使用人粘附分子-2启动子的衰变加速因子重组基因表达载体的构建及意义

Constructing the eukaryorotic expression vectors of the recombinant human decay accelerating factor gene using the human intercellular adhesion molecule-2 promoter

目的 :构建含人粘附分子 -2(ICAM -2)启动子的人衰变因子 (DAF)重组基因的真核细胞表达载体 ,运用于转基因动物克服异种器官排斥的研究。方法:双酶切本室已构建质粒pGEM -7Zf-DAF ,得到含人ICAM -2启动子及DAFcDNA序列的插入片段 (3.7Kb) ;双酶切pcDNA3真核表达载体 ,得到不含病毒启动子、含筛选基因Neo的一段DNA作为载体序列 (4.4Kb) ;两段DNA进行连接反应后转化细菌 ;阳性转化菌落质粒抽提及酶切鉴定。根据人的ICAM -2启动子、DAFcDNA序列 ,设计引物行PCR特异扩增检验。结果:特异性3组酶切重组表达载体 ,产生符合设计的相应条带 ;PCR扩增出特异的318bp及1.7Kb的DNA片段 ,符合设计要求。结论 :含人ICAM

Objective: To construct an eukaryrotic expression vector of the recombinant human decay accelerating factor gene containing the ICAM-2 promoter in xenotransplantation.Methods: Cutting the pGEM-7Zf -DAF plasmid with restriction endonucleases,and obtaining the recombinant human DAF gene which containing ICAM-2 promotor fragment. In the same way, obtaining the pcDNA3 vector fragment. Above two DNA fragments performed recombinant reaction and the production was transformed into reception germs. The plasmids pick-uped from positive transformed germs were identified by the restriction endonucleases and PCR.Results: Obtaining special fragments by different restriction endonucleases.Further, amplifing and obtaining 318bp and 1.7kp special DNA fragments from the positive plasmid by PCR .These results accord with completely the demands of the designs.Conclusion: The eukaryorotic expression vectors of the recombinant DAF gene were consructed successfully.