真核表达载体pcDNA3-ICOSIg的构建及表达

背景：人可诱导共刺激分子0cos）是迄今发现的重要共刺激分子家族成员，可促进激活T细胞的增殖和分泌、调节Th1／Th2细胞的极化、增强依赖T细胞的B细胞功能，阻断ICOS共刺激信号会导致T细胞的克隆失活或克隆无反应，从而诱导肿瘤对机体的免疫逃逸。目的：构建表达ICOSIg的质粒，观察其在小鼠体内的表达。方法：克隆编码ICOS的胞外片段，将其与编码小鼠免疫球蛋白IgG恒定片段（Ig）的基因融合，构建ICOSIg融合基因及其分泌型真核表达载体pcDNA3-ICOSIg，酶切鉴定重组子，测序，利用阳性脂质体载体包被pcDNA3-ICOSlg转染小鼠右侧大腿肌肉组织，Westernblot法检测血清ICOSIg水平。结果与结论：经测序鉴定证实pcDNA3．ICOSIg质粒目的基因片断与Genbank上公布的ICOS序列完全一致，说明质粒构建成功。脂质体载体包被pcDNA3-ICOSIg转染小鼠7d，在小鼠血清中检测到ICOSIg的阳性表达，说明pcDNA3．ICOSlg能够在小鼠肌细胞内表达。证实利用基因合成和重组技术可成功构建真核表达载体pcDNA3-ICOSIg。f201

Construction and expression of eukaryotic expression vector pcDNA3-1COSIg

BACKGROUND： So far, inducible co-stimulator is the important costimulatory molecule family member. Inducible co-stimulator can promote the activation of T cells proliferation and secretion, regulate Thl/Th2 cell polarization dependence, enhance B cell function which depend on the T cells： So, blocking the inducible co-stimulator may result the inactivation and no reaction of cloning in T cells, thus inducing the immune escape of tumor on the body. OBJECTIVE： To build a plasmid expression of inducible co：stimulator Ig, in order to observe the expression in rat body. METHODS： cDNA encoding the extracellular domain of human inducible co-stimulator was prepared. The encode of the domain was fused with the gene of immunoglobulin IgG constant fragment （Ig） of encoding mouse, in order to build the inducible co-stimulator Ig fusion gene and the secreted eukaryotic expression vector pcDNA3-inducible co-stimulator Ig. Enzyme digestion of the recombinant and sequencing was performed, and then the positive liposome coated pcDNA3-inducible co-stimulator Ig was transferred into the muscle tissue of mouse right thigh. Western blot was used to detect the level of inducible co-stimulator Ig.