VCAM-1基因的克隆及VCAM-1-pcDNA3.1(+)真核表达载体的构建  

Cloning of VCAM-1cDNA gene and constructing eukaryotic expression vector of VCAM-1

作　　者：高蕾[1] 陈幸华[1] 张曦[1] 彭贤贵[1] 孔佩艳[1] 刘林[1] 刘红[1] 张怡[1] 刘耀[1] 

机构地区：[1]第三军医大学新桥医院血液科,重庆400037

出　　处：《重庆医学》2005年第9期1310-1312,共3页Chongqing medicine

基　　金：国家自然科学基金资助课题(30070327);第三军医大学科研基金资助项目(XG200347)

摘　　要：目的克隆人血管细胞黏附分子(vascular cell adhesion molecule-1,VCAM-1)cDNA全长,并构建VCAM-1-pcD-NA3.1(+)真核表达载体。方法采用半巢式RT-PCR从人脐静脉内皮细胞系HUV-EC-C中分三段扩增得到VCAM-1cDNA片段,采用重叠延伸剪切技术(SOE)并经SacⅠ酶切、T4连接酶连接获得VCAM-1完整片段,克隆至pcDNA3.1(+)真核表达载体,得到VCAM-1-pcDNA3.1(+)真核表达载体。结果酶切及测序结果证明VCAM-1-pcDNA3.1(+)真核表达载体构建成功。结论成功扩增人VCAM-1基因,构建VCAM-1-pcDNA3.1(+)真核表达载体,为探索VCAM-1修饰人脐血基质细胞(human umbilical cord blood stromal cell,hUCBSC)重建造血微环境奠定了实验基础。Objective To clone VCAM-1cDNA gene and to construct eukaryotic expressive vector pcDNA3.1 （＋） VCAM-1. Methods The three cDNA segments of VCAM-1 were amplified from HUV-EC-C cell line by semi-nested RT-PCR. Technique of splicing by overlapping extension（SOE）,Sac Ⅰ cut and T4 DNA ligase were used to obtain VCAM-1 segment. Then,VCAM-1 cDNA was cloned into eukaryotic expressive vector pcDNA3. 1 （＋）. Results DNA sequencing and restriction endonuclease digestion analysis indicated that the eukaryotic expression vector pcDNA3. 1 （＋）- VCAM-1 had been constructed successfully. Conclusion VCAM-1cDNA was completely cloned and pcDNA3. 1（＋）- VCAM-1 was successfully constructed. It will provide good experimental basis for further study of the function of VCMA-1 modified hUCBSC.

关 键 词：血管细胞黏附分子 真核表达载体 构建 人脐血基质细胞 

分 类 号：Q78[生物学—分子生物学]