重组双功能域补体受体Ⅰ型分子在Vero细胞中的稳定表达及其抑制补体活化的初步研究

构建包含人重组双功能域人补体受体Ⅰ与绿色荧光蛋白(GFP)的重组质粒,观察融合蛋白在非洲绿猴肾细胞(Vero)内表达并检测其抑制补体活化的能力。PCR方法扩增出重组双功能域CR1分子,限制性内切酶XhoⅠ和SalⅠ将重组分子连入真核表达载体pEGFP-N2中,构建出重组质粒pEGFP-N2/CR1-2D,脂质体转染Vero细胞中。新霉素G418筛选出稳定表达细胞克隆,荧光显微镜下观察绿色荧光融合蛋白在细胞内的表达。用Vero细胞和免疫小鼠获得的抗Vero细胞多克隆抗体激活补体后,通过检测乳酸脱氢酶的释放来分析重组蛋白抑制补体活化的功能。结果显示pEGFP-N2/CR1-2D质粒经酶切及测序分析证实载体构建正确。转染细胞后,荧光显微镜下观察到重组质粒pEGFP-N2/CR1-2D在Vero细胞中能够大量表达,G418筛选出了稳定表达细胞克隆,乳酸脱氢酶活性检测显示,与对照组相比CR1-2D能够显著的抑制补体的活化(P0.05),初步证实了其能够抑制补体的活化。

Stable expression of the recombinant complement receptor type 1 molecule in Vero cell and the preliminary analysis on its inhibitory effect of complement activation

To construct a plasmid encoding the recombinant human complement receptor 1(CR1-2D) and the green fluorescent protein(GFP) and to investigate the expression of the recombinant CR1-2D/GFP fusion protein in Vero cells in order to detect its inhibitory effect on complement activation,the recombinant CR1-2D gene was amplified using PCR method,digested by XhoⅠand SalⅠ,and then cloned into plasmid pEGFP-N2.After being confirmed by enzyme digestion and sequencing,the recombinant vector was transfected in toVero cells,and G418 selective culture method was used to obtain the stable expression of single-cloned cells and the green fluorescence were observed by fluorescent microscopy.Meanwhile,the inhibitory effect on the complement activation of Vero cells transfected with plasmid pEGFP-N2/CR1-2D after initiation with anti Vero cell polyclone antibodies was analyzed through detecting lactate dehydrogenase released by these cells.The experimental results showed that the recombinant plasmid pEGFP-N2/ CR1-2D had been confirmed to be correct constructed after digestion and sequencing analysis.Following successful transfection to Vero cells with the plasmid,the recombinant pEGFP-N2/CR1-2D could express in the Vero cells in large amount.The stable expression of the single-cloned cells was obtained by G418 selective culture method.The lactate dehydrogenase released by cells transfected with pEGFP-N2/ CR1-2D was greatly reduced(P<0.05) compared with the mock group cells.These results confirmed that the recombinant protein expressed in the Vero cells can inhibit the activation of complement.