MAGE-3原核表达载体的构建和表达

通过RT-PCR扩增957bp的MAGE-3全长编码序列，将该片段克隆至Pgex-4T-2原核表达载体，转化大肠杆菌BL-21，经IPTG诱导表达，并经12%SDSPAGE凝胶电泳，考马斯亮蓝染色及Western blot鉴定，证明了目的基因的有效表达，目的蛋白高达细菌总蛋白的32%。表达产物经Glutathione Sepharose 4B 纯化后，每100mL菌液最终可获得3mg的目的蛋白，蛋白纯度在90%以上。纯化的GST-MAGE-3蛋白在体外冲击树突状细胞，能诱导特异性CTL杀伤肿瘤细胞活性。

Construction of MAGE-3 Prokaryotic Expression Plasmid and Its Expression in Escherichia coli

To express the GST MAGE 3 protein in E.coli , and investigate the antitumor immune responses induced by Dendri tic cells(DCs) pulsed with GST MAGE 3 protein, the recombinant expression plasmid pGEX MAGE 3 was constructed by ligating MAGE 3 gene, which was amplified by RT PCR and confirmed by sequencing, and the pGEX 4T 2 vector. The recombinant plasmid was transformed into BL 21 E.coli . The expression of GST MAGE 3 was induced with IPTG. The GST MAGE 3 protein expressed as high as 32% of the total cellular protein. After purification with Glutathione Sepharose 4B, the purity of the protein was more than 90%, and 3mg GST MAGE 3 was obtained from 100 mL BL 21 lysate. Dendritic cells from gastric carcinoma patients were pulsed with GST MAGE 3 protein, and these DCs were used to stimulate the autologous T lymphocytes. After 7 days, the T lymphocytes cocultured with DCs pulsed with GST MAGE 3 antigen exhibited specific cytotoxicity against MAGE 3 positive SGC 7901 cells. It is concluded that the GST MAGE 3 protein are able to present antigen to T lymphocytes, activate antigen specific CTLs and induce special antitumor immune responses in vitro . Our results lay the groundwork for further research of the MAGE 3 vaccine.