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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(P<0.05),初步证实了其能够抑制补体的活化。
To construct a recombinant plasmid containing human recombination bifunctional human complement receptor I and green fluorescent protein (GFP), and to observe the expression of the fusion protein in Vero cells and to examine its ability to inhibit complement activation. PCR method to amplify the recombinant bifunctional domain CR1 molecule, restriction endonucleases Xho I and Sal I into the eukaryotic expression vector pEGFP-N2, the recombinant plasmid pEGFP-N2 / CR1-2D, liposome transfer Dye Vero cells. Neomycin G418 screened stable expression of cell clones, observed under a fluorescence microscope green fluorescent protein expression in the cell. After activation of complement with anti-Vero cell polyclonal antibody obtained from Vero cells and immunized mice, the function of the recombinant protein to inhibit complement activation was analyzed by detecting the release of lactate dehydrogenase. The results showed that pEGFP-N2 / CR1-2D plasmid was confirmed by restriction enzyme digestion and sequencing analysis. The transfected cells were observed under a fluorescence microscope recombinant plasmid pEGFP-N2 / CR1-2D in Vero cells can be expressed in large numbers, G418 clones were selected stable expression, lactate dehydrogenase activity test showed that compared with the control group CR1 -2D can significantly inhibit the activation of complement (P <0.05), initially confirmed that it can inhibit the activation of complement.