巨噬细胞特异性表达载体的构建及鉴定

目的 构建巨噬细胞特异性白喉毒素受体过表达载体并检测其在巨噬细胞中的表达情况。方法 将白喉毒素受体基因Hbegf克隆入pcDNA3.1载体中扩增Hbegf与BGHpolyA尾片段，与克隆入 pUCm-T载体中的巨噬细胞特异性启动子相连接，分别转染巨噬细胞与其他细胞，通过荧光定量PCR鉴定其表达情况。结果 特异性表达载体通过测序以及酶切鉴定构建成功，在巨噬细胞中的表达高于未转染的巨噬细胞以及转染的非巨噬细胞。结论 构建了巨噬细胞特异性表达人白喉毒素受体载体，瞬时转染后可在巨噬细胞中特异性表达白喉毒素受体，为动脉粥样硬化易损性斑块的建立以及斑块内与凋亡相关的其他研究提供了一定依据。

Construction and Identification of Macrophage-specific Expression Vector

Objective To construct a macrophage-specific diphtheria toxin receptor overexpression vector and detect its expression in macrophages. Methods Diphtheria toxin receptor gene Hbegf was digested and inserted into pcDNA3.1 vector to amplify the Hbegf and BGHpolyA. Hbegf and BGHpolyA was inserted into pUCm-T vector ligated with macrophage-specific promoter zone to transfect macrophages and other cells.The expression of macrophage-specific human diphtheria toxin receptor expression vector was detected by using quantitative real-time PCR (qRT-PCR). Results Macrophage-specific human diphtheria toxin receptor expression vector was identified by sequencing and restriction enzyme digestion,and its expression in macrophages was higher than non-transfected macrophages and transfected non-macrophages. Conclusion Macrophage-specific human diphtheria toxin receptor expression vector was successfully constructed. It can specifically express diphtheria toxin receptor in macrophages after transient transfection.It is an important method to establish atherosclerotic vulnerable plaque, and lays a foundation for Other research related to apoptosis.