GPI-CTLA4Ig嵌合分子的构建和在CHO-dhfr-细胞膜上的表达

目的：研制GPI锚定修饰的新型免疫抑制分子GPI—CTLA4Ig。方法：通过将从促衰变因子(DAF)来源的GPI修饰性信号序列与CTLA4Ig嵌合，获得CPI修饰的CTLA4Ig分子。将编码GPI—CTLA4Ig嵌合分子的基因克隆到真核表达载体pCI—dhfr中，并用脂质体法转染CHO—dhfr^-细胞，用氨甲喋呤(MTX)进行筛选。重组蛋白的表达用RT—PCR、ELISA、细胞免疫荧光和Western blot进行鉴定。最后采用Protein A亲和层析纯化。结果：构建了GPI—CTLA4Ig嵌合分子，并在CHO—dhfr^-细胞膜上稳定表达。结论：GPI修饰的CTLA4Ig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。

Construction of GPI-CTLA4Ig chimeric molecule and its expression on CHO-dhfr- cells

AIM: To develop a novel immunosuppressant GPI CTLAIg modified by glycosyl phosphatidylinositol(GPI). METHODS: GPI modified CTLAIg was produced by linking up of CTLA4Ig with GPI modification signal sequences from decay accelerating factor (DAF). Chimeric molecule GPI CTLA4Ig gene was cloned into eukaryotic expression vector pCI dhfr. Using lipofectine mediated gene transfer technique, pCI GPI CTLA4Ig was transfected into CHO dhfr cells, and the transfectants were screened by methotrexate (MTX). Expression of the recombinant protein was assessed by RT PCR, ELISA, cell immunofluorescence staining and Western blot, and the purification of expressed protein was performed by protein A affinity chromatography. RESULTS: The chimeric molecule GPI CTLA4Ig has been constructed and stablely expressed on CHO dhfr cells. CONCLUSION: GPI modified CTLAIg will may be used as novel immunosuppressant for suppressing reaction in graft rejection.