摘要核酸酶是以核酸为底物，催化磷酸二酯键水解的一类酶。一般核酸酶可以分为DNA酶、RNA酶和非特异性核酸酶三类，其中非特异性核酸酶专一性较低，能非特异性地降解几乎所有形式的核酸，包括单链、双链、线状、环状和超螺旋形式的DNA和RNA，且对核酸的序列没有要求。本实验以来源于Yersinia enterocolitica subsp。Palearctica的非特异性核酸酶（简称Y。e。p非特异性核酸酶）为研究对象，通过与来源于 Serratia marcescens的核酸酶的氨基酸序列进行比对，预测Y。e。p非特异性核酸酶形成两对二硫键，即$1：C18-C22和$2：C208-C252。定点突变形成这两对二硫键的4个半胱氨酸，在蛋白质浓度一致的情况下，将突变体与野生型核酸酶进行热稳定性比较，研究二硫键对Y。e。p非特异性核酸酶热稳定性的影响。最后初步得出结论：Y。e。p非特异性核酸酶中存在两对二硫键，并对维持Y。e。p非特异性核酸酶的热稳定性起重要作用。71955

Abstract Nuclease is a class of enzymes that can degrade DNA and/or RNA molecules by cleaving the phosphodiester bonds that link adjacent nucleotides。 According to consensus criteria, nucleases can be classified into RNase, DNase, and non-specific nuclease。 Non-specific nucleases, with low specificity, can degrade various DNA and RNA, whether it is single-stranded, double- stranded, linear, cyclic, or supercoiled form。 In this experiment, Non-specific nucleases from Yersinia enterocolitica subsp。Palearctica was researched。 Amino acid sequences of nucleases from Yersinia enterocolitica subsp。Palearctica and Serratia marcescens were compared。 The results showed there are two pairs of disulfide bond, $1：C18-C22和$2：C208-C252, which were mutated by site-directed mutagenesis。  In the case protein concentration is consistent, thermal stability of wild type nuclease and mutations was measured and compared to study the effect of disulfide bond on thermal stability of Y。e。p non-specific nuclease。 Finally it concluded that there are two pairs of disulfide bond in Y。e。p non-specific nuclease and disulfide bond plays a key role in thermal stability of Y。e。p non-specific nuclease。

毕业论文关键词： 核酸酶、定点突变、二硫键、热稳定性

Keyword: nuclease, site-directed mutagenesis, disulfide bond, thermal stability

目录

引言 4

1。材料和方法 5

1。1实验材料 5

1。1。1 菌株与质粒 5

1。1。2 酶与试剂盒 5

1。1。3 主要试剂 5

1。1。4 培养基和工作液 5

1。1。5 实验仪器 5

1。2 实验方法 6

1。2。1二硫键预测 6

1。2。2二硫键质谱分析 6

1。2。3 定点突变 6

1。2。3。2 定点突变引物设计 6

1。2。3。3 定点突变基因的获得 7

1。2。4 突变质粒的提取 8

1。2。5 突变酶的诱导表达 9

1。2。6 突变酶蛋白质含量的测定 9

1。2。7 热稳定性分析 10

2。结果与分析 10

2。1二硫键预测 10

2。2二硫键的质谱分析