定点突变探究盐桥对核酸酶热稳定性的影响

菜单

摘要核酸酶是一类能够降解DNA和RNA分子的酶，在遗传机制研究和生产应用方面都起着重要作用，并具有很高的经济价值。我国具有丰富的核糖核酸（RNA）资源和先进的核酸生产技术，但在推广应用中还存在着一些问题，其中较为突出的是热稳定性问题。故而为了保证核酸酶的高效利用率，提高酶的热稳定性是一种有效的措施。许多研究发现，盐桥是影响蛋白质热稳定性的重要因素，盐桥在蛋白质结构和功能方面都扮演着重要的角色。本实验以Y.e.p非特异性核酸酶为研究对象，定点突变了Y.e.p非特异性核酸酶中含盐键数目最多的盐桥R63Q和含盐键数目最少的盐桥D166Q。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，从而研究盐桥对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：盐桥在Y.e.p非特异性核酸酶热稳定性中起到了关键作用，而且盐键的数量可能与Y.e.p非特异性核酸酶的热稳定性高低成正比。46833

Abstract Nuclease is a kind of enzyme which can degrade DNA and RNA. It plays an important role in the genetic mechanism and production application, and it has very high economic value. Our country has rich resources and advanced nucleic acid (RNA) production technology, but there are still some problems in application, especially in thermal stability. Therefore, in order to ensure high efficient utilization of nuclease, increasing thermal stability of enzyme is a kind of effective measures. Salt bridge has long been considered as an important factor that affects the thermal stability of protein, and plays an important role in protein’s structure and function. In this experiment, the salt bridges mutation of Y.e.p non-specific nuclease, R63Q and D166Q, containing the largest number of salt bond and the least number of salt bond were obtained 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 salt bridges on thermal stability of Y.e.p non-specific nuclease. Finally it concluded that salt bridge plays a key role in thermal stability of Y.e.p non-specific nuclease, and the thermal stability of Y.ep non-specific nuclease is probably proportional to the number of salt bond.

毕业论文关键词：核酸酶、定点突变、盐桥、热稳定性

Keyword: nuclease, site-directed mutagenesis, salt bridge, thermal stability

1 材料与方法.4

1.1 实验材料.4

1.1.1 菌种与质粒4

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.5

1.2.1 盐桥分析..5

1.2.2 突变位点选择.5

1.2.3 定点突变..5

1.2.3.1 定点突变引物设计.5

1.2.3.2 定点突变基因的获得 .6

1.2.4 突变质粒的提取..7

1.2.5 突变酶的诱导表达7

1.2.6 突变酶蛋白质含量的测定7

1.2.7 热稳定性分析 8

2 结果与分析 8

2.1 软件预测结果.9

2.2 突变位点选择.9

2.3 蛋白含量的测定..9

2.4 热稳定性分析.9

3 讨论..10

核酸酶是一类能够通过裂解相邻核苷酸之间的磷酸二酯键，来降解DNA和RNA分子的酶。核酸酶可以分为核糖核酸酶、脱氧核糖核酸酶和非特异性核酸酶。非特异性核酸酶是核酸酶的一种，它可以降解各种DNA和RNA，无论是单链、双链、线状、环状还是超螺旋形式，而且对核酸的序列没有要求。以往的研究表明，核酸酶在遗传机制的不同方面起着重要作用。如防止基因突变，参与DNA修复，DNA复制和重组，冈崎片段起始RNA的去除，凋亡DNA的降解等[1-10]。