daintainaif1y54a突变体的构建表达及其在细胞raw264.7中的功能word格式论文.docxVIP

本研究的主要目的是探讨 Daintain/AIF-1 中的 54 位酪氨酸突变对于巨噬细胞 Raw264.7 的生理功能的影响，从而确定 Da inta in/AIF-1 中的 54 位酪氨酸是否有可能 是酪氨酸激酶磷酸化位点。本实验完成的工作主要有以下几个方面：1.采用免疫沉淀的方法特异性富集稳转 pcDNA-Dainta in 的 Raw264.7 细胞系中Dainta in/AIF-1 蛋白。2.使用 PY20 抗体检测 Dainta in/AIF-1 内酪氨酸发生磷酸化。3.运用 生 物 信 息 学 的 方法 确 定 Dainta in/AIF-1 中的 54 位酪氨酸残基在 Dainta in/AIF-1 的功能调节中有重要作用。4.使 用 重 叠 延 伸 PCR 的 方 法 克 隆 出 Dainta in/AIF-1(Y54A) 基因，构建 pET23a(+)-Da inta in/AIF-1(Y54A)载体。表达纯化 Dainta in/AIF-1(Y54A)蛋白。比较 天然Dainta in/AIF-1和 突 变 体Dainta in/AIF-1(Y54A) ，发现 Dainta in/AIF-1(Y54A)促进 Raw264.7 细胞的增殖及 NO 的释放。比较发现，天然 Dainta in/AIF-1 和突变体 Dainta in/AIF-1(Y54A)对 Raw264.7 细 胞的增殖及 NO 释放的影响无明显差异。故 Y54 位酪氨酸没有发生磷酸化。关键词：酪氨酸磷酸化，原核表达，免疫沉淀，Daintain/AIF-1，Dainta in/AIF-1(Y54A)AbstractDainta in，a new bioactive polypeptide was isolate d and c haracterized from porcine intestines by Chen et al in 1994. The amino acid seque nce is 90% homologous with Allograft infla mmatory factor-1(AIF-1) whic h was cloned by Utans et al from activated macropha ges of rat atherosclerotic alloge nic heart grafts. Therefore, we call the polypeptide as ―Da intain/AIF-1‖. This polype ptide is e xpressed in macrophages and secreted from these cells . It has been characterized as a nove l multifunctiona l inflammatory factor taking part in vasculopathy, allograft rejection, cancer, and autoimmune diseases.Dainta in/AIF-1 is a 17kD cytokine whic h is consisted with 146 a mino acid. It has been definited as a member of EH domain family with two EF-hand structures. Some study reveals one of the EF-hand has the ability of binding Ca2+. MutantDainta in/AIF(D95A) lost the ability of binding Ca2+, which resulted in the loss ofVSMC immigration ability. In addition, Daintain/AIF-1 has a motif which can combine with PDZ domain, tyrosine phosphorylation site(site 37, 54, 103, 124), a –KR–KK–GKR– motif .The tyrosine phosphorylation is an important post-translational modification. It plays an important role in cell signa l transduction. The freque