人黑色素瘤特异性抗原基因的克隆及其在原核系统中的表达

目的：克隆及表达人黑色素瘤特异性抗原(PRAME)基因的cDNA片段。方法：从人急性髓细胞白血病细胞系HL-60提取总RNA，用逆转录-聚合酶链反应(RT-PCR)从中扩增出PRAME基因cDNA片段。将PRAME基因cDNA片段插入载体pGEM-T-easy中，经全自动序列分析仪测序正确后，再克隆至表达载体pGEX-4T-2中，构建高效表达克隆，并转化大肠杆菌进行表达。结果：1mmoL／L异丙基-β—D-硫代半乳糖苷(IPTG)诱导5h，PRAME蛋白表达即达高峰。结论：PRAME基因在极少数正常组织中低表达，而在白血病患者高表达，并编码被自体细胞毒T淋巴细胞识别的抗原，所以该基因有望成为肿瘤免疫治疗的新成员。

Cloning of human preferentially expressed antigen of melanoma gene and its expression in E.coli

Objective: To clone and express the preferentially expressed antigen of melanoma (PRAME) gene in E.coli. Methods: The cDNA encoding human PRAME gene was extracted from human AML cell line HL-60 and amplified by RT-PCR, then the PRAME gene was inserted into plasmid PGEM-T-easy. After sequenced, it was cloned into the prokaryotic expression vector pGEX-4T-2 to construct a clone with high level expression and the recombinant plasmid was cloned in E.coli. Results:The protein product reached the highest level at 5 h after IPTG induction. Conclusion: Since PRAME is only expressed at low levels in a few normal tissues and encodes an antigen recognized by autologous cytotoxic T lymphocytes, while expressed at high levels in patients with leukemia, it might be a new targeting candidate for tumor immunotherapy.