E－选择素cDNA克隆和表达  

CLONING AND EXPRESSION OF E-SELECTION cDNA

作　　者：胡美茹[1] 汲言山[1] 舒翠玲[1] 马民慧 董家新[1] 孙涛[1] 沈倍奋[1] 

机构地区：[1]军事医学科学院基础医学研究所

出　　处：《中国免疫学杂志》1996年第3期143-146,共4页Chinese Journal of Immunology

基　　金：863青年基金

摘　　要：从内皮细胞总ＲＮＡ中用ＲＴ－ＰＣＲ方法扩增出Ｅ－选择素ｃＤＮＡ全长编码区，并将其克隆到ｐＵＣ１８载体中，构建了重组质粒ｐＵＣ１８／Ｅ－ｓｅｌｅｃｔｉｎ，对此质粒进行了酶切及ＤＮＡ序列分析鉴定。将Ｅ－ｓｅｌｅｃｔｉｎｃＤＮＡ插入到天坛株痘苗病毒载体ＳｍａＩ位点，构建了表达质粒ｐＪＳＡ１１７５／Ｅ－ｓｅｌｅｃｔｉｎ。采用Ｌｉｐｏｆｅｃｔｉｎ方法，ｐＪＳＡ１１７５／Ｅ－ｓｅｌｅｃｔｉｎ转染ＴＫ－１４３细胞，用ＢｕｄＲ和ＬａｃＺ双筛选，获得带有人Ｅ－选择素ｃＤＮＡ的重组痘苗病毒。经活细胞荧光染色和ＡＰＡＭ检测，重组痘苗病毒能有效地表达人Ｅ－选择素ｃＤＮＡ。Adhesion molecules is a kind of molecules which mediate the interaction of cell with other cell and the extracellular matrix,E-selectin,as a member of adhesion molecule family,has an important role in inflammatory response,lymphocyte homing and tumor metastasis.For studying the relationship of E-selectin function and its structure,had amplified and isolated the full-length cDNA of E-selectin by RT-PCR from endothelial cellular total RNA.The recombinant plasmid pUC18/E- selectin was obtained by inserting the cDNA into pUC18,and was identified by endonuclease and DNA sequencing;The expression plasmid named pJSA1175/E-selectin was constructed by inserting the cDNA into Sma I site of vaccinia virus vectors of Tian Tan strain. Using the lipofectin method and double selection of BudR and LacZ,recombinant vaccinia virus were obtained by transfecting TK-143 cells with pJSA1175/E-selectin.The recombinant vaccinia virus could express the human E-selectin through IFA and APAAP analysis.

关 键 词：E-选择素 粘附分子 痘苗病毒 基因克隆 CDNA 

分 类 号：Q523[生物学—生物化学]