摘要
目的:克隆及表达特异性抗原(PRAME)基因的cDNA片段。方法:从人急性髓细胞白血病细胞系HL60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pGEX4T2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βD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。
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.
出处
《医学研究生学报》
CAS
2005年第4期298-301,共4页
Journal of Medical Postgraduates
基金
国家自然科学基金资助项目(批准号:30100218)
关键词
人黑色素瘤特异性抗原基因
白血病
微小残留病灶
基因克隆
表达
Preferentially expressed antigen of melanoma gene
Leukemia
Minimal residual disease
Gene cloning
Expression
