溶酶体参与蓖麻毒素A链降解途径的研究

目的:研究溶酶体在蓖麻毒素A链(RTA)细胞内转运过程中的作用.方法:用DNA重组技术,将溶酶体的靶向信号肽KFERQ连接在RTA的羧基端;将构建好的重组质粒pKK223.3-RTA和pKK223.3-RTA-KFERQ转化感受态大肠杆菌JM109,经IPTG诱导表达RTA和RTA-KFERQ蛋白;重组蛋白质用Blue-Sepharose6B亲和柱纯化,以MTT法分别测定纯化后的RTA与RTA-KFERQ蛋白对体外培养的HEPG2(人肝癌细胞)、Hela(人宫颈癌细胞)、A549(人肺癌细胞)3种细胞的毒性作用.结果:在大肠杆菌中成功表达了RTA和RTA-KFERQ.与RTA相比较,纯化的重组蛋白在体外有相似的蛋白质合成抑制活性,RTA-KFERQ对HEPG2、Hela和A549细胞的毒性分别减少了约49.87%、54.18%、88.68%.结论:RTA不能完全被溶酶体灭活,RTA可能存在溶酶体外的降解途径.

Study on lysosomes degradation of ricin A chain

Objective: To study lysosomes involvement in the degradation of ricin A chain. Methods: A lysosome-targeted singal KFERQ was added to the C terminus of rRTA by DNA recombinant technology.A pKK223.3 expression system in E.coli was used to produce recombinant ricine A chain (rRTA) and rRTA-KFERQ.Recombinant proteins were purified by affinity chromatography using Blue-Sepharose 6B.The cytotoxicity of recombinant proteins was measured by the MTT method. Results: Recombinant RTA-KFERQ was 49.87%,54.18% and 88.68% less cytotoxic than RTA itself on the threecell lines HEPG2,Hela and A549,respectively. Conclusion: Lysosomes can degrade,but not completely inactivate RTA in different cells,suggestingcells may have other degradation pathways for RTA.