重组人黑色素瘤抗原MAGE3/HSP70融合蛋白工程菌的高密度发酵及其免疫学活性

High cell-density fermentation of engineered E.coli and antitumor immunity of recombinant human MAGE3/HSP70 in vivo

目的:实现重组人黑色素瘤抗原MAGE3/HSP70融合蛋白工程菌的高密度发酵,并测定MAGE3/HSP70融合蛋白在体内的抗肿瘤免疫效应.方法:利用自控发酵罐发酵,采用溶氧反馈,分批补料的方法对MAGE3/HSP70融合蛋白工程菌进行了高密度发酵,从菌体中纯化MAGE3/HSP70融合蛋白,并用所纯化的MAGE3/HSP70融合蛋白免疫小鼠,运用酶联免疫斑点法(ELISPOT)和细胞毒性杀伤实验(LDH)检测了融合蛋白激活机体细胞免疫反应的状况.结果:高密度发酵取得成功,发酵菌中MAGE3/HSP70融合蛋白的表达量与摇瓶中的相当,摸索出发酵水平纯化MAGE3/HSP70融合蛋白工艺,ELISPOT和LDH显示MAGE3/HSP70可以激活机体免疫反应,并产生针对MAGE3的CTL,特异性杀伤表达MAGE3的肿瘤细胞.结论:MAGE3/HSP70融合蛋白工程菌成功实现高密度发酵,MAGE3/HSP70融合蛋白可以有效激活机体免疫反应,产生针对特异性抗原MAGE3的CTL,免疫学活性良好,为新型抗肿瘤疫苗临床前研究奠定了良好基础.

AIM： To realize high cell-density fermentation of MAGE3/HSP70 engineered E. coli, and to evaluate the antitumor immunity of MAGE3/HSP70 fusion protein in vivo. METHODS ： High cell-density fermentation of MAGE3/HSP70 enginered E. coli was carried out with constant soluble oxygen and continuous cultivation in self-control fermentor. The mouse was immunized by purified MAGE3/HSP70 fusion protein in E. coli, and the antitumot immunity was detected by ELISPOT and LDH release assay. RESULTS： High cell-density fermentation of MAGE3/HSP70 engineered E. coli was successful, and the expression of MAGE3/ HSP70 in fermented bacterial was the same as the flask culture. The results of ELISPOT and LDH release assay showed that MAGE3/HSP70 generated tumor specific cytotoxic T lymphocyte （CTL） to damage tumor cell. CONCLUSION： High celldensity fermentation of MAGE3/HSP70 engineered E. coli was realized and MAGE3/HSP70 fusion protein can generate specific CTL to kill tumor cell.