TAG1抗原HLA-A2限制性表位预测及Hsp70融合蛋白的表达纯化

目的: 对在恶性黑素瘤(MM)组织中表达率极高并具有强免疫原性的新型睾丸肿瘤(C/T)抗原TAG1表位的HLA-A2限制性细胞毒性T淋巴细胞(CTL)表位进行预测,并表达、纯化所预测表位与热休克蛋白70(Hsp70)融合蛋白,为特异性主动免疫治疗MM打下物质基础. 方法: 利用SYFPEITHI 法和多项式方案结合预测TAG1的HLA-A2限制性CTL表位,并将预测所得表位基因片断双串连后进行生物合成,克隆入带有GST标签并含Hsp70基因的原核表达载体,在IPTG诱导表达后,用GST蛋白纯化系统纯化. 结果: 预测并得到了三个TAG1的HLA-A2限制性表位,成功地构建了表位与Hsp70的原核表达质粒(PGEX-bw-HSP70);表达和纯化得到了三个表位与Hsp70的可溶性融合蛋白. 结论: 成功获得三个预测表位与Hsp70的融合蛋白,为进一步研究其免疫功能、研制TAG1对MM的特异性治疗肽疫苗奠定了基础.

Prediction of HLA-A2 restricted cytotoxic T lymphocyte epitopes of TAG1 and expression and purification of fusion proteins of these epitopes and Hsp70

AIM: To predict HLA-A2 restricted cytotoxic T lymphocyte (CTL) epitopes of the novel cancer/testis antigen TAG1 and to express and purify the fusion proteins of these epitopes and Hsp70 in prokaryotic system. METHODS: Long-distance prediction system SYFPEITHI and polynomial method were used to predict the HLA-A2 restricted CTL epitopes of TAG1. The epitopes were then cloned into the expression vector after synthesizing and annealing the nucleotide sequences of these epitopes. After induced with IPTG, the expressed fusion proteins were purified with GSTrap FF column. RESULTS: Three HLA-A2 restricted CTL epitopes of TAG1 were selected. The fusion genes PGEX-bw-Hsp70 were constructed and the fusion proteins were acquired successfully. CONCLUSION: The fusion proteins of Hsp70 and three HLA-A2 restricted CTL epitopes are expressed and purified successfully, which will be helpful in studying their immune functions and develop new peptide vaccines of malignant melanoma.