摘要:弹性蛋白酶是一种以水解不溶性弹性蛋白为特征的广谱肽链内切酶,广泛应用于医疗卫生、食品加工、日用化工及环境治理等行业,具有极大的经济价值和应用前景。本研究主要是通过利用基因定点突变等分子生物学手段去除N-糖基化位点I38T、A69T和N266T,来研究N-糖基化位点对毕赤酵母表达的重组弹性蛋白酶活性的影响,并得到酶活更高的突变类型I38T。53162
毕业论文关键词:N-糖基化,弹性蛋白酶,定点突变,毕赤酵母
Abstract: Elastase is a kind of broad spectrum endonuclease peptide chain, which is characterized by hydrolysis of insoluble elastin. It is widely used in many industries such as health care, food processing, household chemicals, and environmental management. It has great economic value and application prospect. Mainly through the gene site-directed mutation and other molecµLar biological methods to remove N-glycosylation sites I38T, A69T, and N266T, this paper attempts to study N-glycosylation sites′ influences on the recombinant elastase activity of Pichia pastoris yeast expression and to obtain a mutant type I38T with higher enzyme activity.
Keywords: N-glycosylation , elastase, site-directed mutagenesis, Pichia pastoris
目录
1 前言 6
2 材料和方法 7
2.1 材料 7
2.1.1 质粒和菌株 7
2.1.2 培养基 7
2.1.3 酶和试剂 7
2.2 方法 8
2.2.1 含有目的基因质粒的提取 8
2.2.2 引物设计和PCR扩增 8
2.2.2.1 引物设计 8
2.2.2.2 PCR基因定点突变 8
2.2.3 感受态的制备和质粒转化与扩增 9
2.2.3.1 感受态的制备 9
2.2.3.2 质粒转化 9
2.2.4 构建其余突变菌株 10
2.2.5 构建毕赤酵母表达质粒pPIC9K及转化筛选 10
2.2.5.1 构建毕赤酵母表达质粒pPIC9K 10
2.2.5.2 转化筛选 11
2.2.6 重组后pPIC9K对毕赤酵母进行转化 11
2.2.7 重组毕赤酵母菌株的诱导表达与产酶的纯化浓缩 12
2.2.8 SDS-PAGE检测及酶活测定 12
3 结果和讨论 12
3.1 含有目的基因的克隆质粒PCR定点突变 12
3.2 克隆质粒转入受体细胞大量繁殖 13
3.3 乙醇沉淀DNA法浓缩克隆质粒 13
3.4 重组质粒的双酶切鉴定 14
3.5 重组表达质粒pPIC9K的鉴定 14
3.6 重组毕赤酵母的转化和诱导产酶 15
3.7 酶液的纯化和SDS-PAGE的鉴定 15
3.8 转化子酶活测定及高产菌株的筛选 16
结论 16
参考文献 18
致谢 20
1 前言