能和Taq DNA聚合酶作用的蛋白质的筛选
时间:2017-05-25 17:09 来源:毕业论文 作者:毕业论文 点击:次
| 摘要Taq DNA Polymerase是一种来源于嗜热菌Thermus aquaticus的高度热稳定的DNA聚合酶。是从Thermus aquaticus中分离获得了Taq DNA Polymerase并以其耐高温、较高的特异性、灵敏度等优良特性被广泛应用在PCR扩增中。 热启动是在聚合酶链反应(PCR)中先将模板同引物在高于两者变性温度的条件下处理一段时间，然后再加入酶和其他成分进行扩增反应，用以消除非特异性扩增产物的方式。在较低温度时Taq DNA聚合酶和其它蛋白作用，酶作用降低，减少非特异性的PCR产物，只有当热(一般到90℃以上)使蛋白和Taq DNA聚合酶作用解除后，才能进入PCR扩增的热循环。 在以下的实验研究中，将会研究在反应中可与Taq DNA聚合酶作用的蛋白质，并且通过对于上述蛋白质的研究，从中筛选出能够作为和DNA聚合酶的作用蛋白质，通过反应时解除对于Taq DNA聚合酶结合作用，从而达到蛋白热启动的效果 首先，运用基因工程手段将Avi-tag蛋白标签结合到到Taq DNA聚合酶尾端。Avi-tag蛋白标签的表达可以让宿主菌表达的Taq DNA聚合酶尾端出现15个氨基酸短肽标签方便Taq DNA聚合酶的提纯和筛选。 其次用链霉亲和柱纯化该酶，利用SDS-PAGE电泳判断除吸附在链霉亲和柱上的Taq DNA聚合酶外是否还有其它吸附的蛋白质。 然后，通过混合不含Avi-tag标签的DNA聚合酶菌液和含有Avi-tag标签的DNA聚合酶菌夜，重复上述步骤来检测另外两种菌夜中是否含有和Taq DNA聚合酶作用的蛋白质 最后，比较含有混合DNA聚合酶菌液和不含混合液的Taq DNA聚合酶在PCR反应中非特异性的改变，验证所筛选蛋白是否可以适用作为热启动蛋白。 论文初步解决了筛选和Taq DNA聚合酶作用的蛋白质的实验操作流程，并且，做出了相关验证和理论猜想。9168 关键词：PCR，Taq DNA聚合酶，链霉亲和柱，SDS-PAGE Abstract Taq DNA Polymerase is a derived from the Thermus aquaticus bacterium, a high degree of heat stable DNA polymerase. It is isolated from Thermus aquaticus Taq DNA Polymerase .Because of its high working temperature, high specificity, sensitivity, and other excellent features it is widely used in the PCR amplification. At first the hot start is using template in the polymerase chain reaction (PCR) with primers in the conditions above the denaturation temperature of two primers, and then adding enzymes and other components of the amplification reaction, to eliminate the non-specific amplification. Taq DNA polymerase and other protein get together at lower temperature with enzymes reducing non-specific PCR product. Only when the heat (usually more than 90 ℃) , the Taq DNA polymerase isn’t combining with the protein any more, the PCR amplification increases thermal cycling. In the following experimental study will examine in the reaction with Taq DNA polymerase and for these proteins from the filter can be used as the role of protein and DNA polymerase, by the reaction time to lift for Taq DNA polymerase binding role, so as to achieve the effect of protein hot start First, by the use of genetic engineering, Avi-tag protein tag is showed to the end of the Taq DNA polymerase. The expression of the avi-tag protein label which is 15 amino allows the end of the Taq DNA polymerase expression by the host bacterium .The label is used to facilitate the purification of Taq DNA polymerase Then, we use the SDS-PAGE to judge whether there were other proteins attached by the Streptavidin column for purifying of the enzyme Then, through mixing Avi-tag label-free DNA polymerase bacteria with having a Avi-tag-label DNA polymerase bacteria ,then repeating the above steps to detect whether it contains any protein which can combine with Taq DNA polymerase. Finally, By comparing with pure DNA polymerase which do not contain mixture protein , how the mixture protein performs in the PCR reaction nonspecific changes. It shows that the filtered protein can be applied as a hot start protein. (责任编辑：qin) |