子生物学试剂 6
2.1.3.2质粒提取试剂 6
2.1.3.3琼脂凝胶电泳所需试剂 7
2.1.3.4GFP荧光强度所需试剂 7
2.1.4仪器设备 7
2.2实验方法 7
2.2.1质粒DNA的提取和酶切电泳鉴定 7
2.2.1.1大肠杆菌质粒DNA的少量提取 7
2.2.1.2质粒DNA的电泳鉴定 9
2.2.1.3质粒DNA的酶切验证 9
2.2.2质粒的转化 10
2.2.3细菌的细胞表面定位实验方法 11
2.2.3.1绿色荧光蛋白（GFP）荧光活性的测定 11
2.2.3.2大肠杆菌表面属性体系IPTG诱导浓度的变化 11
2.2.3.4大肠杆菌表面展示体系IPTG诱导温度的变化 11
2.2.3.4大肠杆菌表面展示体系IPTG诱导时间与菌体生长关系 11
2.2.3.5蛋白酶降解实验 11
2.2.3.4细菌生长曲线的测定 11
3结果与分析 13
3.1重组菌MB108的构建 13
3.1.1大肠杆菌质粒PTrcHisc-inpN-gfp的提取结果 13
3.1.2大肠杆菌质粒PTrcHisc-inpN-gfp的酶切验证 13