摘要：目的：通过定点突变，在重组弹性蛋白酶氨基酸序列中引入三个N-糖基化位点，分析N-糖基化对该弹性蛋白酶热稳定性和有机溶剂稳定性的影响。方法：以构建好的克隆质粒pGH-T/lasB为模板，通过定点突变（I38T、A69T和N266T），在重组弹性蛋白酶氨基酸序列中引入三个N-糖基化位点。利用突变引物进行PCR扩增以得到突变质粒；通过基因剪切与连接将突变的lasB基因亚克隆到表达质粒pPIC9K上；电转化到毕赤酵母中并诱导表达。经纯化获得重组弹性蛋白酶，并对N-糖基化修饰与重组弹性蛋白酶稳定性之间的关系进行了研究。结果：I38T和N266T突变对重组弹性蛋白酶的N-糖基化水平显著地提高了。而I38T突变降低了该酶的热稳定性，N266T突变对此却基本没有影响；I38T和N266T突变无明显影响对酶在有机溶剂中的稳定性。54933

毕业论文关键词：N-糖基化，定点突变，弹性蛋白酶，毕赤酵母

 Abstract: Objective: Three N-glycosylation sites were introduced into rPAE by site-directed mutagenesis. Whether N-glycosylation had an effect on the thermal stability and solvent stability of rPAE was analyzed. Methods: Using plasmid pGH-T/lasB as template, three N-glycosylation sites (I38T, A69T and N266T) were introduced into rPAE by site-directed mutagenesis;Mutation primers were used to obtain the plasmid mutation; The mutated lasB was subcloned into the expression plasmid pPIC9K by gene shear and connection; The plasmid was then transformed into Pichia pastoris by Electroporation. Purified recombinant elastase was collected, and the relationship between N-glycosylation and the thermal stability and solvent stability of rPAE was analyzed. Results: Compared with the wild-type rPAE, the glycosylation levels of N266T and I38T mutant protease were considerably enhanced. The I38T mutation reduced the thermal stability of the enzyme, while N266T mutation affected that slightly. Both I38T and N266T mutations had no significant effects on the rPAE solvent stability.  

Key words: N – glycosylation, Site-directed mutagenesis, Pseudolysin, Pichia pastoris

目  录

引言： 5

1 实验材料及仪器 8

1.1 酶、菌株与质粒 8

1.2 试剂与溶液 8

1.3 实验仪器 8

2 实验方法 8

2.1 定点突变 9

2.2 目的基因的亚克隆 10

3 实验流程 11

3.1 基因定点突变 11

3.1.1 引物设计 11

3.1.2 原突变位点附近基因序列 11

3.1.3 引物序列 12

3.1.4 聚合酶链式反应 12

3.2 感受态制备和目的基因的转化 13

3.2.1 大肠杆菌感受态制备 13

3.2.2 目的基因的转化 13

3.2.3 提取质粒并测序 13

3.3 目的基因的亚克隆 13

3.3.1 乙醇沉淀DNA 13

3.3.2 酶切 14

3.3.3 目的基因的连接 14

3.3.4 再次转化筛选 14

3.4 诱导产酶 14

3.4.1 毕赤酵母电转化方法