人4-1BB转基因细胞的构建及体外生物学功能的研究

目的构建人共刺激分了4-1BB转基因细胞并探讨4-1BB的体外生物学功能。方法利用RT-PCR方法从人外周血淋巴细胞总RNA中克隆出人4-1BB基因，经双酶切装入逆转录病毒载体pGEZ-Term中，重组逆转录病毒载体4—1BB／pGEZ-Term与两个辅助病毒载体用脂质体法共转染包装细胞293T．用含有完整病毒颗粒的293T细胞的培养上清感染晕新铺板的293T细胞，加入Zeocin选择性培养基进行筛选。经RT-PCR、Western blot、流式细胞仪表型检测等方法鉴定转基因细胞。^3H-TdR掺入法分析转基因细胞对T细胞的作用；DCs培养过程中，加入转基因细胞与激发型CI40单抗5C11．流式细胞仪分析DCs表面分子CD80、CD83和CD86的表达。结果成功构建了能稳定表达人4-1BB蛋白的转基因细胞4-1BB／293T，该转基因细胞与T细胞共培养可抑制其体外增殖，可协同5C11促进DCs的体外成熟，上凋DCs表面分子CD80、CD83和CD86的表达。结论稳定表达4—1BB蛋白的转基因细胞株的建立为该基因功能的后续研究和单克隆抗体的研制奠定了基础。

Construction of Human 4-1BB Transfectant Cell Line and the Study of Its Bioactivity in Vitro

Objective To construct the transfectant cell line 4-1BB/293T and explore the function of 4-1BB in vitro. Methods The gene encoding entire human 4-1BB molecule was obtained by RT-PCR using the mRNA of T cells as templates. Digested with EcoRI and BamH, the PCR product was cloned into corresponding region of pGEZ-Term vector, The recombinant plasmid 4-1BB/pGEZ-Term was transfected into 293T cell line with lipofection and then selected by zeocin. RT-PCR, Western blot and flow cytometry analysis were used to assay the expression stability and efficiency of the target molecule. ^3H-TdR incorporation was used to analyze the effect of 4-1BB/293T on T cells. CD80, CD83, CD86 were tested using FACS after the co-culturing of DCs and 4-1BB/293T cell with the existing of CD40 mAb. Results A stable cell line expressing the human 4-1BB was established successfully. In the presence of anti-CD3 mAb, 4- 1BB/293T was verified to have a potency to inhibit T cell proliferation in vitro. 4-1BB/293T could co-promote the maturation of DCs with CD40 and up-regulate the expression of CD80, CD83 and CD86 on DCs. Conclusion Construction of human 4-1BB transfectant cell line has a great value for the further study of 4-1BB and can provides for the preparation of monoclone antibody of 4-1BB.