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GPI锚固型Met-RANTES真核表达载体的构建和表达

Construction and Eukaryotic Expressing of GPI-Anchored Met-RANTES
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摘要 目的:在仓鼠卵巢细胞(CHO细胞)中高效表达糖基磷脂酰肌醇(GPI)锚定修饰的Met-RANTES融合蛋白,以研制新型免疫抑制分子GPI锚固型Met-RANTES。方法:构建真核表达载体PEF/GPI-Met-RANTES,利用电转化法转染CHO-DHFR-细胞,氨甲喋呤(MTX)筛选抗性克隆。用流式细胞仪、细胞免疫荧光和免疫金标记电镜检测细胞膜上GPI锚固型Met-RANTES融合蛋白的表达情况。结果:构建了阅读框完整的GPI锚固型Met-RANTES嵌合分子,经测序是正确的,并在CHO-DHFR-细胞膜上稳定表达。结论:在CHO细胞表面高效表达GPI修饰的Met-RANTES融合蛋白,GPI锚固型Met-RANTES分子有可能作为新型的免疫抑制剂用于器官移植中,抑制移植排斥反应。 Objective ： To express glycosyl-phosphatidylinositol （GPI） modified Met-RANTES fusion protein on Chinese hamster ovary （CHO） cells and to develop a novel immunosuppressant GPI anchored form of Met-RANTES. Methods： The eukaryotic expression vector PEF/GPI-Met-RANTES were constructed and transfected into CHO cells by electroporation. The transfectants were selected with methotrexate （MTX）. Expression of the recombinant protein was assessed by flow cytometric analysis, cell immunofluorescence staining and immunogold electron microscopy. Results ： The chimeric molecules of GPI anchored form of Met-RANTES including the whole reading frame were constructed, and the sequence was identical to the designed sequence. GPI anchored form of Met-RANTES Was stably expressed on CHO-DHFR^- cells. Conclusion： A large amount of GPI modified Met-RANTES fusion protein was expressed on CHO cells. GPI anchored form of Met-RANTES may be used as novel immunosuppressant for suppressing reaction in graft rejection.
作者 李臻 夏峰 张远强
机构地区 第四军医大学组织学与胚胎学教研室
出处 《中国生物工程杂志》 CAS CSCD 北大核心 2006年第6期6-11,共6页 China Biotechnology
基金 国家自然科学基金资助项目(30400250)
关键词 GPI锚 Met-RANTES CHO-DHFR-细胞 真核表达 GPI-anchor Met-RANTES CHO-DHFR^- cell Eukaryotic expression
分类号 Q782 [生物学—分子生物学]
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参考文献12

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二级参考文献10

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中国生物工程杂志
2006年 第6期

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