人4-1BB-hIgG1Fc融合蛋白真核表达载体的构建

Construction of human 4-1BB-hIgG1 fusion protein eukaryotic expressing vector

目的：构建人４１ＢＢ胞膜外区ｈＩｇＧ１Ｆｃ融合蛋白真核表达载体。方法：ＰＣＲ扩增人４１ＢＢ胞膜外区和ｈＩｇＧ１Ｆｃ基因片段，用ＨｉｎｄⅢ、ＰｓｔＩ酶切４１ＢＢ胞膜外区，ＰｓｔＩ 、ＥｃｏＲ Ｉ 酶切ｈＩｇＧ１Ｆｃ ，ＨｉｎｄⅢ、ＥｃｏＲＩ 酶切ｐＣＤＮＡ３，纯化后的酶切产物经Ｔ４ＤＮＡ连接酶连接，转化大肠杆菌ＤＨ５α，氨苄青霉素筛选阳性克隆，ＰＣＲ及测序鉴定。结果：连接反应产物转化大肠杆菌ＤＨ５α，氨苄青霉素筛选出多个阳性克隆；分别以针对４１ＢＢ胞膜外区和ｈＩｇＧ１Ｆｃ的引物进行ＰＣＲ扩增，电泳后观察到一５５８ｂｐ、７０８ｂｐ的特异性条带，与４１ＢＢ胞膜外区和ｈＩｇＧ１Ｆｃ相符，提示构建成功。进一步测序分析证明克隆在ｐＣＤＮＡ３ 质粒中的４１ＢＢ和ｈＩｇＧ１Ｆｃ序列和读码框架正确，无基因突变。结论：成功构建人４１ＢＢ胞膜外区ｈＩｇＧ１Ｆｃ融合蛋白真核表达载体，为表达４１ＢＢ融合蛋白奠定基础。

Objective:To construct a recombinant eukaryotic e xpression vector containing human 4 1BB hIgG1Fc fusion genes.Methods: Extracellular domain of 4 1BB and hIgG1Fc were obtained by PCR.The PCR pro ducts of 4 1BB and hIgG1Fc were digested by HindⅢ and PstI, PstI and EcoRI, r espectively. These digested DNA fragments were purified and recombined with pCDN A3 digested by HindⅢ and EcoRI.The recombinant DNA was transformed into E.col i DH5α. Positive clones were selected with ampicilin and identified by PCR and DNA sequencing .Results:A 558bp specific fragment for 4 1BB and a 7 08bp specific fragment for hIgG1Fc were detected b y PCR in selected clones. DNA sequencing identified that sequence and reading fr ame of 4 1BB and hIgG1Fc in recombined pCDNA3 vector were correct.Conc lusion: The recombinant vector containing human 4 1BB hIgG1Fc fusion ge ne was successfully constructed.