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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。
Objective To construct and express the eukaryotic expression vector of extracellular domain of vascular endothelial cell adhesion molecule (VCAM-1). Methods Total RNA was extracted from mouse NIH / 3T3 cells and used to amplify the extracellular domain (D1-D4 domain) cDNA of VCAM-1 by RT-PCR. The extracellular region of VCAM-1 was obtained by PCR, and the pMD19-T vector was ligated to sequence the gene. The target fragment of VCAM-1 D1-D4 was inserted into eukaryotic expression vector pIRES2-AcGFP1-Nuc to construct recombinant eukaryotic expression plasmid pIRES2-AcGFP1-Nuc-VCAM-1. Double enzyme digestion identified the extracellular region of VCAM-1 gene eukaryotic expression vector construction of the success or failure. PIRES2-AcGFP1-Nuc-VCAM-1 was introduced into human B lymphoid leukemia cell line (Raji) by liposome. Results The gene sequencing results showed that the extracellular region of VCAM-1 gene was successfully amplified. Double enzyme digestion showed that the recombinant eukaryotic expression plasmid pIRES2-AcGFP1-Nuc-VCAM-1 was successfully constructed. Western blot results showed that VCAM-1 was overexpressed in Raji cells transfected with pIRES2-AcGFP1-Nuc-VCAM-1 plasmid. Cell-binding experiments showed that the expressed VCAM-1 specifically binds to VLA-4 on the surface of pre-B cells (70Z / 3). Conclusion The construction and expression of VCAM-1 eukaryotic expression vector is successful, providing experimental evidence for the formation of pre-B cell clone and the research of B cell differentiation and development.