异种移植转基因用含粘附分子-2启动子的人CD59 重组基因的构建

目的：构建含人粘附分子－2（ICAM－2）启动子的人补体调节蛋白CD59重组基因,并钭其用于异种器官移植转基因。方法：利用PCR方法从人血基因组扩增得到ICAM－2启动子、CD59基因的第一内含子片段,经电泳分离、切胶回收纯化得到上述片段,分别双酶、单酶切这两条片段,纯化回收备用;双酶切pcDNA3-CD59真核表达载体,经电泳分离,切胶回收纯化得到不含病毒启动子、含筛选基因Neo的一段pcDNA3-CD59cDNA作为载体序列;ICAM－2启动子片段先与载体进行定向克隆连接反应后转化细菌,阳性转化蓖质粒抽提及酶切鉴定,得到pcDNA3-ICAM2-CD59质粒;再单酶切此质粒,纯化该载体与CD59第一内含子片段连接,转化细菌、抽提质粒,用脂质体将该质粒转染猪血管内皮细菌,流式细胞仪检测CD59蛋白表达。结果：得到pcDNA3-Enhancer-ICAM2-CD59质粒,以EcoR I消化此重组质粒,产生5．5及0．5kb两片段,以Hind Ⅲ消化重组质粒,得到5．45及0．55kb两片段,以Kpn I/Hind Ⅲ双酶切质粒时,出现5．0、0．55、0．4kb 3条片段,对插入子分别进行测序,上述结果完全符合设计要求及正确序列。流式细胞仪检测重组基因表达阳性。结论：含人ICAM－2启动子的CD59重组基因表达载体获得成功,该重组基因构建成功为转基因应用奠定了基础。

Construction of recombinant human CD59 using ICAM-2 promoter for endothelial-specific expression in xenotransplantation

OBJECTIVE: To construct a recombinant human CD59 gene containing intercellular adhesion molecule-2 promoter for high level endothelial-specific expression in xenotransplantation. METHODS: ICAM-2 promotor fragment and CD59-intron 1 fragment were produced by PCR from the human blood genome, and then clone these fragments into a pcDNA3-CD59 eukaryotic expression vector which was followed by digestion with the specific restricted endonuclease (for example: EcoRI, Hind III). The ICAM-2 promoter and CD59-intron 1 fragments were identified by PCR, and sequencing. The recombinant was then transfected into pig aorta endothelial cells with Lipofection, and the expression was measured by flow cytometer. RESULTS: Products of the sequences measured were in accord with the frames of the gene bank. The expression of the protein of this recombinant was positive. CONCLUSION: The CD59 recombinant gene is constructed successfully, providing a basis for transgenic research.