血管内皮细胞黏附分子胞外区基因真核表达载体构建及表达

Construction and expression of the eukaryotic expression vector of VCAM-1 extracellular domains

目的构建并表达血管内皮细胞黏附分子(VCAM-1)胞外区基因真核表达载体。方法从小鼠NIH/3T3细胞提取总RNA,以其为模板通过RT-PCR扩增VCAM-1胞外区(D1-D4结构域)cDNA。利用PCR获得VCAM-1胞外区基因,连接pMD19-T载体,进行基因序列测序。将VCAM-1 D1-D4目的片段插入到真核表达载体pIRES2-AcGFP1-Nuc中,构建重组真核表达质粒pIRES2-AcGFP1-Nuc-VCAM-1。经双酶切鉴定VCAM-1胞外区基因真核表达载体构建的成功与否。利用脂质体把pIRES2-AcGFP1-Nuc-VCAM-1导入至人B淋巴性白血病细胞株(Raji)内。结果基因测序结果表明成功扩增出VCAM-1胞外区基因,双酶切鉴定表明重组的真核表达质粒pIRES2-AcGFP1-Nuc-VCAM-1构建成功。Western blot结果显示导入pIRES2-AcGFP1-Nuc-VCAM-1质粒的Raji细胞中VCAM-1高表达。细胞结合实验表明,表达的VCAM-1与前B细胞(70Z/3)表面的VLA-4特异性结合。结论 VCAM-1真核表达载体构建及表达成功,为前B细胞克隆形成机理以及为B细胞分化发育研究提供实验依据。

As we know that the extracellular domains from D1 to D4 of vascular cell adhesion molecule 1(VCAM-1) play important roles during early B cell differentiation.To express VCAM-1 extracellular domains from D1 to D4(VCAM-1 D1-D4),the cDNA segments of VCAM-1 were amplified from NIH/3T3 cell line by PCR,and then VCAM-1 cDNA was cloned into eukaryotic expressive vector pIRES2-AcGFP1-Nuc-VCAM-1.With DNA sequencing and restriction endonuclease(NheⅠ and EcoRⅠ) digestion analysis,it is confirmed that the eukaryotic expression vector pIRES2-AcGFP1-Nuc-VCAM-1 had been constructed successfully.After transformation of pIRES2-AcGFP1-Nuc-VCAM-1 vector to Raji cells,the expression of VCAM-1 was detected in VCAM-1 transformed Raji cells.In cell binding assay,the expressed VCAM-1 was specially interacted with very late antigen-4(VLA-4) on 70Z/3 cells.Our results suggested that the expressed VCAM-1 D1-D4 will provide a experimental basis for further study on B cell differentiation and colony formation.