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

作　　者：万兵[1] 张利宁[1] 马春红[1] 宋静[1] 曹英林[1] 李长菲[1] 孙汶生[1] 

机构地区：[1]山东大学医学院免疫学研究所

出　　处：《山东医科大学学报》2002年第1期17-18,22,共3页Acta Academiae Medicinae Shandong

基　　金：山东省自然科学基金资助课题(413769)

摘　　要：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.

关 键 词：T淋巴细胞受体 基因表达 载体蛋白质类 人4-1BB 

分 类 号：Q782[生物学—分子生物学]