MAGE-3原核表达载体的构建和表达

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

通过RT PCR扩增 95 7bp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 2 1,经IPTG诱导表达 ,并经 12 %SDS PAGE凝胶电泳 ,考马斯亮蓝染色及Westernblot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GlutathioneSepharose 4B纯化后 ,每 10 0mL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 。

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.