摘要：目的：N-糖基化是毕赤酵母中蛋白质翻译后修饰时对蛋白质正确折叠和表达的一种常见形式，而铜绿假单胞菌弹性蛋白酶的前导肽在其中起着不可或缺的作用。通过定点突变，在重组弹性蛋白酶前导肽中引入四个N-糖基化位点，分析在前导肽中引入N-糖基化位点对该弹性蛋白酶在毕赤酵母中表达的影响。方法：以构建好的克隆质粒pGH-T/lasB为模板，通过定点突变（D11N、K53S、A95S和K129S），在重组弹性蛋白酶前导肽中引入四个N-糖基化位点。利用突变引物进行PCR扩增以得到突变质粒；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，并对在前导肽引入N-糖基化位点是否促进弹性蛋白酶的表达进行了研究。结果：K53S和A95S突变促进了弹性蛋白酶在毕赤酵母中的表达,D11N和K129S突变抑制了其在毕赤酵母中的表达。66649

毕业论文关键词：N-糖基化，定点突变，弹性蛋白酶，毕赤酵母，前导肽，蛋白质表达

Abstract: Objective: N-Glycosylation is a common form of protein post-translational modification in Pichia pastoris and greatly affects folding and secretion. The propeptide of the Pseudomonas aeruginosa elastase (PAE) is indispensable for its proper folding and secretion. Four N-glycosylation sites were introduced into the propeptide of the rPAE by site-directed mutagenesis. The effect on the enhanced expression of recombinant elastase in Pichia pastoris through addition of N-glycosylation sites to the propeptide was analyzed. Methods: Using plasmid pGH-T/lasB as template, four N-glycosylation sites (D11N, K53S, A95S and K129S) were introduced into the propeptide of the recombinant PAE (rPAE) by site-directed mutagenesis; Mutation primers were used to obtain the plasmid mutation; the mutated lasB was subcloned into the expression plasmid pPIC9K; the plasmid was then transformed into Pichia pastoris by electroporation. Purified recombinant elastase was collected, and the relationship between N-glycosylation and the expression of rPAE was analyzed. Results: Compared with the wild-type rPAE, the expression levels of K53S and A95S mutant were considerably enhanced, while D11N or K129S mutation inhibiting rPAE production levels in Pichia pastoris.

Key words: N-glycosylation, Site-directed mutagenesis, Pseudomonas aeruginosa elastase, Pichia pastoris，Propeptide，Protein expression

目   录

引言： 4

1 实验材料及仪器 7

1.1 酶、菌株与质粒 7

1.2 试剂与溶液 7

1.3 实验仪器 8

2 实验方法 8

2.1 定点突变 8

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 12

3.2.1 大肠杆菌感受态制备 12

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