删除C末端结构域的基质细胞衍生因子-1重组内质网定位片段的真核表达载体构建及其活性作用

THE RECONSTRUCTION EUKARYOTIC EXPRESSION VECTOR OF STROMAL CELL-DERIVED FACTOR-12 WITH DELETION OF C-TERMINAL DOMAIN COMBINATED WITH ER RETENTION SIGAL AND ITS ACTIVATION DETECTION

目的:构建人基质细胞衍生因子(SDF-1)C末端α螺旋结构域缺失及嵌合内质网定位片段的SDF-1α/54/KDEL重组真核表达载体,并考察其对Molt-4细胞的影响。方法:用PCR法扩增SDF-1α/54并将其亚克隆至真核表达质粒pcDNA3.1构建成真核重组表达载体pcDNA3.1/SDF-1α/54/KDEL。脂质体法转染Cos-7细胞后用Western blot检测SDF-1α/54/KDEL的表达。电穿孔法瞬时转染Molt-4细胞,并用流式细胞仪检测CXCR4含量以及其对细胞趋化性的影响。结果:经DNA测序验证目的片段插入方向和碱基组成顺序均准确无误,Western blot证实融合蛋白能在Cos-7细胞表达。SDF-1α/54/KDEL可以显著降低胞膜表面CXCR4表达量并抑制细胞的趋化作用。结论:SDF-1α/54/KDEL可从胞内抑制Molt-4细胞CXCR4的表达,C末端α螺旋结构域的缺失并不影响其抑制作用,推测其是决定细胞趋化作用的主要结构域。

Objective： To construct a eukaryotic expressive plasmid that codes SDF-1α/54/KDEL gene of which stromal cell-derived factor （SDF-1） C-terminal α helix domain deletion combinate ER retention sigal KDEL,and test its biological activity on Molt-4 cells. Methods：The target gene SDF-1α/54/KDEL was amplifed with PCR from the constructed plasrnid SDF-WT-Gly × 4-Dec/PET-30a（＋） and subcloned into eukaryotic expression vectors pcDNA3. 1 to generate recombinant vector pcDNA3. 1/SDF-1α/54/KDEL and then sequenced. After the recombinant plasmids were transfected into Cos-7 cells by liposome, SDF-1α/54/ KDEL expression was determined by Western blot. The recombinant plasmids were isolated and transiently transfected into Molt 4 cells by electroporation. Results：DNA sequencing could prove the direction and insert target genes accuracy,and Western blot could determine the expression of fusion protein in Cos-7 cells. SDF-1α/54/KDEL could remarkablely reduce the membrane surface expression of CXCR4 on Molt-4 cells and inhibit its chemotaxis. Conclusion：SDF-1α/54/KDEL inhibit CXCR4 expression intracellular, loss of Cterminal α- helix domain does not affect this inhibiting effect, suggesting that the domain has the main role in chemotaxis.