Zhao Kunpeng, Wang Xueyin, Wang Yugang, Ma Yuanfang
Institute of Immunology, Medical College, Henan University, PR China.
Hybridoma (Larchmt). 2011 Jun;30(3):217-21. doi: 10.1089/hyb.2011.0002.
Human tumor necrosis factor related apoptosis inducing ligand (TRAIL) can selectively induce apoptosis in a variety of transformed cells and is currently being developed as a cancer therapeutic drug. Here we expressed the TRAIL protein including extracellular (114-281aa) without any tag protein named TRAIL-NT, and prepared anti-TRAIL polyclonal antibodies (Poly-Ab). The human TRAIL extracellular gene was amplified from PBMC and cloned into pGEM-T-Easy vector for sequence analysis. The expression vector pET-28a/TRAIL was constructed using the DNA recombinant method, and the recombinant protein without any tag protein was expressed in Escherichia coli BL21(DE3). The TRAIL-NT protein was purified by cation ion-exchange column and identified by SDS-PAGE and Western blot analysis. The proliferation inhibition activity of TRAIL-NT was detected by the MTT method, Wright-Giemsa staining assay, and FACS. The polyclonal antibody of TRAIL-NT was obtained after the BALB/C mice were immunized with purificated TRAIL-NT protein. Results showed that the target protein expressed in E. coli BL21(DE3) has the same molecular weight as that expected and could be recognized by anti-TRIAL Poly-Ab. The TRAIL-NT protein could also inhibit proliferation and induced apoptosis of Jurkat cells but no cytotoxicity to human liver cells and PBMC was observed. This preliminary research laid a solid foundation for further research on its biological activity and application in anti-tumor therapy.
人肿瘤坏死因子相关凋亡诱导配体（TRAIL）可选择性诱导多种转化细胞凋亡，目前正作为一种癌症治疗药物进行研发。在此，我们表达了不含任何标签蛋白的细胞外区域（114 - 281aa）的TRAIL蛋白，命名为TRAIL - NT，并制备了抗TRAIL多克隆抗体（Poly - Ab）。从外周血单个核细胞（PBMC）中扩增人TRAIL细胞外基因，并克隆到pGEM - T - Easy载体中进行序列分析。采用DNA重组方法构建表达载体pET - 28a/TRAIL，并在大肠杆菌BL21(DE3)中表达无任何标签蛋白的重组蛋白。通过阳离子离子交换柱纯化TRAIL - NT蛋白，并经SDS - PAGE和蛋白质免疫印迹分析进行鉴定。采用MTT法、瑞氏 - 吉姆萨染色法和流式细胞术检测TRAIL - NT的增殖抑制活性。用纯化的TRAIL - NT蛋白免疫BALB/C小鼠后获得TRAIL - NT多克隆抗体。结果表明，在大肠杆菌BL21(DE3)中表达的目的蛋白分子量与预期一致，且能被抗TRIAL多克隆抗体识别。TRAIL - NT蛋白还可抑制Jurkat细胞增殖并诱导其凋亡，但对人肝细胞和PBMC未观察到细胞毒性。该初步研究为进一步研究其生物学活性及其在抗肿瘤治疗中的应用奠定了坚实基础。
