【摘要】 目的:构建及表达全人源抗肝癌MAGE鄄A1单链抗体(A3)与相思子毒素A链(Abrin鄄A)的原核表达载体,并检测其对人BEL7402肝癌细胞系的杀伤作用。方法:应用PCR方法体外扩增A3基因,经测序后重组入原核表达载体pBAD/gⅢ鄄Abrin鄄A相应位点上,将构建正确的pBAD/gⅢ鄄A3/Abrin鄄A表达质粒转化E.coliTOP10,左旋阿拉伯糖诱导表达。表达产物经蛋白纯化及活性鉴定,采用MTT法检测其对BEL7402细胞的体外杀伤作用。结果:构建的重组免疫毒素表达载体pBAD/gⅢ鄄A3/Abrin鄄A,诱导表达产物主要以包涵体形式存在,分子量约60kD;纯化的pBAD/gⅢ鄄A3/Abrin鄄A对BEL7402细胞的最大杀伤率为70.17%,IC50为3.12μg/ml。结论:构建、表达的全人源抗肝癌A3/Abrin鄄A融合免疫毒素对肝癌细胞有较明显的杀伤作用。更多还原

【Abstract】 Objective: To construct and express a recombinant immunotoxin expression vector composed of a humanized anti-human hepatocellular carcinoma MAGE-A1 single-chain Fv fragment(A3) gene and an Abrin-A gene,and to examine the cytotoxicity of the purified product on human HCC cell line BEL7402. Methods: The A3 gene fragments were amplified by PCR and inserted into corresponding sites of expression vector pBAD/gⅢ-Abrin-A,and the fusion protein expressed in E.coli TOP10 induced by L-arabinose. After purification and identifying its activity,the cytotoxicity of the product on BEL 7402 was evaluated by MTT assay.Results:The new recombinant immunotoxin expression vector pBAD/gⅢ-A3/Abrin-A was constructed successfully. The main product was in inclusion bodies;MTT assay showed that purified pBAD/gⅢ-A3/Abrin-A had obvious effects on BEL7402,the maximum killing rate was 70.17% and IC50 was 3.12 μg/ml. Conclusion: Results of our study on pBAD/gⅢ-A3/Abrin-A and its effects on BEL 7402 show its great potential for the targeted therapy of hepatocellular carcinoma.更多还原