骨肉瘤相关新分子5D3Ag的研究

Identification of a novel osteosarcoma associated antigen 5D3Ag

目的 :自建的抗人骨肉瘤单克隆抗体mAb5D3经鉴定有很好的骨肉瘤细胞特异性 ,并且已利用单抗纯化出相应的骨肉瘤相关抗原 5D3Ag纯品 ,研究目的是测定 5D3Ag氨基酸序列 ,鉴定 5D3Ag是否为一新发现的骨肉瘤相关抗原。方法 :用SDS PAGE法分析 5D3Ag纯度和分子量 ,并做免疫印迹分析 ,用半干式转移法将 5D3Ag转移到氨基酸测序膜上 ,测定其氨基酸序列 ,在蛋白质序列中比较分析 ,初步推断此抗原的性质及是否为新发现的骨肉瘤相关抗原。结果 :5D3Ag经SDS PAGE分析达到了电泳纯 ,分子量为 4 3kD ;WesternBlot分析显示 5D3Ag可以与mAb5D3结合 ;5D3AgN端 10个氨基酸序列为 :门冬酰胺、谷氨酰胺、甘氨酸、丝氨酸、组氨酸、甘氨酸、丝氨酸、谷氨酸、丙氨酸、谷氨酰胺 (NQGSHGSEAQ)。结论 :5D3Ag从其分子量来看与以往文献所报道的骨肉瘤相关抗原的分子量都不相同 ;氨基酸序列结果查询分析 ,与蛋白质库中已知的蛋白质同源性很低 ,由此推断 5D3Ag可能是一种新的骨肉瘤相关抗原。

Objective:To analysis the N-terminal amino acid sequence of 5D3Ag purified by mAb5D3 immunoaffinity column.Methods:SDS-PAGE and Western Blot were used to study the characteristics of 5D3Ag.From SDS-PAGE gel,5D3Ag was transferred to PVDF membrane and its N-terminal amino acid sequence was analysised with an auto amino acid sequencer.Results:SDS-PAGE of 5D3Ag showed a band on the gel with molecular weight of 43 kD.Western Blot study showed that mAb5D3 can recognize 5D3Ag.The N-terminal amino acid sequence was NQGSHGSEEQ.Conclusion:It is sure that 5D3Ag is unique to Osteosarcoma because of the purification method itself and the characteristics of mAb5D3.Compared with other reported Osteosarcoma associated antigen,5D3Ag is different from them in molecular weight and may be a novel Osteosarcoma associated antigen.With the help of N-teminal amino acid sequence,some primers may be designed to clone the whole length gene of 5D3Ag.The identification of 5D3Ag gene may be useful in understanding the biologic mechanics of Osteosarcoma and developing new diagnostic and therapeutic strategies.