人脐静脉内皮细胞E-选择素分子克隆

Molecular cloning of E-selectin from human umbilical vein Endothelial cells

为探讨Ｅ－选择素结构与功能的关系。方法：采用人脐静脉内皮细胞，经分离培养及激活后，提取 总 ＢＮＡ，用 Ｍ－ＭＬＶ逆转录酶将其转录成 ｃＤＮＡ第一链，设计引物行ＰＣＲ扩增。结果：其产物经电泳测定为 １．９ ｋｂ的 片段，即Ｅ－选择素ｃＤＮＡ的全长编码区。先将ｃＤＮＡ连接至ＰＵＣ－１８载体并转化至ＪＭ－１０９中，提取质粒ＤＮＡ。再将 Ｅ－选择素酶切为３个片段进行序列测定，显示人脐静脉内皮细胞Ｅ－选择素与文献报道略有差异：即５３位Ｇ→Ａ；６５５ 位Ｔ→Ｃ而导致氨基酸的改变（亮氨酸→脯氨酸）；１９１４－１９１６位缺失ＡＧＣ（丝氨酸）。结论：为进一步研究Ｅ－选择素 结构与功能的关系，制备探针与单克隆抗体打下基础。

Aim:To study relationship between E-selectin structure and its function.Methods:Human umbilical vein endothelial cells(HUVECs)were isolated,cultered and activated.Furthermore,RNA was extracted and reverse-transcripted to the cDNA first chain with M-MLA reverse transcriptase.A pair of primers was designed for the PCR.Results:The amplified product was1 1.9 bk segment through the electroporesis determination that was the encode domain cDNA of E-selectin.The cDNA was ligated into the vector PUC-18 and transformed into JM-109.The plasmid DNA was extracted and cut-up into three segments with endonuclease to sequence the DNA.E-selectin cDNA.E-selectin cDNA differed slightly from that reported before.Conclusion:This study has established the basis for further studying the structure frunction relationship of E-eslectin and preparing the probe and monoclone antibody.